<<

Characterization of Ribosomes and Ribosome Assembly Complexes by

Mass Spectrometry

A dissertation submitted to the

Graduate School of the University of Cincinnati

In partial fulfillment of

the requirements for the degree of

Doctor of Philosophy (Ph.D.)

In the Department of Chemistry

of the College of Arts and Sciences

2013

By

Romel P. Dator

M.S. Chemistry, University of the Philippines Diliman, 2008

B.S. Chemistry, University of the Philippines Visayas, 2003

Committee Chair: Prof. Patrick A. Limbach Abstract

The biogenesis and assembly of the ribosome involves a coordinated cascade of events including

rRNA processing, folding, and post-transcriptional modifications of rRNA along with the

association of ribosomal proteins (r-proteins). Unraveling the pathways and dynamics of these

complex structural processes is a significant challenge. While conventional biophysical techniques

such as nuclear magnetic resonance (NMR), X-ray crystallography and cryo-electron microscopy

(cryo-EM) provide high-resolution information, these methods are not ideally suited to

characterize transient and heterogeneous ribosome assembly intermediate structures. Here mass

spectrometry-based approaches are being used to gain insights into the composition and structural

organization of ribosomes and ribosome assembly particles in vivo, particularly those particles that

result from perturbations (e.g. deletion of assembly factors, antibiotics).

15N-labeling and data-dependent LC-MS/MS were used to characterize the proteins associated

with pre-30S complexes from E. coli RimM and RbfA deletion strains. RimM and RbfA are

ribosome assembly factors implicated in the maturation of the small 30S subunit in bacteria. The

precise roles of these assembly factors in 30S subunit assembly are unclear. Along with in vivo x-

ray footprinting and mass spectrometry data, detailed molecular mechanisms how RimM and

RbfA facilitate maturation of the 30S subunit in vivo were uncovered.

Although relative quantitation of proteins by 15N-labeling and LC-MS/MS provides information

on the differential expression of proteins in normal and perturbed samples, this approach is limited

to comparing two samples at a time, labeling can be expensive and laborious, and not amenable to

other multicellular organisms. The applicability of a label-free approach, LC-MSE for absolute

“ribosome-centric” quantification of r-proteins was evaluated. Using an additional dimension of

gas-phase separation through ion mobility and multiple endoproteinase digestion allow accurate

and reproducible quantitation of proteins associated with mature ribosomes. The improved LC-

i MSE approach was then extended to characterizing proteins associated with different functional

states of the ribosomes (free 30S, free 50S, 70S and polysomes). The actively translating

ribosomes (polysomes) contain stoichiometric amounts of proteins consistent with their known

stoichiometry within the complex. Significant heterogeneity was found with free subunits as they

are composed of immature complexes and dissociated subunits from 70S. The stoichiometric

measurements among the different classes of ribosomes showed very good run-to-run

reproducibility and biological reproducibility with %CV less than 15% and 35%, respectively.

Finally, the in vivo assembly complexes formed in the presence of the antibiotic erythromycin was

isolated and characterized. A strategy was devised to isolate and purify the erythromycin-induced

50S assembly particle in SK5665 cells grown in the presence of the antibiotic erythromycin.

Quantitative analysis of the proteins associated with the Δ50S particles suggests a heterogeneous

collection of 50S intermediates with different subsets and varying amounts of proteins. The

amounts of the assembly factors, SrmB and DbpA, detected in the Δ50S particle indicate that the

Δ50S particle is immature and is a late assembly intermediate.

ii

iii Acknowledgments

I would like to express my sincerest gratitude to my advisor, Professor Patrick A. Limbach, for giving me this great opportunity to work under his guidance, supervision, and for the countless opportunities to grow and develop as a scientist. Thank you for the enlightening intellectual discussions and for inspiring me to pursue bigger scientific possibilities. I would like to thank my dissertation committee members, Professor Albert Bobst and Professor Joseph Caruso, for their guidance, constructive criticisms to improve my scholarly work, and for being part of my professional development.

A big thank you to my collaborators, Professor Sarah Woodson and Sarah Clatterbuck Soper (Johns Hopkins University) for a very successful collaborative work. To my colleagues and mentors, Dr. Kirk Gaston, Dr. Balu Addepali, and Dr. Anne McLachlan for sharing me their expertise, help for the techniques I am not so familiar with, and most importantly for the fruitful scientific discussions. The current and past Limbach Group members for making my research life fun, enjoyable, and highly stimulating.

I would like to thank Professor Ken Greis, Dr. Wendy Haffey, and Therese Rider (UC Department of Cancer Biology) for allowing me to use their MALDI MS instrument, access to the Mascot Server for my proteomics analysis, and for the technical assistance. To Dr. Stephen Macha and Dr. Larry Sallans (Mass Spectrometry Facility) for their help and guidance when I need to run my samples in the facility’s mass spectrometers.

My heartfelt thanks to Dr. Ann P. Villalobos for being instrumental to my graduate school career and for the enormous help, support and encouragement all these years. To my master’s advisors, Dr. Sonia Jacinto and Dr. Amelia Guevara for motivating me to pursue graduate studies in US and for paving my intellectual curiosity to advance my career in science.

To my best friend, soon to be Dr. Morwena Jane V. Solivio, for being a fun and enjoyable buddy and for the great memories we have had all these years. To my family and friends, for the love and support and for inspiring me to become a better and compassionate person.

I am thankful for the National Institutes of Health (NIH) for the funding and the Chemistry Graduate Student Association (CGSA) for giving me the opportunity to serve and to explore my leadership capabilities.

My thanks and appreciation to the UC Department of Chemistry, faculty and staff and the University of Cincinnati for giving me this once in a lifetime opportunity to pursue graduate studies here and for being part of my scientific aspirations.

iv Table of Contents

Abstract……………………………………………………………………………...... i

Acknowledgments………………………………………………………………….…………...... iv

List of Tables………..…………………………………………………………………...... vii

List of Figures……………………………….……………………………………...... ix

List of Schemes………………………………….……………………………………………………xiv

List of Abbreviations…………………………….…………………………………………...... xv

Chapter 1. Introduction and Background…………………………………………………………....1

1.1 Ribosome Structure, Function, and Antibiotic Action……………………….……….…....1

1.1.1 Ribosome Structure…………………………………………………………...... 1

1.1.2 Ribosome Function……………………………………………………………...4

1.1.3 Ribosome as Target of Antibiotics…………………………………….………...5

1.2 Bacterial Ribosome Biogenesis and Assembly………………………………………...... 6

1.2.1 Posttranscriptional Modifications of Ribosomal RNA………………………….7

1.2.2 Posttranslational Modification of Ribosomal Protein…………………….……..9

1.2.3 In Vitro and In Vivo Studies of Ribosome Assembly……………………….....10

1.2.4 Ribosome Assembly Factors………………………………………………...... 14

1.2.5 Ribosome Assembly as Target of Antibiotics…………………………...……..17

1.3 Biophysical and Biochemical Methods to Study Ribosome Assembly………...... 19

Chapter 2. Literature Review…...... 22

2.1 Characterization of Ribosomal Proteins by Mass Spectrometry……………………….....22

2.1.1 Identification of Ribosomal Proteins Their Posttranslational Modifications…..25

2.1.2 Analysis of Ribosomal Proteins to Probe Ribosome Topology………………..28

2.2 Mass Spectrometry-Based Quantitative Analysis of Ribosomal Proteins………………...29

2.2.1 Stable Isotope Labeling for Relative and Absolute Quantitation…………...... 31

2.2.2 Label-Free Approaches for Relative and Absolute Quantitation…………...... 32

v 2.3 Purpose of the Work Presented……………………………………………………...... 35

Chapter 3. Analysis of 30S Ribosomal Subunit Assembly Particles in RimM and RbfA

Escherichia coli Deletion Mutants by Mass Spectrometry

3.1 Introduction……………………………………………………………...... 38

3.2 Experimental………………………………………………………………………...... 38

3.3 Results and Discussion……………………………………………………………...... 43

3.4 Conclusion………………………………………………………………………...... 53

Chapter 4. Quantitation of Bacterial Ribosomal Proteins by LC-MSE

4.1 Introduction……………………………………………………………………………...... 54

4.2 Experimental………………………………………………………………………...... 57

4.3 Results and Discussion………………………………………………………………….....61

4.4 Conclusion…………………………………………………………………...... 71

Chapter 5. Characterization of Ribosomal Proteins Associated with the Different Functional

States of the Ribosome

5.1 Introduction……………………………………………………………………………...... 73

5.2 Experimental…………………………………………………………………………...... 74

5.3 Results and Discussion………………………………………………………………...... 75

5.4 Conclusion…………………………………………………………………...... 87

Chapter 6. Mass Spectrometry-Based Characterization of the Erythromycin-Induced Ribosome

Assembly Particles in Escherichia coli

5.1 Introduction…………………………………………………………………………..…....88

5.2 Experimental…………………………………………………………………………...... 88

5.3 Results and Discussion………………………………………………………………...... 92

5.4 Conclusion…………………………………………………………………...... 104

Chapter 7. Conclusions and Future Directions…………………………………………………...106

References…………………………………………………………………………...... 115

vi List of Tables

1.1 Summary of ribosomal proteins and their associated functions in protein translation…...... 3

1.2 Posttranscriptional modifications of ribosomal RNA in Escherichia coli………………...... 9

1.3 Posttranslational modifications of ribosomal proteins in Escherichia coli…………………10

1.4 Protein complement of in vitro and in vivo 30S assembly intermediates…………….…...... 13

1.5 Protein complement of in vitro and in vivo 50S assembly intermediates……………….…..14

1.6 Summary of protein factors that are involved in regulation of ribosome biogenesis and protein synthesis…………………………………………………………………………….16

2.1 Strategies and approaches in mass spectrometry-driven proteomics………………………..23

2.2 Properties of Escherichia coli K12 30S and 50S ribosomal proteins……………………….26

2.3 Quantitative mass spectrometry-based quantitative proteomics…………………………….31

3.1 Quantification of S5 acetylation from various sources. Recombinant S5 was overexpressed in E. coli cells and purified before analysis. K12 proteins were isolated from sucrose gradient fractions as indicated. Pre-30 proteins were obtained from oligonucleotide affinity- purified complexes…………………………………………………………………………..48

5.1 Run-to-run reproducibility of the stoichiometric measurements of small 30S subunit proteins in wild-type polysomes……………………………………………………………………...83

5.2 Run-to-run reproducibility of the stoichiometric measurements of large 50S subunit proteins in wild-type polysomes……………………………………………………………………...84

5.3 Biological reproducibility of the stoichiometric measurements of small 30S subunit proteins in wild-type polysomes……………………………………………………………………...85

5.4 Biological reproducibility of the stoichiometric measurements of large 50S subunit proteins in wild-type polysomes……………………………………………………………………...86

6.1 Large 50S ribosomal subunit proteins detected in wild-type K12 50S and SK5665 50S with and without erythromycin and Δ50S particle……………………………………………….98

6.2 Non-ribosomal proteins detected in mature 50S of wild-type K12 and SK5665 with and without erythromycin and Δ50S particle……………………………………………………99

6.3 Comparison of the protein complement of in vitro and in vivo 50S intermediates with that of the erythromycin-induced Δ50S particle…………………………………………………..100

6.4 Small 30S ribosomal subunit proteins detected in wild-type K12 30S and SK5665 30S with and without erythromycin and Δ50S particle……………………………………………...104

vii List of Tables (continued)

7.1 Large 50S ribosomal subunit proteins detected in AD2291 tagged 23S rRNA complexes harvested at different time points…………………………………………………………..112

viii List of Figures

1.1 Escherichia coli 70S ribosome. Escherichia coli 30S (PDB 2AVY) and 50S (PDB 2AW4) subunits modeled using PyMOL software……………………………………...... 1

1.2 Cartoon representation of the bacterial rrnB operon showing one primary transcript for the 16S, 23S, and 5S rRNA. The rRNA and tRNA components, promoters (P1 and P2), and terminators (T1 and T2) are shown…………………………………………………………...7

1.3 Assembly map of the 30S subunit as deduced by Nomura. Ribosomal proteins are categorized as primary binding proteins, secondary binding proteins, and tertiary binding proteins………………………………………………………………………………………11

1.4 Assembly map of the 50S subunit. R-proteins associated with the reconstitution intermediates, RI50(1) and RI50(2) are shown……………………………………………….12

1.5 Cartoon representation of bacterial ribosome assembly. This process requires a multitude of components necessary in the maturation of the small and large ribosomal subunits……….15

1.6 Model of the bacterial 50S subunit assembly inhibition by erythromycin. In the presence of the drug, resistant cells can form mature 50S subunit (top), however in sensitive cells (bottom), assembly stalls creating an improperly folded assembly particles, which is then targeted for degradation by cellular ribonucleases. By inactivation of RNase E at non- permissive temperatures, isolation of assembly particles that can be detected in sucrose gradients is possible…………………………………………………………………………18

1.7 Absolute quantification of proteins by LC-MSE. This method is based upon the observation that the average signal response of the three most intense peptides per mole of protein is constant with (CV) of less than 10% evaluated for proteins with molecular weight od 14 kDa to 97 kDa………………………………………………34

2.2 Overview of the LC-MSE approach for absolute quantification of proteins………………..34

3.1 Relative quantities of r-proteins in wild-type (K12) 30S ribosomes, ΔrimM and ΔrbfA (L+H) pre-30S complexes. For each protein, the ratio of unlabeled (L) peptide in the test sample to 15N-labeled (H) peptide from MRE600 TP30 was determined by data-dependent LC-MS/MS and data processing on Mascot Distiller. Relative quantities are normalized to protein S8. Error bars represent the of three technical replicates. R-proteins are organized by their position on the Nomura map and colored by their location in the structure: white, 5’ domain (body); light gray, central domain (platform); dark gray, 3’ domain (head); black, S1……………………………………………………………………44

3.2. MALDI-TOF MS analysis of intact proteins from wild-type K12 total 30S proteins. Proteins S5 and S18 are fully acetylated in mature 30S subunit……………………………………..46

3.3 MALDI-TOF MS analysis of intact proteins from ΔrimM pre-30S complexes. ProteinS18 is mostly unmodified in ΔrimM complexes……………………………………………………47

3.4. MALDI-TOF MS analysis of intact proteins from ΔrbfA pre-30S complexes. Protein S18 is mostly modified in ΔrbfA complexes……………………………………………………….47

ix List of Figures (continued)

3.5 (A) Selected ion chromatograms (SIC) of the acetylated (m/z 548) and non-acetylated (m/z 537) peptide fragments from ΔrimM TP30. (B) Mass spectrum of the 4+, 3+, and 2+ charge states of the acetylated peptide from ΔrimM TP30. (C) Mass spectrum of the 4+, 3+, and 2+ charge states of the non-acetylated peptide fragment from ΔrimM TP30………………..…49

3.6 (A) Selected ion chromatograms (SIC) of the acetylated (m/z 548) and non-acetylated (m/z 537) peptide fragments from ΔrbfA TP30. (B) Mass spectrum of the 4+, 3+, and 2+ charge states of the acetylated peptide from ΔrbfA TP30. (C) Mass spectrum of the 4+, 3+, and 2+ charge states of the non-acetylated peptide from ΔrbfA TP30……………………………...50

3.7 Collision-induced dissociation (CID) fragmentation of (A) acetylated and (B) non-acetylated peptide fragments of S5 from ΔrimM TP30………………………………………………...51

3.8 Collision-induced dissociation (CID) fragmentation of (A) acetylated and (B) non-acetylated peptide fragments of S5 from ΔrbfA TP30………………………………………………….52

4.1 Plot of the molecular weight of bacterial ribosomal proteins. More than 50% of the proteins have molecular weight less than 14 kDa, a cut-off range that was originally determined in quantifying proteins by LC-MSE……………………………………………………………56

4.2 Quantitation of small subunit 30S proteins and large subunit 50S proteins by LC-MSE as described by Silva et al. (2006). R-proteins are not uniformly distributed and are detected in less than 1 copy per ribosome. Amounts of small subunit proteins and large subunit proteins are normalized to protein S4 and protein L3, respectively. Error bars represent the standard deviation of 3 multiple measurements………………………………………………………62

4.3 Comparing single protease (trypsin) digestion with and without ion mobility separation. Plots of the number of peptides identified for (A) 30S subunit proteins and (B) 50S subunit proteins. Plots of protein sequence coverage of (C) 30S subunit proteins and (D) 50S subunit proteins. Additional dimension of gas-phase separation through ion mobility increases the number of peptides detected and sequence coverage of the proteins.………………...... …..64

4.4 Quantitation of (A) small subunit 30S proteins and (B) large subunit 50S proteins by LC- MSE with additional dimension of gas-phase separation through ion-mobility. Error bars represent the standard deviation of 3 multiple measurements……………………………....65

4.5 Comparing single protease (trypsin) digestion with IMS and dual protease (trypsin and Asp- N) digestion with IMS. Representative plots of the number of peptides identified for (A) 30S subunit proteins and (B) 50S subunit proteins. Representative plots of protein sequence coverage of (C) 30S subunit proteins and (D) 50S subunit proteins. Dual protease digestion and using ion mobility increase the number of peptides detected and sequence coverage of the proteins……………………………………………………………………………..……67

4.6 Comparing dual protease (trypsin and Asp-N) digestion with and without ion mobility separation. Representative plots of the number of peptides identified for (A) 30S subunit proteins and (B) 50S subunit proteins. Representative plots of protein sequence coverage of (C) 30S subunit proteins and (D) 50S subunit proteins. Ion mobility separation increases the number of peptides detected and sequence coverage of the proteins generated upon dual protease (trypsin and Asp-N) digestion…………..………………………………………....68

x List of Figures (continued)

4.7 Quantitation of small subunit 30S proteins using ion mobility separation and dual protease digestion. Protein levels are normalized to protein S4……………………………………...69

4.8 Quantitation of large subunit proteins using ion mobility separation and dual protease digestion. Protein levels are normalized to protein L3……………………………………...69

4.9 The number proteins identified in database searching dramatically increased with ion mobility separation and dual protease digestion…………………………………………….70

4.10 Quantitation of small subunit 30S proteins and large subunit proteins using ion mobility separation and multiple endoproteinase digestion. A uniform distribution of proteins is observed which is in agreement with their known stoichiometry within the complex. Amounts of small subunit proteins and large subunit proteins are normalized to protein S4 and protein L3 respectively. Error bars represent the standard deviation of 3 multiple measurements………………………………………………………………………………..71

5.1 10-50% sucrose gradient profile of crude cell lysate from wild-type E. coli K-12 cells in non-dissociating conditions. The different functional states of the ribosomes including the free subunits (30S and 50S), single ribosomes (70S), and polysomes are shown…………..76

5.2 Quantitation of large 50S subunit and small 30S subunit r-proteins associated with wild-type polysomes. Protein amounts were normalized to protein L3 and protein S4 for the large subunit and small subunit, respectively. Data represent the of the normalized protein amounts of two biological preparations of polysomes with three technical replicates each. Error bars represent the standard deviation of the 6 trials…………………………………..28

5.3 Quantitation of large 50S subunit and small 30S subunit r-proteins associated with wild-type 70S ribosomes. Protein amounts were normalized to protein L3 and protein S4 for the large subunit and small subunit, respectively. Data represent the mean of the normalized protein amounts of two biological preparations of 70S ribosomes with three technical replicates each. Error bars represent the standard deviation of the 6 trials…………………………………..77

5.4 Plot of log2 fold change of r-proteins in K12 70S ribosomes over K12 polysomes. Protein amounts were normalized to protein L3 and protein S4 for the large subunit and small subunit, respectively. Positive values indicate that the amounts of proteins are reduced in 70S ribosomes compared to K12 polysomes while negative values indicate otherwise. Data represent the mean of the normalized protein amounts of two biological preparations of 70S ribosomes with three technical replicates each. Error bars represent the standard deviation of the 6 trials……………………………………………………………………………………78

5.5 Quantitation of large 50S subunit and small 30S subunit r-proteins associated with wild-type free subunits. Protein amounts were normalized to protein L3 and protein S4 for the large subunit and small subunit, respectively. Data represent the mean of the normalized protein amounts of two biological preparations of 70S ribosomes with three technical replicates each. Error bars represent the standard deviation of the 6 trials…………………………………..80

xi List of Figures (continued)

5.6 Plot of log2 fold change of r-proteins in free subunits over K12 polysomes. Protein amounts were normalized to protein L3 and protein S4 for the large subunit and small subunit, respectively. Positive values indicate that the amounts of proteins are reduced in free subunits compared to K12 polysomes while negative values indicate otherwise. Data represent the mean of the normalized protein amounts of two biological preparations of free subunits with three technical replicates each. Error bars represent the standard deviation of the 6 trials……………………………………………………………………………………81

6.1 Growth curves of SK5665 cells grown in 2xYT media (25°C) with and without erythromycin. Final erythromycin concentration used was 18 µg/mL……………………...93

6.2 0-45% sucrose gradient profile of crude ribosome from SK5665 cells (A) with erythromycin (Ery) and (B) without erythromycin. Cells grown with erythromycin showed an abnormal particle that sediments ~35-40S in sucrose gradients. This particle is absent in cells grown without the antibiotic………………………………………………………………………..94

6.3 Representative LC-MS/MS spectral data of the tryptic digests from K12 50S proteins at 15 ug starting total protein concentration. (A) Total ion chromatogram (TIC) of the tryptic digests (B) MS spectrum of the peak eluting at 37 min showing a base beak at m/z 840.8. (C) MS/MS spectrum obtained by collision-induced dissociation (CID) of m/z 840.8. The fragmentation pattern corresponds to a peptide fragment from the E. coli large ribosomal subunit protein L6…………………………………………………………………………...97

6.4 Quantitation of r-proteins in Δ50S particle and mature 50S subunit. (A) Plot of log10 (fold change) of proteins from mature SK5665 50S over wild K12 50S subunit. (B) Plot of log10 (fold change) of proteins from Δ50S particle over wild K12 50S subunit. R-proteins are grouped into early binders, moderate binders, and late binders as previously described. Protein amounts were normalized to protein L3. Measurements were done in triplicate and error bars represent standard deviations of the three trials………………………………...101

6.5 Quantitation of ribosome assembly factors DbpA and SrmB detected in Δ50S particle….103

7.1 E. coli model system used to characterize the events during the biogenesis and assembly of the 50S subunit. A streptavidin binding aptamer (red segment) is incorporated into the functionally neutral helix 25 of the 23S rRNA allowing purification of pure ribosomal 23S rRNA complexes…………………………………………………………………………...109

7.2. Expression of tagged rRNA from the phage λ promoter is induced by raising the temperature from 25°C to 42°C. As the transcription and assembly of the tagged rRNA proceeds, 23S rRNA complexes are captured at different time points by addition of rifampicin………...110

7.3 Streptavidin affinity purification of tagged 23S rRNA complexes. The aptamer-tagged complexes were captured at different time points after induction. Streptavidin affinity purification allows isolation of pure ribosomal particles. Elution of the mutant ribosomes from streptavidin beads was carried out by incubation of the mixture with 25mM biotin...110

xii List of Figures (continued)

7.4 Reverse transcription-PCR analysis to confirm the presence of streptavidin binding aptamer. 2.5% agarose gel profile of PCR products. The wild-type and tagged 23S rRNAs were reverse transcribed into their corresponding cDNA sequences. PCR of the resulting cDNA using appropriate primers confirmed the presence of the aptamer in the mutant rRNA (~436 bp product). The aptamer was absent in uninduced cells or wild-type 23S rRNA (~359 bp product)…………………………………………………………………………………….111

7.5 Determination of the minimum inhibitory concentration (MIC) determination of different antibiotics against AD2291 cells. The 50S inhibitors, erythromycin and chloramphenicol, and the 30S inhibitors, neomycin and paromomycin were used…………………………..113

7.6 Example of protein binding progress curve generated by plotting the scaled protein level at various time points. Protein levels will be obtained by quantifying the proteins using the LC- MSE approach……………………………………………………………………………...114

xiii List of Schemes

2.1 Diagram of mass spectrometry-based approaches used to characterize ribosomes and ribosome assembly complexes in vivo.……………………………………………………...37

4.1 Multiple endoproteinase digestion of total proteins from E. coli wild-type polysomes…….60

6.1 Isolation scheme used to purify the assembly particle in cells grown with the antibiotic. The differences between the sedimentation profiles of the subunits and Δ50S particle under dissociating and non-dissociating conditions were exploited as to isolate and purify the assembly particle………………………………………………………………………...95

xiv List of Abbreviations

RNP ribonucleoprotein

RNA ribonucleic acid

DNA deoxyribonucleic acid

cDNA complimentary deoxyribonucleic acid

rRNA ribosomal ribonucleic acid

r-protein ribosomal protein

Da daltons, 1 Da = 1 amu (atomic mass unit)

tRNA transfer ribonucleic acid

mRNA messenger ribonucleic acid

RRF ribosome recycling factor

PTC peptidyl transferase center

SD Shine-Dalgarno

IF1 initiation factor 1

IF2 initiation factor 2

IF3 Initiation factor 3

fMet-tRNAfmet formyl methionine transfer ribonucleic acid

PC/QMS pulse chase/quantitative mass spectrometry

MALDI matrix assisted laser desorption/ionization

MS mass spectrometry

DMS dimethyl sulfate

DSP discovery single particle

F3CS fluorescence triple correlation spectroscopy

PAGE polyacrylamide gel electrophoresis

ESI electrospray ionization

SRM selected reaction monitoring

xv List of Abbreviations (continued)

MRM multiple reaction monitoring

PTM posttranslational modification

XIC extracted ion chromatogram

SIM selected ion chromatogram

CID collision-induced dissociation

ECD electron capture dissociation

ETD electron transfer dissociation

TOF time-of-flight

LC liquid chromatography

UV ultraviolet

SILAC stable isotope labeling of amino acid

ICAT isotope-coded affinity tag

ITRAQ isobaric tag for relative and absolute quantitation

TMT tandem mass tag

APEX absolute protein expression

PAI protein abundance index

RSD relative standard deviation

2D two-dimensional

LC-MSE enhanced liquid-chromatography mass spectrometry

CV coefficient of variation

FT-ICR Fourier transform-ion cyclotron resonance

NMR nuclear magnetic resonance

EM electron microscopy

TFA trifluoroacetic acid

CAM chloramphenicol

xvi List of Abbreviations (continued)

DTT dithiothreitol

IA iodoacetamide

SC sequence coverage

IMS ion mobility separation

ODU optical density unit

HPLC high performance liquid chromatography

LTQ linear trap quadrupole

RT-PCR reverse transcription-polymerase chain reaction

MIC minimum inhibitory concentration

xvii Chapter 1. Introduction and Background

1.1 Ribosome Structure, Function, and Antibiotic Action

1.1.1 Ribosome Structure

The ribosome is a ribonucleoprotein (RNP) particle responsible for catalyzing protein synthesis in

living organisms. The bacterial 70S ribosome (2.6-2.8 MDa, ~200-250 Å in diameter) is composed of

two unequal subunits defined by their relative sedimentation coefficients. The large 50S ribosomal

subunit consists of two ribonucleic acid (rRNA) molecules (23S rRNA, ~2900 nucleotides and 5S

rRNA, ~120 nucleotides) plus 34 ribosomal proteins (r-proteins) (L1-L36). Although the

nomenclature suggests there are 36 r-proteins, there are only 34 bona fide large subunit proteins.

Protein S8 is an aggregation artifact, while L26 is the same as protein S20. The small 30S ribosomal

subunit is composed of only one rRNA molecule (16S rRNA, ~1500 nucleotides) and 21 r-proteins

(S1-S21). R-proteins are present in stoichiometric amounts within the complex, that is, one copy per

70S ribosome except for proteins L7/L12, which are present in two copies each.

L18 S10 S9 L15 S14

S3 L9 S7 L25 L27 L30 L4 S11 L21 L24 L31 S21 S2 L20 L29 S18 S5 S4 L6 L13 L22 S6 S8 S16 S15 L32 L23 S17 L3 L17 S20

Large 50S subunit 70S Small 30S subunit

Figure 1.1 Escherichia coli 70S ribosome. Escherichia coli 30S (PDB 2AVY) and 50S (PDB 2AW4) subunits modeled using PyMOL software.

1 In eukaryotic systems such as yeast, the 80S ribosome is composed of four rRNA molecules (28S,

5.8S, 5S and 18S) and more than 70 r-proteins. It is more complex due to an increase in size of the

rRNA, an additional rRNA molecule (5.8S rRNA), and around 20-30 extra r-proteins, but it maintains

a similar structural form and function as its prokaryotic counterpart.

Detailed X-ray crystal structures of the ribosome have revealed a wealth of information that increased

our understanding of ribosome structure, function and antibiotic action. The structures of the

individual 30S and 50S subunits and their complexes have been solved, and were the basis for

elucidating the structures of the 70S complexes later [1-6]. Soon thereafter, the structures of intact

bacterial 70S ribosomes in complex with mRNA and tRNA, or tRNA analogs were elucidated [7-10].

These high-resolution structures provide insights into various aspects of RNA and protein structure,

RNA-protein interactions, antibiotic action, ribosome assembly, and the overall topology of the

ribosome.

The 70S particle is made up of approximately two-thirds rRNA (by mass) and one-third proteins (by

mass) and constitutes more than 50% of the cells’ total dry mass [11,12]. Proteins are distributed

unevenly within the complex and the biological significance of this structural organization is not

clearly understood. The rRNA and r-proteins are intertwined by various RNA-protein interactions that

determine the overall architecture of the complex. Major rRNA-protein contacts are through

electrostatic interactions between the basic arginine and lysine amino acids in proteins and the

phosphate backbone of rRNA. Likewise, base recognition occurs along the minor and major grooves

of RNA helices, as well as through hydrophobic binding pockets that capture bulged nucleotides and

through insertion of amino acid residues into the hydrophobic crevices in the rRNA [13]. The rRNA

provides a scaffold for proteins to bind while r-proteins stabilize inter-domain interactions necessary

in maintaining subunits’ structural integrity. In general, r-proteins are very basic (average pI~10.1),

suggesting their role to counteract the negative charges of the phosphate residues in the rRNA [14].

2 Aside from stabilization of the whole complex, r-proteins are also implicated in a variety of

ribosome-associated functions. Table 1.1 summarizes the roles and functions implicated for several r-

proteins during protein translation.

Table 1.1 Summary of ribosomal proteins and their associated functions in protein translation [14]. Protein Functions S1 Suggested to bring the mRNA into the proximity of the ribosome during initiation. Translational feedback regulation of the S1 operon. S3, S4, S5 Form the mRNA entry pore and may have a helicase activity to unwind mRNA secondary structure encountered during translation. S4 Mutations (ram) increase the error during the decoding process; role in rRNA transcription antitermination and translational feedback regulation of the alpha operon. S5 Probably facilitates changes of rRNA conformations that alters the selection from accurate to error prone and vice versa; mutations confer resistance against streptomycin and spectinomycin; ram mutations S12 Involved in decoding of the second and third codon positions at the A site. Mutations in S12 confer resistance against streptomycin, increase accuracy of the decoding process and, in most cases, concomitantly decrease the rate of translation. The lack of S12 in reconstituted particles also increases accuracy. L1 Probably involved in the removal of deacylated tRNA from the E site. Translational feedback regulation of L11 operon. L4 Mutations in L4 can confer resistance against macrolide antibiotics such as erythromycin by indirectly interfering with drug binding; role in rRNA transcription antitermination. L7/L12 Involved in elongation-factor binding and GTPase activation. Together with L10, involved in translational feedback regulation of L10 operon. L9 Mutations in L9 affect the of translational bypassing. L11 Mutations in L11 or the lack of complete protein confer resistance against thiostrepton, an antibiotic that blocks ribosomal transition from the pre- to post-translocational state and vice versa. During the stringent response this protein senses the presence of a deacylated tRNA in the A site; mutations or the absence of the protein can cause relaxed phenotype (relC) resulting from loss of stringent control. L16 May be involved in the correct positioning of the acceptor stem of A- and P-site tRNAs as well as RRF (ribosome recycling factor) on the ribosome. Mutations in L16 confer resistance to the orthomycins avilamycin and evernimicin. L22 May interact with specific nascent chains to regulate translation. Furthermore, deletion of three amino acids in L22 confers erythromycin resistance without interfering with the binding of the drug. L23 Present at the exit tunnel site and has been shown to be a component of the chaperone trigger factor binding site on the ribosome. L27 Bacterial-specific protein implicated in the placement of the acceptor stem of the P-site tRNA and binding of the ribosome recycling factor on the 50S subunit. L29 Is located close to the tunnel exit site and may constitute part of the binding site for the signal recognition particle.

3 Because of the highly cooperative nature of RNA-protein interactions, and interactions between

proteins themselves during translation, it is difficult to discern specific individual roles for these

proteins [14]. Nevertheless, r-proteins are essential in maintaining the overall architecture of the

complex and for efficient and optimal ribosome functions.

1.1.1 Ribosome Function

One of the vital functions of the ribosome is to decode the genetic information encoded in the

messenger RNA (mRNA) into sequences of amino acids and proteins. The large ribosomal subunit

contains the peptidyl transferase center (PTC), which catalyzes the formation of peptide bonds. It also

serves as a binding site for initiation, elongation, and termination factors [3]. Likewise, the small

ribosomal subunit contains the decoding center, which interacts with the mRNA via recognition of the

Shine-Dalgarno (SD) sequence and mediates the between mRNA, transfer RNAs (tRNAs)

and initiation factors [2,15].

During translation initiation in bacteria, the 30S subunit interacts with the SD sequence on the mRNA

that is complementary to the 3’-end sequence on the 16S rRNA. This process is facilitated by the

three initiation factors IF1, IF2, and IF3 [15]. Each of these factors has distinct roles in the initiation

step of protein synthesis. IF3 prevents premature association of the 50S subunit by binding to the 30S

subunit and helps selection of the initiator tRNA (fMet-tRNAfMet). IF2, which is a GTPase, binds

preferentially to fMet-tRNAfMet, and its affinity is enhanced by IF1. IF1 binds to the A-site of the 30S

subunit and prevents tRNA binding [2,15]. Once the pre-initiation complex is formed, the process of

elongation starts.

Each subunit has three distinct binding sites for tRNA. The A site (aminoacyl) accepts the incoming

aminoacylated tRNA; the P site (peptidyl) holds the tRNA with the nascent polypeptide chain; and

4 the E site (exit) holds the deacylated tRNA before it is dissociated from the ribosome. Both the 30S

and 50S subunits are involved in the translocation process wherein the mRNA and tRNA move

precisely one codon at a time within the ribosome with the help of other protein factors such as the

GTPases. Once the stop codon in the mRNA enters the A-site, release factors recognize these codons

and bind to the ribosome to trigger hydrolysis and release of the polypeptide chain [9,15]. The 70S

ribosomes are dissociated into free subunits ready for the next cycle of translation.

1.1.1 Ribosome as Target of Antibiotics

Because of the complex structural processes involved during protein synthesis, ribosomes are an ideal

target for antibiotics. These antimicrobial drugs interfere with various aspects of protein synthesis

namely, initiation, ternary complex formation, elongation, fidelity of translation, translocation,

peptide bond formation, progression of the nascent polypeptide chain, termination, recycling and

trans-translation [16,17-21]. For example, the antibiotics kasugamycin, edeine, pactamycin and the

oxalidinones are known inhibitors of the initiation step of protein translation. Kasugamycin and

edeine inhibit initiator tRNA binding to the 30S subunit [5]. The exact mechanism of pactamycin is

unclear but it is speculated to prevent association of the pre-initiation complex to the 50S subunit [22].

Likewise, puromycin, sparsomycin, and chloramphenicol are known as peptidyl transferase (PTC)

inhibitors. These compounds bind to the peptidyl transferase center and inhibit the formation of

peptide bonds, thus protein synthesis is halted [21]. The macrolides including erythromycin,

azithromycin, and tylosin are potent inhibitors of polypeptide chain progression. These drugs bind to

the exit tunnel on the 50S subunit of the ribosome causing premature termination of protein synthesis

[18,24-26].

Although there are current arsenals of antimicrobial drugs used to fight infections, microorganisms

can develop resistance to a variety of these antibiotics. Several mechanisms of drug resistance have

been proposed, including antibiotic modification, blockade of transport, target dilution, and bypassing

5 and alteration of the target site [27,28]. Therefore, new natural and synthetic antibiotics are

continually being developed to overcome these potentially harmful drug-resistant microorganisms. In

addition, new targets of antibiotics are also explored to increase efficacy and effectiveness of

antimicrobial agents. One promising and emerging new target of antibiotics is the formation and

assembly of ribosomal components [29-33]. Understanding how the ribosome is being formed from

its constituent rRNA and proteins can lead to the development of small molecule drugs or ligands

specific to bacterial ribosome assembly.

1.2 Bacterial Ribosome Biogenesis and Assembly

Although much is known on the intimate details and molecular mechanisms involved in each step of

protein translation, less is understood about how the ribosome is being assembled from its component

rRNA and proteins to form active and functional particles. Bacterial ribosome biogenesis and

assembly involves a complex and coordinated cascade of events, which starts with the transcription of

rRNA molecules (16S, 23S, 5S rRNA) as a single transcript from the rRNA operon (Figure 1.2)

[34,35]. The transcript is processed by various processing enzymes, mostly endonucleases, which

cleave intervening sequences to produce mature 16S rRNA and 23S rRNA. As transcription and

processing occurs, r-proteins are also being synthesized, a process that is highly coordinated with

rRNA production. Folding of rRNA and binding of proteins occurs co-transcriptionally at a rapid rate

as transcription progresses, which leads to formation of local secondary structures and the emergence

of binding sites for ribosomal proteins and protein factors. Final processing and maturation of the

transcripts occurs in polysomes, where most of the processing and modifying enzymes act upon on

[35-37].

6 23S 16S rRNA rRNA G ? ? T III III E III III III III 5’ P1 P2 T1 T2 3’ E E ? T

5S rRNA tRNAGlu2

Figure 1.2 Cartoon representation of the bacterial rrnB operon showing one primary transcript for the 16S, 23S, and 5S rRNA. The rRNA and tRNA components, promoters (P1 and P2), and terminators (T1 and T2) are shown.

1.2.1 Posttranscriptional Modifications of Ribosomal RNA

Along with rRNA folding and r-protein binding to the assembling subunit, posttranscriptional

modification of rRNA takes place, which is catalyzed by various RNA modifying enzymes. Table 1.2

summarizes the modified nucleosides identified in Escherichia coli. There are 11 modified

nucleosides in 16S rRNA, most of which are incorporated late during in vivo 30S subunit assembly.

Likewise, there are 25 modified nucleosides in 23S rRNA, where most of these modifications occur at

early stages, suggesting their essential role during 50S subunit assembly [38].

Chemical modifications of rRNA are localized in either the nucleotide bases or in the ribose moiety.

Base methylations and pseudouridylations are the most common posttranscriptional modifications in

rRNA. The exact role of most of these modifications, however, is largely unknown. The hydrophobic

methyl groups have been suggested to modulate rRNA maturation and affect the stability of the

complex [39]. The hydrophilic pseudouridines have been proposed to function as molecular glue and

tighten rRNA conformation [38]. Majority of the rRNA modifications are clustered towards the

7 functional regions of the ribosome and are thought to fine tune and stabilize structural motifs rather

than be involved in ribosome function [39-45]. For instance, the two adjacent and universally

6 conserved m 2A residues at positions A1518 and 1519 in 16S rRNA are shown to improve the

formation of the initiation complex during the initiation stage of protein translation [34,46]. These

methylated adenine residues are modified by KsgA, a universally conserved methyltransferase [46].

KsgA probably have a checkpoint role in the biogenesis and assembly of the small 30S subunit,

where methylation by KsgA marks the completion of 30S assembly in vivo [46]. The absence of this

modification confers resistance to the antibiotic kasugamycin [46,47]. Likewise, analysis of sub-

ribosomal particles in E. coli revealed stages at which the rRNA nucleotides are modified during

bacterial ribosome assembly [48]. Seven of the 11 modified nucleosides in 16S rRNA are late

assembly specific events. In , 16 of the 25 modified nucleosides in the 23S rRNA are

incorporated early during 50S subunit assembly [48].

8 Table1.2 Posttranscriptional modifications of ribosomal RNA in Escherichia coli [34]. Modification Modification Modifying enzyme Phenotype when lacking Site gene 16S rRNA 516 ψ rsuA 527 m7G rsmGb 966 m2G rsmD, yhhFc 967 m5C rsmB 1207 m2G rsmC 1402 m4Cm 1407 m5C rsmFd 1498 m3U rsmEe 1516 m2G 6 1518 m 2A ksgA, rsmA 6 1519 m 2A ksgA, rsmA

23S rRNA 745 m1G rrmA 746 ψ rluA 747 m5U rumB 955 ψ rluC 1618 m6A 1835 m2G rlmG, ygjOf 1911 ψ rluD Effect on growth due to assembly defect 1915 m5ψ rluD Effect on growth due to assembly defect 1917 ψ rluD Effect on growth due to assembly defect 1939 m5U rlmD Effect on growth due to assembly defect 1962 m5C 2030 m6A 2069 m7G 2251 Gm rlmB 2445 m2G rlmL, ycbYg Slow growth in competition ; necessary for in vitro assembly 2449 hU Necessary for in vitro assembly 2457 ψ rluE Necessary for in vitro assembly 2498 Cm Necessary for in vitro assembly 2501 Unknown C Necessary for in vitro assembly 2503 m2A Necessary for in vitro assembly 2504 ψ rluC Necessary for in vitro assembly 2552 Um rlmE, rrmJ Deficiency in assembly 2580 ψ rluC 2604 ψ rluF 2605 ψ rluB

1.2.2 Posttranslational Modifications of Ribosomal Proteins

Aside from modifications of rRNA, r-proteins are modified as well (Table 1.3). Methylation,

acetylation, and loss of terminal methionine are common posttranslational modifications found in

bacterial r-proteins. For instance, proteins S11, L3, L7/L12, L16, and L33 contain one methyl group

9 while L11 contains nine methyl groups. Proteins S5, S18, and L12 are acetylated. Acetylation of L12

generates L7, and depending on the growth conditions, the ratio of these two proteins varies. About

half of the protein S11 molecules contain an isoaspartate residue in addition to a methyl group.

Protein S12 has been identified to have a methylthio-aspartate, while L16, aside from being

methylated, has an additional unknown modification [47]. It is also necessary to point out that there

may be other modifications present but are not detected by conventional methods. The exact role of

these modifications, however, is not yet fully understood. These modifications may fine-tune the

interactions of r-proteins to the rRNA and may also provide contacts for translation factors [34].

Absence of methylation of the r-protein L3, for instance, led to a mutant having slow growth and

cold-sensitive phenotype that accumulates subunit assembly precursors [50]. Possible roles for these

modifications include stabilization of RNA structure or RNA-protein interactions, modulation of

translation factor recruitment, and they probably serve as a checkpoint during ribosome assembly [34].

Table 1.3 Posttranslational modifications of ribosomal proteins in Escherichia coli [49]. r-protein Modification

S5 Acetylation S6 Glutamic acid residues S11 Monomethylation; partial modification with isoaspartate S12 Methylthio-aspartate S18 Acetylation L3 Monomethylation L7/L12 Monomethylation L12 Acetylation L11 Nine methylations; unknown modification L16 Monomethylation L33 Monomethylation

1.2.3 In Vitro and In Vivo Studies of Ribosome Assembly

In vitro reconstitution experiments performed by Nomura and Nierhaus 40 years ago provided

valuable insights on the mechanistic details of ribosome structure, function, and ribosomal subunit

10 assembly [49,50]. Assembly maps of 30S and 50S subunits were deduced depicting hierarchy and

cooperativity of protein binding into the rRNA (Figures 1.3 and 1.4).

5’ domain Central domain 3’ domain

17 4 8 15 7 1° binding proteins

20

9 19 16 18 6 2° binding proteins

11 5 13

10 14 21 12 3° binding proteins

3

2

Figure 1.3 Assembly map of the 30S subunit as deduced by Nomura [51]. Ribosomal proteins are categorized as primary binding proteins, secondary binding proteins, and tertiary binding proteins.

Assembly of the small ribosomal subunit 30S in vitro from 16S rRNA and 21 proteins requires an

ordered binding of ribosomal proteins to the rRNA. Primary binding proteins (S4, S7, S8, S15, S17,

and S20) bind first, nucleating the association of the secondary and tertiary binding proteins. The

hierarchy of protein association to the assembling subunit follows the co-transcriptional folding of the

rRNA molecule as proteins bind first to the 5’-end and then to the 3’-end. Only one in vitro 30S

ribosome assembly intermediate is observed sedimenting at 21S. Reconstituted ribosomes in vitro are

active in cell-free polypeptide synthesizing systems [51]. Likewise, the ordered binding and hierarchy

of protein association in the large 50S subunit was also deduced using in vitro experiments.

11 5’ 13S 8S 12S 3’

20 4 2 23 9 1

13

24 22 17 3

29

21 5 34

7/12 11 33

10 15 18 5S RI50 (1)

RI50 (2) 16 25 27 30 19 32 14 28 31 6

Figure 1.4 Assembly map of the 50S subunit [52]. R-proteins associated with the reconstitution

intermediates, RI50(1) and RI50(2) are shown.

In the large 50S subunit assembly, the same mechanism follows. A subset of proteins binds to the 23S

rRNA yielding two distinct precursor particles, which sediment at 32S and 43S. R-proteins L4, L13,

L20, L22, and L24 are essential for RI50(1) to RI50*(1) conformational change [52]. Protein L3 plays a

stimulatory role during this transition but are non-essential. Proteins L20 and L24, although essential

for the conformational change, are dispensable during late steps of the assembly and are not

functionally important. Binding of the 5S rRNA to the complex is mediated by proteins L5, L15, and

L18 [52,53]. The assembly of the large subunit is more complex compared to the small subunit

because of the larger rRNA molecules and much more proteins are being assembled sequentially. In

vivo studies of the assembly sequence information of the 30S and 50S formation are in good

agreement to that found in vitro despite the discrepancies found [53,54].

12 Differences in the protein complement of in vitro and in vivo ribosome assembly intermediates for

both small and large ribosomal subunits were observed (Tables 1.4 and 1.5). This is largely due to the

highly dynamic processes occurring in vivo and the differences in the conditions used for in vitro

studies. In vitro conditions used do not mimic exact and relevant physiological conditions in vivo or

inside the cells. Assembly of the ribosomal subunits in vivo is fast and efficient suggesting that other

factors are involved during the assembly of the subunits. These factors include RNA chaperones,

RNA helicases, ribosome-dependent GTPases, and other maturation factors.

Table 1.4 Protein complement of in vitro and in vivo 30S assembly intermediates [54]. Protein In vitro reconstitution In vivo p130S (21S) intermediate (21S) S1 − + S2 − − S3 − − S4 + + S5 + + S6 + − S7 + − S8 + + S9 + − S10 − − S11 + − S12 + − S13 + + S14 − − S15 + + S16 + + S17 + + S18 + − S19 + − S20 + + S21 − +

13 Table 1.5 Protein complement of in vitro and in vivo 50S assembly intermediates [54]. Protein In vitro reconstitution In vivo p150S (32S) In vivo p250S (43S) intermediate (33/41S) L1 + + + L2 + − − L3 + − + L4 + + + L5 + + + L6 − − − L7/L12 + − + L9 + + + L10 + + + L11 + − + L13 + + + L14 − − + L15 + − + L16 − − − L17 + + + L18 + + + L19 − − + L20 + + + L21 + + + L22 + + + L23 + − + L24 + + + L25 − + + L27 − + + L28 − − − L29 + + + L30 − + + L31 − − − L32 − − − L33 + − + L34 + U U L35 U U U L36 U U U

1.2.4 Ribosome Assembly Factors

A multitude of ribosome biogenesis factors are involved in the maturation of ribosomal subunits into

functional complexes in vivo. Figure 1.5 depicts a cartoon representation of the different array of

assembly factors facilitating efficient and proper assembly of ribosomal components before they can

actively participate in protein synthesis. There are approximately ~170 assembly factors identified

that are thought to be involved in yeast ribosome biogenesis and assembly [55,56]. However, a

limited number have been characterized in bacteria. The assembly factors, Era, RbfA, RimJ, RimM,

14 RimP, and RsgA are involve in 30S subunit assembly while CsdA, DbpA, Der, and SrmB are involve

in 50S subunit assembly [54]. The exact roles of these assembly factors in the maturation of

ribosomes are poorly understood as depicted by the gray areas in Figure 1.5. Understanding the roles

of these factors could lessen these gray areas as more sophisticated tools are being developed. Table

1.6 summarizes the protein assembly factors identified and characterized with their corresponding

functions. These factors include nucleases, RNA modifying enzymes, protein modifying enzymes,

structural factors, GTPases and helicases.

r- proteins r- proteins

16S rRNA Other factors Structural factors 30S subunit rRNA r-protein Endonucleases modifying modifying factors Helicases factors GTPases

23S rRNA 5S rRNA r- proteins r- proteins 50S subunit

Figure 1.5 Cartoon representation of bacterial ribosome assembly. This process requires a multitude of components necessary in the maturation of the small and large ribosomal subunits. Figure adapted from [41].

15 Table 1.6 Summary of protein factors that are involved in regulation of ribosome biogenesis and protein synthesis [12]. Factor Possible functions CsdA (DeaD) Cold shock Dead A (CsdA) is an ATP-dependent RNA helicase that binds to large ribosomal subunit to mediate the unwinding of 23S rRNA during assembly. DbpA DEAD box protein A (DbpA) is an ATP-dependent RNA helicase that mediates unwinding of 23S rRNA during assembly. Termed YxiN in Bacillus subtilis. EF-3 (yeast) Elongation factor 3 is a yeast specific ATPase that promotes release of the E-tRNA from the ribosome, upon binding of the ternary complex EF1-aminoacylated tRNA- GTP to the A site. EF-P (IF-4) Elongation factor P structurally mimics tRNA, binds to the ribosome and facilitate translation initiation by stimulating formation of the first peptide bond; is a homologue of eIF5A and has been renamed as IF-4. EngA (Der/YphC) Unique G-protein with tandem G-domains and RNA binding KH domain. Probably involved in assembly of the large ribosomal subunit. Also known as Der/YfgK (E. coli) or YphC (B. subtilis). EngB (YihA, YsxC) E. coli YihA and the B. subtilis homologue YsxC are essential proteins that appear to have a role in assembly of the large ribosomal subunit. Era E. coli Ras-like protein is a GTPase that binds to pre-30S subunit to facilitate processing of the 3’-end of the 16S rRNA precursor. Ortholog in B. subtilis termed Bex. Hsp15 Heat shock protein 15 (Hsp15) is encoded by the yrfH/hslR gene and is involved in recycling of nascent polypeptide containing free large ribosomal subunits. LepA (EF4) Leader peptidase A, renamed to Elongation Factor 4 (EF4). Shown to bind to POST state ribosomes and induce back translocation. Obg (CgtA) SpoOB-associated GTP-binding protein (OBG) binds ppGpp and appears to monitor levels of G-nucleotide in the cell. Obg also binds the large ribosomal subunit and may provide a link the stress response, DNA and ribosome assembly. Also known as CgtA or YhbZ. pY (YfiA, RaiA) Protein Y binds and inactivates ribosomes under conditions of cold shock. Previously known as YfiA and Ribosome-associated inhibitor A (RaiA). RbbA (YhiH,W) Ribosome-bound ATPase has been proposed to facilitate ejection of E-tRNA from the ribosome, analogous to yeast EF-3. The yhiH gene encodes RbbA and the truncated form was originally termed W. RbfA (P15B) Ribosome binding factor A binds to 30S subunit to facilitate subunit assembly. Overexpression suppresses cold sensitive C23U mutation in the 16S rRNA. Previously termed P15B. RbgA (YlqF) Ribosome biogenesis GTPase A is involved in a late assembly step of B. subtilis 50S subunit. Previously called YlqF. Not present in E. coli. RelA The stringent factor RelA binds to ribosomes containing uncharged or deacylated tRNA RelE at the A site and synthesizes the alarmone (p)ppGpp. RimM (21K, YfjA) RelE is a toxin that binds to ribosomes and cleaves mRNA in the A site. The antitoxin RelB inactivates RelE. Ribosome maturation factor M binds to head of small ribosomal subunit to facilitate assembly. Previously called 21K or YfjA. RimN (YrdC) Ribosome maturation factor N has been suggested to bind 16S rRNA to promote proper processing. RMF Ribosome modulation factor (RMF) binds to stationary phase 70S ribosomes to induce dimerization (100S formation).

16 Table 1.5 (continued) Factor Possible functions RsgA (YjeQ/YloQ) Ribosome small subunit-dependent GTPase A (RsgA) has a putative role in small subunit ribosomal assembly. Previously called YjeQ (E. coli). Homologues include YloQ/YqeH (B. subtilis) or YawG (yeast). SRA (S22) Stationary phase induced ribosome-associated protein (SRA) binds to ribosomes in stationary phase. Previously identified as ribosomal protein S22. SmpB Small protein B (SmpB) binds tmRNA and is involved in the trans-translation system for rescue of ribosomes stalled on truncated mRNAs. SrmB Suppressor of temperature-sensitive mutation in ribosomal protein L24. SrmB is a DEAD box RNA helicase involved in ribosome biogenesis. Tet(O) A ribosome protection protein (RPP) that binds to tetracycline-stalled ribosomes to release the drug and allow translation to continue. YciH Bacterial ortholog of eiF1/SUI1. Not present in many bacterial genomes. YhbH Found bound to 100S ribosome dimers in stationary phase cells. Putative role in stabilization and preservation of ribosomes. YhbY Has a similar fold to the C-terminal domain of IF3, suggesting potential interaction with RNA. Reported to associate with 50S subunit.

1.2.4 Ribosome Assembly as Target of Antibiotics

The ribosome is a popular target of various medically important antibiotics. These agents inhibit

important ribosome functions such mRNA decoding, peptidyl transfer, elongation, and inhibition of

ribosome-associated GTPases. Microorganisms, however, can develop resistance to these

antimicrobial agents. Therefore, new targets should be improved and developed. One emerging and

important new target of antibiotics is the biogenesis and assembly of the ribosome. Erythromycin is a

macrolide antibiotic known to inhibit protein synthesis in bacteria. The drug interacts with ribosomal

components at the exit tunnel of the ribosome where it blocks the elongation of the nascent

polypeptide chain. The large subunit proteins L4, L15, L16, and L22 are implicated in erythromycin

binding [25,57]. Alterations in proteins L4 and L22 led to erythromycin-resistant strains and the

effect of L22 mutations are more pronounced than that of protein L4 [18,27,58,59]. Champney and

co-workers previously examined the effects of erythromycin on the formation of ribosomal subunits

in both wild-type and RNase E mutant cells using pulse-chase labeling techniques [33]. They showed

that 50S subunit formation is inhibited by erythromycin without affecting the rate of the small

17 ribosomal subunit 30S. Hence, a second novel mechanism of erythromycin has been proposed. Figure

1.6 shows the sequence of 50S formation in vivo. In the presence of erythromycin, drug–resistant

cells can still form mature and functional 50S subunits while in sensitive cells, 50S assembly stalls

and an assembly particle accumulates. This particle is then targeted for degradation by cellular

ribonucleases [29-31,33]. Inactivation of the RNase E complex promotes stabilization of the assembly

particle and can be isolated and characterized.

+ proteins + proteins E E Erythromycin resistant cells

32S 43S 50S + E 5’

+ proteins 23S rRNA 5S rRNA RNase E Erythromycin E sensitive cells

stalled intermediate 23S and 5S rRNA fragments

E = erythromycin

Figure 1.6 Model of the bacterial 50S subunit assembly inhibition by erythromycin [33]. In the presence of the drug, resistant cells can form mature 50S subunit (top), however in sensitive cells (bottom), assembly stalls creating an improperly folded assembly particles, which is then targeted for degradation by cellular ribonucleases. By inactivation of RNase E at non-permissive temperatures, isolation of assembly particles that can be detected in sucrose gradients is possible.

Siibak and co-workers also examined the effects of erythromycin in protein translation and inhibition

of subunit assembly. They showed that the 50S assembly particles accumulated in cells in the

presence of either erythromycin or chloramphenicol are the result of the secondary effects of the

antibiotics in protein synthesis [60]. These antibiotics inhibit protein synthesis leading to an

18 imbalance between rRNA and r-protein production. Because there is no sufficient supply of r-proteins

in the cells, rRNA could not assemble and fold properly leading to incompletely assembled 30S and

50S subunits.

1.3 Biophysical and Biochemical Methods to Study Ribosome Assembly

The biogenesis and assembly of the ribosome involves a coordinated cascade of events including

rRNA processing, folding, and post-transcriptional modifications of rRNA along with the association

of r-proteins. Unraveling the pathways and dynamics of these complex structural processes is a

significant challenge. While conventional biophysical techniques such as nuclear magnetic resonance

(NMR), X-ray crystallography and cryo-electron microscopy (cryo-EM) provide high-resolution

information, these methods are not ideally suited to characterize transient and heterogeneous

ribosome assembly intermediate structures. Along with the availability of high-resolution crystal

structures of the ribosomes and the application of various biophysical and biochemical techniques,

details of the dynamics and energetics of the biogenesis and assembly of the ribosome are now being

realized.

The use of mass spectrometry has gained widespread application in studying ribosome assembly by

monitoring the binding of many proteins simultaneously into the assembling subunit. Williamson and

co-workers developed an elegant approach to monitor 30S subunit assembly kinetics using pulse-

chase monitored by quantitative mass spectrometry (PC/QMS) [61]. This approach involves binding

of 15N-labeled 30S proteins into the 16S rRNA and at various time points chasing with unlabeled 30S

proteins. The ratio of the labeled and unlabeled proteins is determined by matrix assisted laser

desorption/ionization mass spectrometry (MALDI MS) and progress curves are generated for each

protein so their binding kinetics can be analyzed. This approach yielded insights into how the 30S

assembles through diverse pathways with various conformational transitions converging to mature

subunits. This approach has also been used in studying the dynamics and kinetics of ribosome

19 assembly intermediates that accumulate in cells under perturbed conditions such as in the presence

antibiotics [62].

RNA chemical footprinting is another powerful tool to characterize RNA-folding events and protein

binding reactions during ribosome assembly. Wooodson and co-workers used time-resolved X-ray

hydroxyl radical footprinting to visualize RNA folding events during 30S ribosome assembly. X-ray

beams generate hydroxyl radicals, which cleave the exposed regions of the RNA backbone. Primer

extension experiments are performed where RNA cleavage sites are revealed by strong stops on

sequencing gels of cDNA products [63]. This technique showed that early steps during assembly are

attributed to stable rRNA structure while later steps are dominated by induced fit between proteins

and rRNA. Likewise, Culver and co-workers used chemical modification and primer extension to

map structural differences between 30S ribosomal subunit assembly intermediates [64]. Instead of

using hydroxyl radicals to cleave exposed RNA backbone, base-specific chemical probes were used

(kethoxal modifies guanines, and dimethyl sulfate (DMS) modifies adenines and cytosines). Through

primer extension of the probed 16S rRNA particles, modified sites in the rRNA are revealed by strong

stops on sequencing gels. This approach demonstrated many changes in the transition of the in vitro

30S intermediates (RI to RI*) are r-protein dependent and localized into functional regions of the 30S.

This further suggests that this transition is a critical step in the formation of functional 30S subunit.

Other approaches have been used to interrogate ribosome assembly including discovery single-

particle profiling (DSP) and fluorescence triple correlation spectroscopy (F3CS) to visualize and

quantify 30S ribosome assembly intermediates [65,66]. DSP, an application of time-resolved electron

microscopy, was used to obtain a million snapshots of the assembling 30S subunit, identify and

visualize their structures, and monitor the population flux of these intermediates over time. The

results of the DSP were combined with mass spectrometry data to deduce the first ribosome assembly

mechanism incorporating binding dependencies of proteins, rate constants, and structural

20 characterization of populated intermediates [65]. Likewise, fluorescence triple-correlation

spectroscopy was used to quantify ten 30S ribosomal assembly intermediates by identifying ternary

complexes. These ternary complexes are detected by coincident fluctuations in three-color

fluorescence data. Ten 30S ribosome assembly intermediates were quantified and this approach can

be applied as well in characterizing macromolecular complexes too large to be analyzed by

conventional techniques [66].

The ribosome is an intricate macromolecular complex responsible for synthesizing proteins within a

cell. Although the structures of the different complexes of the ribosome have been elucidated, many

aspects of ribosome biogenesis and assembly remain poorly understood. This lack of understanding is

largely due to the limited methods suitable for characterizing these oftentimes flexible and

heterogeneous ribosome assembly intermediates. Biochemical and biophysical approaches have been

used and developed to study ribosome assembly. These techniques revealed intimate details of RNA

folding and kinetics during formation of both 30S and 50S ribosomal subunits in vitro and in vivo.

These techniques, however, do not reveal information on the exact composition and stoichiometry of

proteins associated with in vivo ribosome assembly complexes. Therefore, methods that allow robust

and rapid characterization of these complexes will be valuable in elucidating the pathways of

ribosomal subunit formation from its constituent RNA and r-proteins.

21 Chapter 2. Literature Review

2.1 Characterization of Ribosomal Proteins by Mass Spectrometry

Polyacrylamide gel electrophoresis (PAGE) is a popular technique used to characterize r-proteins.

Comparison of the relative mobilities of proteins isolated from mutant and wild-type cells in 2-

dimensional gel systems enabled differentiation of mutated or posttranslationally modified proteins.

For example, mutations in proteins L4 and L22, which confer resistance to the antibiotic

erythromycin were detected by their reduced first dimension mobilities [31]. 2D-PAGE also allowed

identification of 21 r-proteins in the 30S subunit and 34 r-proteins in the 50S subunit in E. coli [67].

This method however, is not sensitive enough to allow identification and localization of mutations or

modifications in a particular protein.

Mass spectrometry is a powerful analytical tool used in proteomics analysis [68,69]. It involves the

generation of gas-phase ions of analytes followed by separation of the ions based on their mass-to-

charge ratios. Two commonly used techniques are matrix assisted laser desorption ionization mass

spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS) [70,71].

MALDI-MS has been extensively used to study post-translational modifications in proteins because

of its inherent simplicity of mass spectral data interpretation. The spectrum is dominated by singly

charged ions making it possible to analyze complex mixtures of proteins. On the other hand, the more

elaborate electrospray ionization (ESI) generates multiply charged ions extending the mass range and

improving fragmentation yield in tandem mass spectrometry [72]. Multiple charging, however,

creates complication in data interpretation due to increased mass spectral complexity.

22 There are several approaches that are being used in mass spectrometry-driven proteomics. Table 2.1

summarizes the different strategies employed to obtain comprehensive proteome information in a

wide variety of biological samples.

Table 2.1 Strategies and Approaches in Mass Spectrometry-Driven Proteomics [73]. Approach Qualitative Quantitative Bottom-up Internal peptide sequencing Selected reaction monitoring/multiple reaction monitoring (SRM/MRM) proteotypic transtions

Protein identification-mass fingerprint Label-free extracted ion chromatogram/selected ion chromatogram (XIC/SIM)

Single posttranslational modifications Isotope labeling (metabolic and chemical) (PTMs) sequencing

Middle-down Internal peptide sequencing SRM/MRM proteotypic transtions

Multiple PTMs sequencing Label-free (XIC-SIM)

Isotope labeling (metabolic and chemical)

Top-down PTMs code Label-free XIC (relative or absolute; dependent of standard availability

Intact sequencing (dependent on molecular Area of electrospray ionization (ESI) dimensions) spectrum deconvolution N-terminus identification C-terminus identification

The bottom-up approach is a popular and widely used technique in proteomics. It involves enzymatic

digestion of proteins into peptides before MS analysis [73-75]. The peptides identified from either

database searching or de novo sequencing are used to infer the presence of the proteins. This

approach typically requires collision-induced dissociation (CID) of peptides to gain sequence

information and PTMs of proteins. However, because a large number of proteolytic peptides is

generated upon digestion in the bottom-up approach, front-end separation is necessary to reduce

23 sample complexity before CID during mass analysis. Major drawbacks of this approach include lower

sequence coverage by identified peptides, some proteins are not amenable to enzyme digestion, loss

of labile PTMs, and ambiguity in peptide identification due to redundant peptides and different

protein isoforms.

The top-down approach, on the other hand, is based on direct analysis of intact proteins and their

product ions for identification [75,76]. This method requires high- resolution mass spectrometers to

resolve overlapping signals due to complex spectra generated upon CID fragmentation of intact

proteins. Typically, electron capture dissociation (ECD) and electron transfer dissociation (ETD) are

used to yield complete sequencing information while at the same time retaining labile PTMs.

Advantages of this approach include higher sequence coverage of proteins and retention of labile

PTMs information. In addition, the top-down approach is known to have an improved reliability in

protein quantification [77]. This approach, however, has several limitations including larger amount

of sample required for analysis, challenging front-end separation, requirement of high mass

measurement accuracy instruments, and complex MS/MS spectral interpretation.

Another technique that is being used to interrogate high-mass proteomes is the “middle down”

approach [73,78]. This approach is based on size-dependent protein fractionation and robust but

restricted proteolysis of proteins. Restricted enzymatic proteolysis is done using the outer membrane

protease T (OmpT) to generate large peptides (>6.3 kDa on average) for mass spectrometric analysis.

This approach identified 3,697 unique peptides from 1,038 proteins in pre-fractionated high-mass

HeLa proteins. Observation of various posttranslational modifications and differentiation of closely

related protein isoforms are enabled using large OmpT peptides [78].

24 2.1.1 Identification of Ribosomal Proteins and Their Posttranslational Modifications

Mass spectrometry is extensively used in characterizing the composition and posttranslational

modifications of proteins in macromolecular complexes like the ribosome using top-down, bottom-up

or a combination of both approaches. Reily et al. (1999) demonstrated the use of MALDI-MS to

identify r-proteins and their post-translational modifications in the E. coli 70S ribosome. A total of 55

out 56 proteins were identified and their posttranslational modifications were established consistent

with observed modifications in proteins involving N-terminal methionine loss, methylation,

thiomethylation, and acetylation [49]. Table 2.2 summarizes the small subunit and large subunit r-

proteins and their PTMs associated with E. coli K12 ribosomes. MALDI-MS in combination with

ESI-TOF/MS was also used to characterize the r-protein composition of defective particles associated

with an E. coli mutant strain [79]. The presence or absence of r-proteins from the aberrant particle

was determined by comparing the MS signal intensities of the tryptic peptides with that of the mature

subunits. The low molecular weight proteins escaping identification using the approach were further

analyzed by MALDI TOF-MS and nano-ESI TOF-MS, which allowed observation of all r-proteins

and ribosome assembly factors associated with the defective and mature particles [79].

25 Table 2.2 Properties of Escherichia coli K12 30S and 50S ribosomal proteins [49]. Protein Isoelectric Amino acid Methionine Expected Observed Mass Modification pH (pI) residue in Loss? MW MW (Da) Difference (Da) position 2 (Da) S1 4.7 Thr Y 61159 S2 6.7 Ala Y 26613 S3 10.7 Gly Y 25852 S4 10.4 Ala Y 23338 S5 10.6 Ala Y 17472 17516 -43 acetylated S6 4.7 Arg N 15704 15445 258 S7 10.8 Pro Y 19888 17474 2414 S8 9.8 Ser Y 13996 S9 11.3 Gln Y 14725 S10 10.2 Gln N 11736 S11 11.7 Ala Y 13714 13728 S12 11.3 Ala Y 13606 13655 -49 β-methylthiolated S13 11.2 Ala Y 12968 S14 11.6 Ala Y 11449 S15 10.8 Ser Y 10138 S17 11.0 Thr Y 9573 S18 10.1 Ala Y 8855 8896 -41 acetylated S19 10.9 Pro Y 10299 S20 11.0 Ala Y 9553 S21 11.6 Pro Y 8369 L1 10.1 Ala Y 24599 L2 11.4 Ala Y 29729 L3 10.4 Ile N 22243 22259 -16 methylated L4 10.2 Glu N 22086 L5 9.8 Ala Y 20170 L6 10.1 Ser Y 18773 L7 4.4 Ser Y 12164 12207 -43 acetylated L9 6.2 Gln N 15769 L10 9.5 Ala Y 17580 L11 10.1 Ala Y 14744 14871 -127 9 methylations L12 4.4 Ser Y 12163 12176 -13 methylated L13 10.3 Lys N 16019 L14 10.9 Ile N 13541 L15 11.6 Arg N 14980 14966 14 L16 11.6 Leu N 15281 15325 -44 unknown L17 11.5 Arg N 14365 L18 10.8 Asp N 12769 L19 11.1 Ser N 13002 L20 11.9 Ala Y 13366 L21 10.3 Tyr N 11564 L22 10.7 Glu N 12226 L23 10.4 Ile N 11199 L24 10.7 Ala Y 11185 L25 10.0 Phe N 10693 L27 11.1 Ala Y 8993 L28 11.8 Ser Y 8875 L29 10.5 Lys N 7273 L30 11.4 Ala Y 6410 L31 9.8 Lys N 7871 L32 11.4 Ala Y 6315 L33 10.7 Ala Y 6255 6255 -15 methylated L34 13.4 Lys N 5380 L35 12.2 Pro Y 7158 L36 11.2 Lys N 4364

26 Aside from identifying r-proteins and PTMs in mature ribosomes and defective particles, MALDI-

MS has been a method of choice in characterizing r-proteins of closely related and unsequenced

organisms for phylogenetic studies. Suh et al. (2005) previously developed and extended

identification of ribosomal proteins to unsequenced bacterial strains by MALDI-MS. Nearly 60% of

the r-proteins were found identical among the three strains of Thermus thermophilus and over 94% of

these proteins were successfully identified using this technique [80] Further, a combination of top-

down and bottom-up approaches were used to analyze the proteins from ribosomes of gram-negative

α-proteobacterium Caulobacter crescentus [81] and Rhodopseudomonas palustris [82]. R-proteins

were identified from direct analysis of intact proteins. Common posttranslational modifications like

N-terminal acetylation (S9 and S18), near absence of L7, and extensive modification of L11 were

observed from the analysis of proteolytic peptides.

These approaches were also applied to more complex ribonucleoproteins (RNPs) of eukaryotic and

mammalian systems. Lee and co-workers reported the direct analysis of intact proteins of the yeast

large ribosomal subunit using capillary liquid chromatography-Fourier transform ion cyclotron (LC-

FTICR) mass spectrometry. Forty-two of the forty-three core large subunit r-proteins and 58 out of 64

large subunit r-protein isoforms were identified in a single analysis. Posttranslational modifications of

proteins including methionine loss, acetylation, methylation, and proteolytic maturation were

observed [83]. Furthermore, LC-ESI MS was used to examine the posttranslational modifications of

40S subunit proteins from Rat-1 fibroblasts [84]. Thirty-two rat 40S r-proteins were identified

consistent with their known sequences. PTMs including N-terminal methionine loss and/or

acetylation were observed for S7, while proteins S5 and S27 were internally formylated and

acetylated, respectively. Protein S23 is internally hydroxylated or methylated. Mass spectral changes

other than the known PTMs were also observed possibly representing novel posttranslational

modifications. Following posttranslational modifications by MS and the structural changes of the

stalk proteins L7/L12 on intact 70S ribosome was also described [85]. Increased acetylation of L12

27 was observed from cells harvested during stationary phase grown under optimal conditions. N-

terminal acetylation increased the stability of the stalk complex in the gas phase. Solution deuterium

exchange was further used to monitor the incorporation of deuterium on proteins L9, L10, L11, L12,

and the acetylated form of L12 (L7). Deuterium incorporation was slower for L7 relative to L12.

Modification of L12 is also predominant in minimal media suggesting that acetylation is probably one

of the cells’ strategy to increase stability of the stalk complex under non-optimal conditions.

2.1.2 Analysis of Ribosomal Proteins to Probe Ribosome Topology

Ribosomal proteins can be used as probes to gain insights into the overall architecture and structural

organization of ribosomes. Yamamoto et al. (2006) investigated the structural flexibility of 70S

ribosomal proteins using a combination of hydrogen/deuterium exchange and mass spectrometry.

Based on the deuterium incorporation map, they showed the structure-flexibility-function relationship

of the ribosome at the level of its constituent proteins. Another approach developed by Suh et al.

(2007) combined limited proteolysis and MALDI-MS to probe protease-accessible sites of r-proteins

in ribosomes from E. coli and T. thermophilus. Results from this showed that a larger

faction of r-proteins from the 50S subunit of both organisms have similar behavior upon limited

proteolysis compared to the 30S subunit. The data were also found to be consistent with high-

resolution structures of the ribosomes from both organisms.

Beardsley and co-workers (2006) used chemical labeling (amidation) and mass spectrometry to probe

the structure of the Caulobacter crescentus ribosome. Fourty-seven of the fifty-four proteins were

detected by labeling and the extent of derivatization is consistent with the solvent accessibility of the

target residues in the proteins. The flexible stalk proteins were labeled extensively, consistent with

their known large solvent accessibility. Proteins known to be highly shielded in the ribosome are

labeled at very few potential sites. These results along with x-ray crystal studies provided insights

into the structural organization of C. crescentus ribosome. This approach was further extended to

28 probe solvent accessibility and reactivity of r-proteins of Deinococcus radiodurans. The extent of

labeling correlated with the solvent-exposed area of the proteins, where lysine residues on the subunit

interface are highly reactive than those residues buried into the rRNA [81]. In addition, ultraviolet

(UV) and chemical cross-linking and mass spectrometry were used to probe details of protein-rRNA

interaction in the 30S and 50S ribosomal subunits of E. coli and B. stearothermophilus [86,87].

Amino acid residues from 13 r-proteins that are cross-linked to rRNA were identified from both

organisms. These studies along with biochemical and structural data revealed functional implications

for these proteins that interact with rRNA.

Aside from the analysis of proteins within the ribosome, mass spectrometry of intact ribosomes was

also described. Robinson and co-workers probed the changes in conformation and interactions of

intact ribosomes during mass spectrometry analysis. They showed that noncovalent complexes can be

maintained both during and after electrospray of these assemblies [88,89]. The masses of both 30S

and intact 2.3 MDa 70S ribosomes from T. thermophilus were determined and revealed that gas-phase

ribosomes consist of a number of discrete populations of particles [90]. Investigation of the

dissociation of proteins from intact ribosomes during electrospray provides structural basis for the

release of these proteins from ribosomes for which no crystal structures are available [89].

2.2 Mass Spectrometry-Based Quantitative Analysis of Ribosomal Proteins

Because r-proteins are present in one copy per ribosome except for proteins L7 and L12, which are

present in two copies each, the ribosome serves as an ideal model system in developing analytical

tools for quantitative analyses. “Ribosome-centric” quantitation of r-proteins is typically

accomplished by gel-based techniques [91-94]. Although these strategies are able to provide both

qualitative and quantitative information, a good separation of differential protein bands is a

prerequisite. In many cases, however, individual spots in a 2D gel may still consist of several proteins

29 and isoforms thereof. Identification and quantitation of proteins are therefore hindered using 2D gel

separation alone in normal and perturbed samples. The advent of protein/peptide sequencing

technologies by mass spectrometry provides a convenient and sensitive approach to identify the

proteins in excised 2D gel bands [95]. Although gel-based coupled to mass spectrometry techniques

are still widely used in proteomics, hyphenated techniques using liquid chromatography and mass

spectrometry offer a versatile approach for both qualitative and quantitative proteomics analyses.

MS-based quantitative strategies can be categorized into two types, relative or absolute. Relative

quantitation determines the differential expression (up-down regulation) of proteins between normal

and perturbed samples and is typically expressed as fold-increase or decrease. Absolute quantitation,

on the other hand, determines the exact concentration or amount and is expressed as nmoles or

nanograms per gram of cells/tissues. Depending on the type of information one wants to obtain from a

particular quantitative proteomics experiment, diverse approaches can be used. Thorough

consideration of each strategy’s corresponding strength and weaknesses is important for successful

quantitation. Table 2.3 represents various strategies that are widely used in quantitative proteomics.

30 Table 2.3 Quantitative Mass Spectrometry-Based Quantitative Proteomics [96]. Approach Dynamic Proteome Quantitative Nature of Number of Quantitation range coverage accuracy quantitation samples to level compare Metabolic labeling 15N 1-2 Medium Precise Relative 2 MS (<10% rsd) SILAC 1-2 Medium Precise Relative 2 or 3 MS (stable isotope labeling of (<10% rsd) amino acids) Chemical labeling ICAT (isotope-coded 1-2 Poor Precise Relative 2 MS affinity tag) (<10% rsd)

ITRAQ (isobaric tag for 2 Medium Medium Relative 2-8 MS2 relative and absolute (10-30% rsd) quantitation), TMT (tandem mass tag)

Standard peptide 2 Poor Precise Relative/ 2-many MS or MS2 Absolute1 Label-free Ion intensities 3 Good Medium Relative Many MS (10-30% rsd)

Spectrum count 3 Good Poor Relative Many MS2 (>30% rsd)

Derived indices (APEX, 3 or 4 Good Poor Relative/ Many MS absolute protein (>30% rsd) Absolute2 expression); (emPAI, exponentially modified protein abundance index) Gels 2D gels 1 to 4 Medium Medium Relative/ Many Image (10-30% rsd) Absolute1 Analysis 1 Absolute quantitation is possible only through relative comparison to a spiked “known” standard. 2 Absolute quantitation is possible only through empirical features and back-calculation using the molecular weight of the protein and total protein amount in the sample.

2.2.1 Stable Isotope Labeling for Relative and Absolute Quantitation

Relative quantification is dominated by stable isotope labeling to obtain information on the

differential expression of proteins in normal and perturbed samples. Introduction of a chemically

equivalent differential mass tags allows comparative quantification. The isotopic labels change the

mass of the peptide without affecting its physicochemical properties. Differential isotopic labels can

be introduced metabolically, enzymatically, or chemically. Commonly used approaches include the

isobaric tags, ICAT [97], iTRAQ [98], SILAC [99], 15N [100], and 18O-labeling [101,102] to mention

31 a few. On the other hand, absolute quantification based on stable isotope incorporation was also

described using synthetic peptides (AQUA peptides) with isotope labels that mimic target peptides

during enzymatic proteolysis [103].

Stable-isotope labeling techniques based on relative quantification are commonly used to determine

the amounts of r-proteins. Williamson and co-workers used 15N-labeling to quantify levels of r-

proteins in ribosome assembly complexes and r-protein turnover in vivo [104,105]. 15N labeling

yields relative protein levels of r-proteins associated with ribosome assembly complexes as compared

to mature subunits. Mature subunits of the ribosome showed uniform distribution of proteins while

some proteins in the assembly particles are underrepresented. These results suggest stages of

ribosome assembly at which these complexes are stalled in the presence of antibiotics or perturbations.

A similar approach was used to quantify proteins in 50S assembly particle in DEAD box protein A

(DbpA) deletion mutant [106] and antibiotic-induced ribosome assembly particles [107]. Likewise,

isobaric tags (iTRAQ) were used for quantification of r-proteins associated with the 30S, 50S,

translating ribosomes isolated at different temperatures (16°C and 37°C) [108], and in vivo assembled

immature 30S subunits from assembly factor deletion strains [109,110]. Although the above

techniques provide excellent quantitative information of proteins in ribosomes and ribosome

assembly complexes, methods based on absolute “ribosome-centric” quantification have not been

implemented. Accurate determination of the absolute quantity of proteins in ribosomes and ribosome

assembly particles will provide useful information relating to complex stoichiometry, biogenesis, and

degree of heterogeneity of ribosomes and ribosome assembly particles, which is assumed but not yet

proven experimentally.

2.2.2 Label-free Approaches for Relative and Absolute Quantitation

Although techniques based on isotope labeling offer excellent quantitative accuracy, the poor to

medium proteome coverage and challenging implementation, hinder their application in high-

32 throughput quantitative analyses. Thus, methods based on label-free quantitation are continuously

being improved and developed. Label-free quantification requires no labeling step, eliminating costly

isotopic labeling reagents, and does not require the multi-step labeling process, which can lead to

irreproducibility and recovery issues. Several label-free approaches are widely used in MS-based

quantitative analyses. These include the emPAI, exponentially modified protein abundance index

[111], peptide ion intensity counting [112], ion accounting [113], absolute protein expression (APEX)

[114], spectral counting [115], and spectra TIC method [116]. Label-free approaches are ideally

suited for high-throughput analysis, because of their high proteome coverage, multiplexing capability

and cheaper experimental cost. These techniques however have relatively lower quantitative accuracy

compared to isotope labeling strategies.

One approach used for absolute quantification of proteins is LC-MSE [113]. This method is based

upon the observation that the average signal response of the three most intense peptides per mole of

protein is constant with CV (coefficient of variation) less than 10% (MW range from 14 kDa- 97

kDa) (Figure 2.1). This technique involves alternating scans of low energy collision-induced

dissociation (CID) and high-energy CID during LC-MS to enable both protein identification and

quantitation in a single experiment (Figure 2.2). Using an internal standard, a universal MS signal

response factor (counts/mole of protein) is determined and then applied to other well-characterized

proteins in the mixture to determine their absolute amounts.

33

Figure 2.1 Absolute quantification of proteins by LC-MSE. This method is based upon the observation that the average signal response of the three most intense peptides per mole of protein is constant with coefficient of variation (CV) of less than 10% evaluated for proteins with molecular weight range of 14 kDa to 97 kDa [113].

Low Energy CID Elevated Energy CID Ion Abundance Ion Abundance

m/z m/z • Accurate mass measurement of • Peptide fragmentation precursor ions • High mass accuracy of fragment ions • Intensity data for quantitation provides increased selectivity and specificity for peptide ion spectra identification

Figure 2.2 Overview of the LC-MSE approach for absolute quantification of proteins.

34 2.3 Purpose of the Work Presented

The biogenesis and assembly of the ribosome into mature and functional complexes is a major

metabolic activity within a cell. Unraveling the structural processes involved in ribosome formation

remains a significant challenge. Although conventional techniques such as nuclear magnetic

resonance (NMR), X-ray crystallography and cryo-electron microscopy (cryo-EM) provide high-

resolution information, these methods are not ideally suited to characterize transient and

heterogeneous ribosome assembly intermediate structures. Therefore, methods that allow robust and

rapid characterization of ribosome assembly complexes in vivo will be valuable in elucidating the

pathways of ribosomal subunit formation from its constituent RNA and r-proteins.

The goal of this dissertation is to use mass spectrometry-based approaches to characterize ribosomes

and ribosome assembly particles in vivo, particularly those particles as a result of perturbations (e.g.

deletion of assembly factors, antibiotics). Major tasks are directed towards identifying the

composition and obtaining quantitative information, and posttranslational modifications of r-proteins

within in vivo ribosomes and ribosome assembly complexes as a result of these perturbations. Scheme

2.1 represents the experimental approaches used to characterize these complexes in vivo by MS-based

methodologies.

First, a combination of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS),

15N-labeling and data-dependent liquid chromatography tandem mass spectrometry (LC-MS/MS)

were used to characterize the proteins associated with E. coli RimM and RbfA deletion strains in

Chapter 3. RimM and RbfA are assembly factors implicated in the maturation of the small subunit

30S in bacteria and their precise roles are poorly understood. The mass spectrometry data presented

here along with in vivo hydroxyl radical footprinting yielded insights how these factors facilitate

maturation, assembly and remodeling of the small ribosomal subunit before they can engage in

protein synthesis. In Chapter 4, I explored the applicability of a label-free approach, LC-MSE for

35 absolute “ribosome-centric” quantification of r-proteins. Because the information obtained in this

experiment is related to the number of peptides identified per protein, I optimized experimental

conditions that allow accurate and reproducible quantitation of r-proteins associated with mature

ribosomes from E. coli. Improvements of the LC-MSE approach using additional dimension of gas

phase separation through ion mobility and the use of multiple endoproteinase digestion significantly

enhanced quantitation of r-proteins. These two strategies allow reproducible and accurate quantitation

of proteins associated with mature ribosomes. The improved approach was then extended in

characterizing proteins associated with different functional states of the ribosomes (free 30S, free 50S,

70S and polysomes) in Chapter 5. Finally, Chapter 6 was devoted towards characterizing in vivo

assembly complexes formed in the presence of antibiotics, erythromycin and chloramphenicol.

Quantitative analysis of these captured complexes provided information relating to the interplay and

dynamics of how these perturbations interfere ribosome assembly.

The mass spectrometry-based approaches used here will enable rapid characterization of RNA-protein

complexes and ribosome assembly complexes resulting from various perturbations. These approaches

are applicable to more complex eukaryotic and mammalian systems as well. Insights gained on how

the ribosome is being formed from its constituent RNA and proteins, and how

antibiotics/perturbations affect this process will be valuable in understanding the dynamics and

detailed molecular mechanisms involved in ribosome formation. Ultimately, deciphering these

complex processes will enable the development and rational drug design of small molecules or

ligands specific to bacterial ribosome assembly.

36 Cell culture!

MALDI TOF r-proteins rRNA MS ! • protein ID ! protease ! • modification profile digestion

ribosomes, Proteolytic ! peptides ribosome assembly particles ! !

LC-MS/MS LC-MSE !

Data analysis ! • protein ID • modification profile • quantitative information

!

Scheme 2.1 Diagram of mass spectrometry-based approaches used to characterize ribosomes and ribosome assembly complexes in vivo.

37 Chapter 3. Analysis of 30S Ribosomal Subunit Assembly Particles in rimM and rbfA

Escherichia coli Deletion Mutants by Mass Spectrometry

3.1 Introduction

The formation of active and functional ribosomes requires a multitude of ribosome assembly factors.

These protein factors ensure efficient production of ribosomal components engaging in protein

synthesis. In yeast, more than 170 non-ribosomal protein factors have been implicated in ribosome

biogenesis. While a relatively few assembly factors have been identified in bacteria, their exact roles

remain obscure. Ribosome maturation factor M (RimM) and ribosome binding factor A (RbfA) are

assembly factors involved in the final processing and maturation of the small ribosomal subunit 30S

in bacteria. Deletion of these proteins accumulates 30S precursor particles in vivo and their precise

roles in 30S subunit assembly are poorly understood. The aim of this chapter is to characterize the

small ribosomal subunit 30S precursor particles in E. coli RimM and RbfA deletion mutants using

15N-labeling and mass spectrometry. A combination of LC-MS/MS, MALDI-TOF MS and LC-MSE

approaches were used to define the composition, modification status and relative amounts of proteins

associated with these particles. Along with the in vivo hydroxyl radical footprinting and mass

spectrometry data, detailed molecular mechanisms how RimM and RbfA facilitate proper maturation

of the 30S subunit in vivo were uncovered. This work is part of a joint publication with Sarah Soper

(Sarah Woodson Lab at Johns Hopkins University), and this Chapter will only focus on experiments I

conducted here at the University of Cincinnati.

3.2 Experimental

3.2.1 Materials

Tryptone and yeast extract were obtained from Fisher Scientific (Fairlawn, NJ). Trypsin (sequencing

grade) and RNase-free DNase were purchased from Promega (Madison, WI). Micrococcal nuclease

38 (MNase) was purchased from New England Biolabs (Ipswich, MA). Endoproteinase Arg-C, Protease

Inhibitor Cocktail, chloramphenicol (CAM), 2-mercaptoethanol, dithiothreitol (DTT), iodoacetamide

(IA) and molecular-grade sucrose were obtained from Sigma (St. Louis, MO). Acids and organic

solvents were HPLC grade or better.

3.2.2 Bacterial Culture

E. coli wild-type K-12 strain was obtained from the American Type Culture Collection (ATCC,

Manassas, VA). The strain was cultured following standard growth conditions, harvesting and

isolation procedures previously described with minor modifications [117]. A single colony of wt K-

12 was inoculated in 10 mL 2xYT media (1.6% Bacto Tryptone, 1% Bacto Yeast Extract, 0.5%

NaCl) and incubated overnight at 37 °C and 200 rpm. One (1) ml of cell suspension was transferred

into 1-liter culture media. At mid-log phase (A600: 0.6-0.8), chloramphenicol was added (0.1 mg/mL,

final concentration) three minutes before harvesting. The cells were rapidly cooled on ice and

harvested by centrifugation at 6,000 rpm, 4 °C for 7 min. The cell pellet was resuspended in lysis

buffer (60 mM KCl, 60 mM NH4Cl, 50 mM Tris-HCl, 6 mM MgCl2, 6 mM 2-mercaptoethanol, pH

7.5-7.7). Cells were passed twice through a French Press and the supernatant was collected. RNase-

free DNase (1 g of cell/2.5 µL) and protease inhibitor cocktail (2 g of cell/0.5 mL) were added and

incubated on ice for 20 min. An equal volume of buffer D (60 mM KCl, 60 mM NH4Cl, 10 mM Tris-

HCl, 12 mM MgCl2, 6 mM beta-mercaptoethanol, pH 7.5-7.7) was added and the mixture was then

clarified by centrifugation at 16,000 rpm, 4 °C for 30 min. The crude lysate was recovered and stored

at -80 °C until further use.

3.2.3 Ribosome Preparation

Following culturing, 30S and 70S ribosomes and polysomes were purified by sucrose density gradient

ultracentrifugation. A 10-50 % sucrose gradient in buffer D (60 mM KCl, 60 mM NH4Cl, 10 mM

39 Tris-HCl, 12 mM MgCl2, 6 mM beta-mercaptoethanol, pH 7.5-7.7) was prepared using a gradient

mixer. Approximately 80-100 ODU of crude lysate were gently layered on top of the gradients. The

gradients were then centrifuged for 15 h at 23,000 rpm at 4 °C using a Beckman SW32 rotor/ SW28

swinging bucket rotor. The fractions were then collected using an ISCO automated fraction collector

with continuous monitoring at 254 nm. Fractions corresponding to the 70S ribosomes or polysomes

were pooled and pelleted by centrifugation for 20 h at 48,000 rpm at 4 °C. A second 0-45% sucrose

gradient in dissociating conditions (60 mM NH4Cl, 10 mM Tris-HCl, 1.1 mM MgCl2, 0.1 mM EDTA,

2 mM beta-mercaptoethanol, pH 7.5-7.7) was performed to purify mature 30S and 50S subunits from

intact 70S ribosomes. The purity of the ribosomal subunits was confirmed on a 0.1% non-denaturing

agarose gel. The purified ribosomal subunits were resuspended in Buffer D and stored at -80 °C until

further use.

3.2.4 Micrococcal Nuclease (MNase) Digestion of RNA and Protein Extraction

To enable dissociation of proteins from ribosomes, the RNA was first digested with MNase (100 µg

ribosomes/100 U MNase) at 37 °C overnight. Proteins were then precipitated by acetone after RNA

extraction with acetic acid. The total protein concentration was determined using the Coomassie Plus

Bradford Assay Kit (Pierce Scientific, Rockford, IL).

3.2.5 Mass spectrometry of mature 30S and pre-30S complexes

Total proteins from ΔrimM and ΔrbfA 30S ribosome assembly complexes and 15N-labeled 30S

proteins (E. coli MRE600) were provided by Prof. Sarah Woodson’s Lab. 15N-labeling and data-

dependent LC-MS/MS were used to determine the relative amounts of proteins associated with

mature 30S and pre-30S complexes {Gouw, 2011 #154}. Equal amounts (1:1) of unlabeled TP30S

proteins from K12, ΔrimM, or ΔrbfA strains were mixed with 15N-labeled TP30S proteins. The

proteins were reduced and alkylated using dithiothreitol (DTT) and iodoacetamide (IAA),

40 respectively. The protein mixture was digested with sequencing grade trypsin (1:30 enzyme: protein

ratio) overnight at 37°C.

Tryptic digests were desalted using C18 ZipTips and analyzed by data-dependent LC-MS/MS on a

Synapt G2 mass spectrometer coupled to a nanoAcquity UPLC (Waters, Milford, MA). Reversed-

phase chromatography on an BEH130 C18 column (1.7 µm, 100 µm x 100 mm, Waters, Milford,

MA) was performed at a flow rate of 1.0 µL min-1 using 0.1% formic acid in water as mobile phase A

and 0.1% formic acid in acetonitrile as mobile phase B. Two (2 µL) microliters of tryptic digest were

injected on column (250 nanograms total protein concentration). Gradient elution was carried out

starting with 5% B for the first min and 5% B min-1 to 10% B for another minute. A linear gradient

from 10% B to 50% B for 58 min was then employed followed by a constant 50% B for 10 min.

Finally, a 4.5% B min-1 to 5% B for 10 min was carried out and the column was re-equilibrated at 5%

B for another 10 min. Mass spectra were recorded in positive ion mode with a source temperature of

100 °C, spray voltage of 3.5 kV. All LC-MS analyses were performed in high-resolution mode with

at least 40,000 mass resolving power (FWHM). All samples were analyzed in triplicate.

3.2.6 Database Searching and Data Analysis

For peptide identification and quantitation, raw mass spectral data from a data-dependent acquisition

mode were uploaded to Mascot Distiller version 2.3.2.0 (www.matrixscience.com) [118]. Raw mass

spectral data were processed into .mgf files and searched against the UniProtKB/Swiss-Pro protein

database. Searches were limited to E. coli and Trypsin/P was selected as the protease. Search

parameters used were monoisotopic masses with peptide mass tolerance of 0.6 Da and fragment mass

tolerance of 0.3 Da. One (1) internal missed cleavage was allowed with cysteine residues being

carbamidomethylated (+57.0215 Da) and methionine being oxidized (+15.9949 Da) as dynamic

41 modifications. Quantification was set to “15N Metabolic [MD]”. A composite decoy search was

performed and specific cut-off scores were determined allowing <1% false discovery rate.

For peptide quantification, the threshold for the extracted ion chromatogram (XIC) was set to 0.1 Da-

0.2 Da. The minimum signal-to-noise (S/N) was 0.1 and the minimum and maximum m/z values were

set from 50 to 100,000 respectively. The correlation threshold for quantification was set to 0.7- 0.9. A

minimum of two light-heavy peptide pairs were used for quantification of the proteins, while manual

inspection of the data was done for proteins with only one light-heavy peptide pair to assess data

quality. Quantitative analysis was performed in triplicate and the L/H ratios were averaged and

standard deviation of these multiple measurements were reported.

3.2.7 MALDI-TOF MS of intact proteins

All matrix assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS)

experiments were performed on a MDS SCIEX 4800 MALDI TOF/TOF analyzer equipped with

Nd:YAG 200 Hz laser (Applied Biosystems, Framingham, MA). Total 30S proteins (TP30) from

wild-type K12, ΔrimM, or ΔrbfA were acidified with trifluoroacetic acid (TFA) and mixed with equal

volume of saturated sinapinic acid as matrix in 33% aqueous acetonitrile with 0.1% trifluoroacetic

acid [119]. One (1) microliter of the mixture was spotted onto a MALDI target plate and analyzed in

linear mode. Mass spectra were collected in positive ionization mode. The MALDI-TOF mass

spectrometer was calibrated using monoisotopic peaks of ubiquitin [M+H]+ 8565.9, cytochrome C

[M+H]+ 12361.0, and myoglobin [M+H]+ 16952.3. The spectra were then internally calibrated using

well-resolved peaks of small subunit proteins of S15, S16, and S20.

42 3.2.8 LC-MSE Analysis of Protein S5 Arg-C Digestion Products

Endoproteinase Arg-C digestion of protein S5 was performed to determine the modification status of

the protein as described elsewhere [120]. Total 30S, 70S, and polysomal proteins from wild-type K12,

ΔrimM and ΔrbfA were digested with Arg-C and the digestion products were analyzed by alternate

scanning mode, LC-MSE [113,121]. Peptide ions corresponding to the acetylated peptide (m/z 548,

[M+4H]4+) and non-acetylated peptide (m/z 537 [M+4H]4+) were extracted from total ion

chromatograms (TIC) and ion abundances were obtained by integrating their corresponding peak

areas. Values in Table 3.1 were obtained by dividing the ratio of the ion abundance of the acetylated

or non-acetylated peptide to the total ion abundance of both acetylated and non-acetylated peptides in

a single LC-MS/MS run. Percent modification was then determined from these values. At least three

independent measurements for each sample were performed. Manual evaluation of collision-induced

dissociation (CID) spectra of the precursor ions and database searching on PLGS (ProteinLynx

Global Server, Version 2.5.2, Waters, Milford, MA) were performed to verify identity and

modification status of protein S5.

3.3 Results and Discussion

3.3.1 Quantitative analysis of proteins associated with pre-30S complexes in rimM and

rbfA deletion mutants

To determine the identity and relative amounts of r-proteins associated with pre-30S complexes

obtained from ΔrimM and ΔrbfA E. coli strains, 15N-labeling and data-dependent LC-MS/MS was

performed. Equal amounts of TP30 from purified pre-30S complexes were mixed with 15N-labeled

TP30 prepared using mature 30S ribosomes from MRE600 cells grown in heavy medium, and the

mixture was analyzed by data-dependent LC-MS/MS [100]. To facilitate comparison of the various

strains, the 14N/15N (L/H) ratios were normalized to that of primary binding protein S8. Protein S8 is

expected to be present in mature 30S subunit, and thus has a copy number of one per 30S ribosome.

43 Figure 3.1 shows the proteins and their relative amounts in mature wild-type 30S, and pre-30S

complexes from ΔrimM and ΔrbfA strains. For the K12 30S control, r-proteins are equally distributed

among primary, secondary and tertiary 30S assembly proteins defined by the Nomura map (Figure

3.1, top). The uniform distribution of proteins is consistent with their known stoichiometry within the

mature 30S subunit [7,122]. The small subunit protein S1, which is not constitutively bound to the

30S subunit, is detected in K12 30S ribosomes suggesting its important role in translation. Protein S1

is indispensable for translation initiation as well as in tmRNA-mediated translation in bacteria

[14,123,124]. Quantitative analysis revealed a stoichiometric amount of S1 in mature K12 30S

subunit. This protein, however, is under-represented in pre-30S complexes from ΔrimM and ΔrbfA

mutants indicating diminished activity of these complexes in protein translation (Figure 3.1, far right

column).

Figure 3.1 Relative quantities of r-proteins in wild-type (K12) 30S ribosomes, ΔrimM and ΔrbfA (L+H) pre-30S complexes. For each protein, the ratio of unlabeled (L) peptide in the test sample to 15N-labeled (H) peptide from MRE600 TP30 was determined by data-dependent LC-MS/MS and data processing on Mascot Distiller. Relative quantities are normalized to protein S8. Error bars represent the standard deviation of three technical replicates. R-proteins are organized by their position on the Nomura map and colored by their location in the structure: white, 5’ domain (body); light gray, central domain (platform); dark gray, 3’ domain (head); black, S1 (Adapted from Soper et al. 2013).

44 The 30S ribosome assembly complexes formed in the absence of RbfA contained all primary and

secondary assembly proteins but lacked some tertiary assembly proteins. Proteins S2, S3, and S21 are

mostly depleted in these complexes. These proteins are the final ones that bind to the 30S subunit

during maturation as defined in the Nomura map (Figure 3.1, bottom). In contrast, 30S ribosome

assembly complexes that resulted from RimM deletion have a more severe effect in the amounts of

proteins than the deletion strain of RbfA. The complexes formed in the absence of RimM were less

complete and most of the secondary as well as tertiary proteins are significantly depleted (Figure 3.1,

middle). The secondary binding proteins S5, S12, and S19 are under-represented in ΔrimM and every

tertiary r-protein is substantially reduced as well, to less than 10%. The proteins, which bind the 3’

domain including the primary binding protein S7 and secondary assembly protein S9, are less

abundant than those proteins binding in 5’ and central domain (Figure 3.1, dark gray bars). These

results are consistent with RimM acting on earlier stages of 30S assembly (than RbfA) and facilitate

assembly and maturation of the head portion of the 30S subunit [109,125-127]. The results further

reveal an unexpected defect in the assembly of the central domain of the 30S complexes in ΔrimM

strain (Soper et al. 2013).

3.3.2 Posttranslational modification (PTM) of small ribosomal subunit 30S proteins

Several bacterial ribosomal proteins are posttranslationally modified [49]. The most common

modifications found in E. coli ribosomal proteins include loss of terminal methionine, acetylation and

methylation. The exact role of these PTMs in ribosome structure and function is unclear [34]. The

small subunit proteins S5 and S18 are posttranslationally modified by the N-acetyltransferases RimJ

and RimI, respectively [128]. To determine whether these modifications are present in the pre-30S

complexes obtained from ΔrimM and ΔrbfA strains as well as in wild-type mature 30S subunit,

MALDI-TOF mass spectrometry was used to analyze intact proteins. Mass spectral analysis revealed

proteins S5 and S18 fully acetylated in E. coli K12 30S proteins (Figure 3.2). Protein S18 is mostly

unmodified in the ∆rimM complexes (Figure 3.3, left inset), and mostly acetylated in ∆rbfA

45 complexes (Figure 3.4, left inset). The modification status of protein S5 needs to be confirmed,

however, as the MALDI mass spectral data could not resolve both the acetylated and non-acetylated

proteins. Although the m/z corresponding to the acetylated version of S5 is detected in the mass

spectra, the +42 Da difference due to acetylation of the protein could not be unambiguously

confirmed. An alternative approach was used to determine and quantify the modification status of S5.

Figure 3.2. MALDI-TOF MS analysis of intact proteins from wild-type K12 total 30S proteins. Proteins S5 and S18 are fully acetylated in mature 30S subunit.

46

Figure 3.3 MALDI-TOF MS analysis of intact proteins from ΔrimM pre-30S complexes. Protein S18 is mostly unmodified in ΔrimM complexes.

Figure 3.4. MALDI-TOF MS analysis of intact proteins from ΔrbfA pre-30S complexes. Protein S18 is mostly modified in ΔrbfA complexes.

47 Because MALDI-TOF MS could not resolve the modification status of protein S5, we used another

approach LC-MSE [113,120] to determine the relative abundance of the modified and unmodified

forms of S5. This was accomplished by selectively monitoring the mass-to-charge values

corresponding to the acetylated (m/z 548) and non-acetylated (m/z 537) peptide fragment ions

generated from endoproteinase Arg-C digestion of S5. By integrating the peak areas in the extracted

ion chromatograms (XICs) of the modified versus the unmodified peptides, the relative abundance of

S5 acetylation status was determined.

Table 3.1. Quantification of S5 acetylation from various sources. Recombinant S5 was overexpressed in E. coli cells and purified before analysis. K12 proteins were isolated from sucrose gradient fractions as indicated. Pre-30 proteins were obtained from oligonucleotide affinity-purified complexes. Protein S5 % non-acetylated % acetylated

Recombinant non-acetylated 96.3±0.4 3.7±0.4 S5 K12 Polysome 1.3±0.1 98.7±0.1

K12 70S 1.0±0.2 99.0±0.2

K12 30S 1.8±0.3 98.2±0.3

ΔrimM 11.6±0.4 88.4±0.4

ΔrbfA 33.5±1.0 66.5±1.0

Protein S5 is almost 99% acetylated in K12 30S and 70S ribosomes and polysomes, while it is mostly

unmodified in recombinant non-acetylated S5 (Table 3.1). Protein S5 is 88% acetylated in TP30 from

∆rimM (Figure 3.5 A-C, Table 3.1) and 66% acetylated in ∆rbfA TP30 (Figure 3.6 A-C, Table 3.1).

Over-expressed S5 is only 4% acetylated, confirming that RimJ acts on the pre-30S complex rather

than free protein [129,130]. Further, RimJ has been reported to function both as r-protein modifying

enzyme as well as ribosome assembly factor in E. coli [130]. Biochemical and footprinting

48 experiments further showed that the extent of protein modification correlates with the formation of

specific rRNA interactions during assembly (Soper et al. 2013). These results suggest that

modification of r-proteins is tightly coupled with the course of ribosome assembly.

A B

C D

Figure 3.5 (A) Selected ion chromatograms (SIC) of the acetylated (m/z 548) and non-acetylated (m/z 537) peptide fragments from ΔrimM TP30. (B) Mass spectrum of the 4+, 3+, and 2+ charge states of the acetylated peptide from ΔrimM TP30. (C) Mass spectrum of the 4+, 3+, and 2+ charge states of the non-acetylated peptide fragment from ΔrimM TP30.

49 A B

C D

Figure 3.6 (A) Selected ion chromatograms (SIC) of the acetylated (m/z 548) and non-acetylated (m/z 537) peptide fragments from ΔrbfA TP30. (B) Mass spectrum of the 4+, 3+, and 2+ charge states of the acetylated peptide from ΔrbfA TP30. (C) Mass spectrum of the 4+, 3+, and 2+ charge states of the non-acetylated peptide from ΔrbfA TP30.

To confirm the peptide sequence containing the acetylated and non-acetylated amino acid alanine (A)

in Arg-C digestion products from ∆rimM and ∆rbfA TP30, collision-induced dissociation of the

+ precursor ions of both the acetylated and non-acetylated peptides was performed (Figure 3.7). The b2

+ ion (m/z 251) confirmed the presence of acetylated peptide while the b2 ion (m/z 209) confirmed the

presence of non-acetylated peptide. The mass difference of 42 Da suggests acetylation.

50 3+ A 730.42 100 ΔrimM

Ac-A H I E K Q A G E L Q E K L I A V N R

679.52

%

4+ I + 2 548.06 + 110.07 y5 + + 2+ b2 y4 572.35 y 2+ + + 6 b3 + 1095.62 I19 251.11 459.28 y6 647.37 2+ 129.10 + y + 2+ y17 2+ b6 7 y16 + + + + y b9 y + + + y2 + b4 10 + 9 b10 y10 y1 y3 y8 0 100 500 1000 1500 m/z

B 761.44 488.32 100 ΔrimM

A H I E K Q A G E L Q E K L I A V N R

% 3+ 716.41

524.83

+ y1 4+ + 537.55 b2 906.45 I + 209.10 + + 19 y 626.40 y 4 6 + + + y + y9 b3 y5 8 2+ y + + 2+ 3 y7 y17 1074.61 0 100 500 1000 1500 m/z

Figure 3.7 Collision-induced dissociation (CID) fragmentation of (A) acetylated and (B) non- acetylated peptide fragments of S5 from ΔrimM TP30.

51 3+ A 730.40 100 ΔrbfA

Ac-A H I E K Q A G E L Q E K L I A V N R

679.52 %

+ I2 + b2 4+ + 2+ y0 2+ y6 2 251.11 + 548.30 y4 + 1095.60 + b + + + y 6 b9 y1 b3 5 y + y + 7 + + + 2 + y9 b10 y10 + b4 + y3 y8 0 100 500 1000 1500 m/z

B 761.44 100 ΔrbfA

A H I E K Q A G E L Q E K L I A V N R

488.32

%

3+ 524.83 716.41

+ b2 4+ + 537.55 I19 209.10 + y + 626.40 + y1 4 + y + y8 9 b + + + y6 2+ 3 y3 y5 + 2+ y7 y17 906.45 1074.61 0 100 500 1000 1500 m/z

Figure 3.8 Collision-induced dissociation (CID) fragmentation of (A) acetylated and (B) non- acetylated peptide fragments of S5 from ΔrbfA TP30.

52 3.4 Conclusions

In this chapter, a combination of MS-based approaches including 15N-labeling and data-dependent

LC-MS/MS, MALDI-TOF MS and LC-MSE were used to define the composition, relative amounts

and posttranslational modifications of proteins associated with pre-30S complexes in rimM and rbfA

deletion mutants. Relative protein levels of 30S proteins in mutant strains were determined by 15N-

labeling and LC-MS/MS analysis of the tryptic peptides compared to that of the wild-type 30S

proteins. Quantitative analysis of the proteins is in agreement with the Nomura assembly map

wherein tertiary binding proteins are under-represented in the complexes suggesting their roles in late

stages of 30S assembly. The lack of RimM has a more profound effect than has the lack of RbfA on

the amount of proteins in the 30S particles, which is in agreement that RimM is needed in a step prior

to RbfA during maturation of 16S rRNA. In addition, the modification status of proteins S5 and S18

was established demonstrating the extent of protein modification correlates with the formation of

specific rRNA interactions during 30S assembly.

53 Chapter 4. Quantitation of Bacterial Ribosomal Proteins by LC-MSE

4.1 Introduction

Relative quantitation of proteins by 15N-labeling and LC-MS/MS provides information on the

differential expression of proteins in normal and perturbed samples. This approach, however, is

limited to comparing two samples at a time, labeling can be expensive and laborious, and not

amenable to other multicellular organisms. Thus, methods based on label-free absolute quantitation of

proteins are continually being improved and developed. One such approach is LC-MSE (enhanced

liquid chromatography-mass spectrometry) developed by Silva et al. (2006). This method has been

described in Chapter 2.

The LC-MSE approach developed by Silva and co-workers has been used for absolute quantification

of proteins in simple and complex mixtures [113,133]. The alternate scanning mode of data

acquisition during LC-MS analysis allows a comprehensive inventory of all precursor ions with their

corresponding time-resolved product ions. This approach does not bias gas phase pre-selection of

precursor ions during CID as compared to that of data-dependent analysis. Thus, all components that

are above the detection limit of the instrument are detected and identified. In addition, two aspects of

the LC-MSE approach make it a potentially viable method for high-throughput qualitative and

quantitative analyses. First, the accurate mass measurements of precursor and their respective product

ions provide confident identifications of large number of proteins with high sequence coverage.

Second, its ability to collect high quality MS data across the entire chromatographic peak width for all

detected peptides allows accurate quantitation of peptides/proteins present in a given sample. These

attributes of the LC-MSE approach enabled quantification of many important analytes in a variety of

biological systems including viral antigens [131], microsomal membrane and bacterial proteome

[132,133], human serum [134], and plant proteins [135,136]. Further, the approach was also evaluated

54 in different instrumental platforms such as on FT-ICR instruments and been found to be robust if at

least three or more peptides are identified and detected per protein [137].

One major limitation of the original LC-MSE approach is the method biased against small molecular

weight r-proteins (under 14 kDa). In addition, this approach has not been evaluated for proteins under

14 kDa. Small molecular weight r-proteins do not generate numerous peptides upon trypsin digestion

and therefore are under-represented in a typical tryptic digest. The magnitude of the error in

quantitation is highly dependent on protein size as there are too few peptides to choose from the best

ionizing peptides generated from small proteins [113]. Because more than 50% of bacterial 70S r-

proteins have molecular weight less than 14 kDa, absolute quantitation of r-proteins by LC-MSE

remains challenging (Figure 4.1). Most of the large subunit proteins (62%, 21 out of 34) and several

small subunit proteins (48%, 10 out of 21) are under 14 kDa and generate few peptides upon

proteolytic digestion. To evaluate the applicability of the LC-MSE approach for “ribosome-centric”

quantitation of proteins, we have investigated strategies to enable accurate and reproducible

quantitation of r-proteins in wild-type E. coli polysomes. Because polysomes are actively

synthesizing complexes, stoichiometric amounts of r-proteins are expected [7,62]. That is, one copy

of each r-protein is present per ribosome except for proteins L7 and L12, which are present in two

copies each. The goal of this current work is to investigate experimental strategies to increase the

number of peptides generated from proteins, enhance peptide detection and quantification by LC-MSE.

55 30000 25000 20000 15000 14 kDa

MW (Da) MW 10000 5000 0 S2 S3 S4 S7 S5 S6 S9 S8 S11 S11 S12 S13 S10 S14 S19 S15 S17 S20 S16 S18 S21 Small subunit proteins

30000 25000 20000 15000 14 kDa MW (Da) MW 10000 5000 0 L2 L1 L3 L4 L5 L6 L9 L11 L11 L10 L13 L16 L15 L17 L14 L20 L19 L18 L22 L21 L23 L24 L25 L27 L28 L31 L29 L35 L30 L32 L33 L34 L36 L7/12 Large subunit proteins

Figure 4.1 Plot of the molecular weight of bacterial ribosomal proteins. More than 50% of the proteins have molecular weight less than 14 kDa, a cut-off range that was originally determined in quantifying proteins by LC-MSE.

The aim of this chapter is to investigate the LC-MSE approach for “ribosome-centric” quantitation of

proteins associated with actively synthesizing ribosomes such as polysomes. Because the information

obtained in these experiments is related to the number of peptides identified per protein (within the

complex), experimental conditions were optimized to provide an unbiased analysis of all low and

high molecular weight components. Experimental strategies including the use of multiple

endoproteinases to digest total proteins and an orthogonal dimension of gas phase separation through

ion mobility were employed to increase the number of peptides generated and detected during MS

analysis. These strategies significantly enhanced the number of peptides detected and protein

sequence coverage, thus allowing accurate and reproducible quantitation of r-proteins. The ability to

accurately determine the absolute quantity of proteins in ribosomes and ribosome assembly particles

will provide useful information relating to complex stoichiometry and biogenesis.

56 4.2 Experimental

4.2.1 Materials

Tryptone and yeast extract were obtained from Fisher Scientific (Fairlawn, NJ). Trypsin (sequencing

grade) and RNase-free DNase were purchased from Promega (Madison, WI). Chymotrypsin

(sequencing grade) was obtained from Protea Biosciences (Morgantown, WV). Micrococcal nuclease

(MNase) was purchased from New England Biolabs (Ipswich, MA). Phosphorylase b (MassPrep

Standard) and Fibrinopeptide b (Glu-Fib) were obtained from Waters (Milford, MA). Protease

Inhibitor Cocktail, endoproteinases Asp-N and Glu-C, chloramphenicol (CAM), 2-mercaptoethanol,

dithiothreitol (DTT), iodoacetamide (IA) and molecular-grade sucrose were obtained from Sigma (St.

Louis, MO). Acids and organic solvents were HPLC grade or better.

4.2.2 Bacterial Culture

E. coli wild-type K-12 strain was obtained from the American Type Culture Collection (ATCC,

Manassas, VA). The strain was cultured following standard growth conditions, harvesting and

isolation procedures previously described with minor modifications [117]. A single colony of wild-

type K-12 was inoculated in 10 mL 2xYT media (1.6% Bacto Tryptone, 1% Bacto Yeast Extract,

0.5% NaCl) and incubated overnight at 37 °C and 200 rpm. One (1) ml of cell suspension was

transferred into 1-liter culture media. At mid-log phase (A600: 0.6-0.8), chloramphenicol was added

(0.1 mg/mL, final concentration) 3 min before harvesting. The cells were rapidly cooled on ice and

harvested by centrifugation at 6,000 rpm, 4 °C for 7 min. The cell pellet was resuspended in lysis

buffer (60 mM KCl, 60 mM NH4Cl, 50 mM Tris-HCl, 6 mM MgCl2, 6 mM 2-mercaptoethanol, pH

7.5-7.7). Cells were passed twice through a French Press and the supernatant was collected. RNase-

free DNase (1 g of cell/2.5 µL) and protease inhibitor cocktail (2 g of cell/0.5 mL) were added and

incubated on ice for 20 min. An equal volume of buffer D (60 mM KCl, 60 mM NH4Cl, 10 mM Tris-

HCl, 12 mM MgCl2, 6 mM beta-mercaptoethanol, pH 7.5-7.7) was added and the mixture was then

57 clarified by centrifugation at 16,000 rpm, 4 °C for 30 min. The crude lysate was recovered and stored

at -80 °C until further use.

4.2.3 Isolation of Free Subunits (30S and 50S), 70S Ribosomes and Polysomes

Following culturing, free subunits (30S and 50S), 70S ribosomes and polysomes were purified by

sucrose density gradient ultracentrifugation. Sucrose gradients (10-50 %) in buffer D (60 mM KCl, 60

mM NH4Cl, 10 mM Tris-HCl, 12 mM MgCl2, 6 mM beta-mercaptoethanol, pH 7.5-7.7) were

prepared using a gradient mixer. Approximately 80-100 ODU (optical density unit) of crude cell

lysate were gently layered on top of the gradients. The gradients were then centrifuged for 15 h at

23,000 rpm at 4 °C using a Beckman SW32 rotor/ SW28 swinging bucket rotor. The fractions were

then collected using an ISCO automated fraction collector with continuous monitoring at 254 nm.

Fractions corresponding to the free 30S subunit, free 50S subunit, 70S ribosomes and polysomes were

pooled and pelleted by centrifugation for 20 h at 48,000 rpm at 4 °C. Non-denaturing agarose gel

(0.1 %) was used to evaluate the purity of the isolated subunits. If necessary, a second sucrose

gradient fractionation is done to further purify the ribosomes. The ribosome pellets were resuspended

in Buffer D and stored at -80 °C until further use.

4.2.4 Micrococcal Nuclease (MNase) Digestion of rRNA and Protein Extraction

To enable dissociation of proteins from ribosomes, the RNA was first digested with MNase (100 µg

ribosomes/100 U MNase) at 37 °C overnight. Proteins were then precipitated by acetone after RNA

extraction with acetic acid. The total protein concentration was determined using the Coomassie Plus

Bradford Assay Kit (Pierce Scientific, Rockford, IL).

58 4.2.5 Enhanced Liquid Chromatography- Mass Spectrometry (LC-MSE)

Quantitation of total polysome r-proteins using LC-MSE was performed as described elsewhere [113].

Five (5 µg) of total 70S proteins were resuspended in 8 M urea, 0.4 M ammonium bicarbonate (pH

7.5-8.5). The protein mixture was reduced and alkylated using DTT and IAA, respectively. Trypsin

(1:30 enzyme: protein ratio) was added and incubated overnight at 37°C to digest the proteins.

Tryptic digests were cleaned-up using C18 ziptips and spiked with pre-digested phosphorylase b (0.5

pmol on column) as internal standard (IS). Tryptic peptides were analyzed on a Synapt G2S mass

spectrometer coupled to a nanoAcquity UPLC (Waters, Milford, MA). Reversed-phase

chromatography on a BEH130 C18 column (1.7 µm, 100 µm x 100 mm, Waters, Milford, MA) was

performed at a flow rate of 1.0 µL min-1 using 0.1% formic acid in water as mobile phase A and 0.1%

formic acid in acetonitrile as mobile phase B. Two (2 µL) microliters of tryptic digest were injected

on column (500 nanograms total protein concentration). Gradient elution was carried out starting with

5% B for the first min and 5% B min-1 to 10% B for another minute. A linear gradient from 10% B to

50% B for 58 min was then employed followed by a constant 50% B for 10 min. Finally, a 4.5% B

min-1 to 5% B for 10 min was carried out and the column was re-equilibrated at 5% B for another 10

min. Mass spectra were recorded in positive ion mode with a source temperature of 100 °C, spray

voltage of 3.5 kV. Lock mass (Glu-Fib, mass-to-charge 875.8426, 2+) was delivered using an

auxiliary pump at 0.5 µL min-1 at 100 fmol µL-1 to the reference sprayer of the NanoLock Spray

source. All LC-MSE analyses were operated in sensitivity mode with at least 10,000 mass resolving

power (FWHM). Continuum and accurate mass LC-MSE spectral data were collected on alternating

acquisition mode of low-energy CID and high-energy CID as previously described [113]. A constant

4 eV was applied at low energy CID mode while an energy ramp from 15-40 eV was used at high

energy CID mode. Scan time was 1 sec for both low energy CID mode and high-energy CID mode.

For ion mobility MS separation (IMS) of peptides, the tri-wave region was operated at constant wave

velocity and wave height of 1600 m/s and 40 V, respectively. All samples were analyzed in triplicate.

59 4.2.6 Multiple Endoproteinase Digestion of Proteins

Multiple proteases including trypsin, chymoytrpsin, Asp-N and Glu-C were used to digest total r-

proteins (Scheme 4.1). The digestion conditions for each of the protease were individually optimized

as described in the manufacturer’s manual. Five (5 µg) of total proteins were digested with each

protease and the proteolytic digests were combined and spiked with IS before LC-MSE analysis.

Identical LC-MS conditions were used as described above.

Trypsin Chymotrypsin Asp-N Glu-C ! ! TP polysome TP polysome TP polysome! TP polysome!

Trypsin Chymotrypsin Asp-N Glu-C

! ! ! ! Proteolytic Proteolytic Proteolytic Proteolytic peptides peptides peptides peptides

Combine proteolytic peptides

! Spike IS

! LC-MSE

!

Identify 3 most intense and unique peptides

Quantitative analysis

!

Scheme 4.1 Multiple endoproteinase digestion of total proteins from E. coli wild-type polysomes.

60 4.2.7 Database Searching and Data Analysis

Continuum LC-MSE spectral data were uploaded to PLGS (ProteinLynx Global Server, Version 2.5.2,

Waters, Milford, MA) for protein identification and quantitation [138]. Searches were limited to E.

coli. Search parameters included use of monoisotopic masses with peptide mass tolerance and

fragment mass tolerance set to automatic. Typical peptide and fragment mass tolerances at high-

resolution mode are less than 5 ppm. One internal missed cleavage was allowed with cysteine

residues being carbamidomethylated (+57.0215 Da) and methionine being oxidized (+15.9949 Da). A

composite decoy search was performed and specific cut-off scores were determined allowing <4%

false positive rate. A minimum of two peptide matches was considered for positive protein

identification. Manual inspection of MS/MS data was performed for proteins having only two peptide

matches to verify sequence assignments. For quantitation of proteins in continuum LC-MSE data, the

calibration protein, phosphorylase b (P00489), was set to 500 fmol. For database searching of LC-

MSE data from multiple protease digestion, trypsin was set as the primary digest reagent while non-

specific protease was set as secondary digest reagent. Manual evaluation and inspection of the unique

peptides and their ion abundances from multiple enzymatic digestion were done to verify peptide

sequences and quantitation of r-proteins.

4.3 Results and Discussion

4.3.1 Quantitation of Proteins in Escherichia coli Polysomes by LC-MSE

Our initial LC-MSE analysis of proteins from wild-type polysomes using the previously described

approach by Silva et al. (2006), revealed a non-uniform distribution of both small subunit and large

subunit proteins associated within the complex (Figure 4.2). The original approach is based upon a

single protease digestion of total r-proteins with trypsin and the corresponding tryptic digests were

analyzed by LC-MSE. To limit run-to-run variability, protein levels were normalized to protein S4

and protein L3 for 30S proteins and 50S proteins, respectively. Proteins S4 and L3 are expected to be

61 present in one copy per ribosome as they are primary binding proteins and essential for ribosome

assembly and function.

Figure 4.2 shows the small molecular weight proteins from both 30S and 50S subunits are under-

represented, that is, they are present in less than one copy per ribosome. These proteins did not yield

good quantitative results because they generate too few peptides upon trypsin digestion. Therefore,

we sought strategies to increase the number of peptides generated from these proteins and enhance

their detection and identification during MS analysis.

2 LC-MSE

1 Amount(pmol) S1 S2 S3 S4 S5 S6 S7 S8 S9 S11 S11 S10 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 2 LC-MSE

1 Amount(pmol) L1 L2 L3 L4 L5 L6 L9 L11 L11 L10 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36 L7/12 r-proteins

Figure 4.2 Quantitation of small subunit 30S proteins and large subunit 50S proteins by LC-MSE as described by Silva et al. (2006). R-proteins are not uniformly distributed and are detected in less than 1 copy per ribosome. Amounts of small subunit proteins and large subunit proteins are normalized to protein S4 and protein L3, respectively. Error bars represent the standard deviation of 3 multiple measurements.

4.3.2 Additional Dimension of Gas Phase Separation Through Ion Mobility Increases the

Number of Peptides Detected and Sequence Coverage (SC) of Proteins

Quantitation of proteins by LC-MSE is based upon the detection of the three most intense peptides for

each protein. Thus, detection and identification of more peptides for each protein should enhance

reproducibility and accuracy of quantitation. Small molecular weight r-proteins generate few

62 proteolytic peptides and some of these peptides could escape detection and identification during LC-

MSE analysis. Therefore, an optimal approach that could detect and identify most, if not all peptides,

emanating from each protein is important to enable reproducible and accurate quantitation of these

proteins. We have investigated the utility of an additional dimension of gas phase separation using ion

mobility prior to collision-induced dissociation of peptides in quantifying r-proteins.

Ion mobility separation provides an additional dimension of gas phase separation prior to CID. It

offers a rapid 2D separation (µs-ms) of ionized precursor ions and the reduction of chemical noise

and ion suppression effects. This technique offers a greater number of observed proteolytically

derived peptides [139]. Valentine and co-workers showed that using ion mobility separation provides

more than an order of magnitude enhancement in component resolution, increasing measurement

sensitivity, dynamic range [140], spectral quality [141], increased number of peptide/protein

identifications/, higher sequence coverage, lower errors in protein abundance measurements and

depth of quantitative measurements [121].

My results reveal that incorporation of an additional dimension of gas-phase separation increases the

number of peptides detected, protein sequence coverage, and protein identifications. Figure 4.3 shows

the number of peptides detected for the small subunit and large subunit proteins after trypsin digestion

without and with ion mobility separation. The number of peptides increases with ion mobility

separation compared to without ion mobility. Similarly, the sequence coverage of the proteins

increases as well with ion mobility separation.

63 A 30 Trypsin without IMS 25 20 Trypsin with IMS 15 10

# of peptides#of 5 0 S1 S2 S3 S4 S5 S6 S7 S8 S9 S11 S11 S10 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 B 30 25 20 15 10

# of peptides#of 5 0 L1 L2 L3 L4 L5 L6 L9 L11 L11 L10 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36

C L7/12 100 80 60

%SC 40 20 0 S1 S2 S3 S4 S5 S6 S7 S8 S9 S11 S11 D S10 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 100 80 60 40 %SC 20 0 L1 L2 L3 L4 L5 L6 L9 L11 L11 L10 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36 L7/12 r-proteins

Figure 4.3 Comparing single protease (trypsin) digestion with and without ion mobility separation. Plots of the number of peptides identified for (A) 30S subunit proteins and (B) 50S subunit proteins. Plots of protein sequence coverage of (C) 30S subunit proteins and (D) 50S subunit proteins. Additional dimension of gas phase separation through ion mobility increases the number of peptides detected and sequence coverage of the proteins.

Some proteins show a dramatic enhancement in the number of peptides detected and protein sequence

coverage with IMS. For instance, protein S1 has an almost 6-fold increase in the number of peptides

identified with IMS. This increase afforded a 3-fold improvement in sequence coverage. Other

proteins, such as proteins S6 and S7, have no appreciable increase either in the number of peptides

detected or the sequence coverage probably because of the limited peptides generated upon trypsin

64 digestion of these proteins. The significant enhancement in the number of peptide detected and

sequence coverage using ion mobility is attributed to the ability of IMS to reduce and eliminate

chemical noise and ion suppression effects, thereby confidently identifying low-abundant peptides,

which are often masked by highly abundant ions in the mixture. Further, IMS allows simultaneous

MS/MS fragmentation of IMS-separated ions, thus enhancing throughput and experimental sensitivity

[139]. Figure 4.4 shows the amounts of r-proteins after incorporating ion mobility separation.

A 2 Trypsin with ion mobility separation

1 Amount(pmol) S1 S2 S3 S4 S5 S6 S7 S8 S9 S11 S11 S10 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 B r-proteins 2 Trypsin with ion mobility separation

1 Amount(pmol) L1 L2 L3 L4 L5 L6 L9 L11 L11 L10 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36 L7/12 r-proteins

Figure 4.4 Quantitation of (A) small subunit 30S proteins and (B) large subunit 50S proteins by LC- MSE with additional dimension of gas-phase separation through ion-mobility. Error bars represent the standard deviation of 3 multiple measurements.

Although I observed a dramatic enhancement in the number of peptides detected and higher protein

sequence coverage using ion mobility separation, several small r-proteins (e.g. S14, S19, S20 for the

small subunit and L18, L23, L29 for the large subunit) are still detected in sub-stoichiometric

amounts (Figure 4.4). Therefore, I investigated another strategy using multiple endoproteinases to

digest total r-proteins to improve quantitative accuracy of the LC-MSE approach to quantify r-proteins,

especially low molecular weight proteins.

65 4.3.3 Multiple Endoproteinase Digestion of Proteins Improves Quantitation by LC-MSE

Even using ion mobility separation, the small subunit proteins S15, S17, S18, S19, S20, S21 and the

large subunit proteins L21, L23, L28, L29, L30, L31, L32, L33, L34, L35 and L36 generate few

detectable tryptic peptides during LC-MSE analysis (Figure 3 A-B). Consequently, these proteins are

more likely to have large quantitation errors as highlighted by Silva et al. (2006). The magnitude of

the error is proportional to the size of the protein as there are too few peptides to choose from the

three best ionizing peptides generated from small proteins [113]. In addition, the three most intense

peptides typically exhibit a wide range of ionization efficiencies that skew the resulting average ion

abundance. In contrast, large molecular weight proteins have many peptides to choose from and

exhibit similar ionization efficiencies for the three best ionizing peptides. Because no significant

improvements were observed in quantifying these r-proteins using tryptic digestion and ion mobility

separation, I used multiple endoproteinases to digest total proteins. In addition to trypsin,

chymotrypsin, Asp-N, and Glu-C were used.

The utility of multiple protease digestion to enhance proteome coverage has been previously reported.

Choudhary et al. (2003) showed that using multiple enzyme digestion (trypsin, Lys-C, and Asp-N),

the sequence coverage of proteins identified by shotgun sequencing approach to proteomic analysis

was significantly increased [142]. Likewise, Swaney and co-workers demonstrated using S. cerevisiae

as the model organism, that a large portion of the proteome is inaccessible using a single protease

digestion alone [143]. Using multiple proteases (trypsin, LysC, ArgC, Asp-N, and Glu-C), the

sequence coverage of the proteins is improved three-fold. Similar results were observed using

multiple enzymatic digestion to enhance sequence coverage of proteins in human cerebrospinal fluid

[144] and ErbB2 tumor receptor [145].

The use of dual protease digestion and ion mobility separation in quantitation of r-proteins was

investigated. Five micrograms of total r-proteins were individually digested with chymotrypsin, Asp-

66 N and Glu-C. These were then combined with equal amounts of proteins digested with trypsin. The

resulting proteolytic peptides were analyzed by LC-MSE with and without ion mobility separation.

My results demonstrated a significant increase in the number of peptides detected and protein

sequence coverage using multiple high specificity enzymes to digest total proteins. Figure 4.5 shows

representative plots of the number of peptides and sequence coverage of r-proteins using trypsin

digestion alone and using trypsin and Asp-N (dual digestion) with ion mobility separation.

A 60 Trypsin 40 Trypsin_Asp-N

20 # of peptides#of 0 S1 S2 S3 S4 S5 S6 S7 S8 S9 S11 S11 S10 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 B 60

40

20 # of peptides#of 0 L1 L2 L3 L4 L5 L6 L9 L11 L11 L10 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36 L7/12 C 100 80 60

%SC 40 20 0 S1 S2 S3 S4 S5 S6 S7 S8 S9 S11 S11 S10 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 D 100 80 60 40 %SC 20 0 L1 L2 L3 L4 L5 L6 L9 L11 L11 L10 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36 L7/12 r-proteins

Figure 4.5 Comparing single protease (trypsin) digestion with IMS and dual protease (trypsin and Asp-N) digestion with IMS. Representative plots of the number of peptides identified for (A) 30S subunit proteins and (B) 50S subunit proteins. Representative plots of protein sequence coverage of (C) 30S subunit proteins and (D) 50S subunit proteins. Dual protease digestion and using ion mobility increase the number of peptides detected and sequence coverage of the proteins.

67 The number of peptides and the sequence coverage of the proteins dramatically increased using dual

protease digestion (e.g. trypsin and Asp-N) and ion mobility. For example, more peptides were

detected and identified by combining the proteolytic peptides from trypsin and Asp-N digestions

compared to trypsin alone. In addition, a similar trend was observed using IMS and dual protease

digestion. Figure 4.6 shows the increase in the number of peptides detected and the sequence

coverage of the proteins as compared to without IMS.

A 60 Trypsin_Asp-N_without IMS 40 Trypsin_Asp-N_with IMS

20 # of peptides#of 0 S1 S2 S3 S4 S5 S6 S7 S8 S9 S11 S11 S10 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 B 60

40

20 # of peptides#of 0 L1 L2 L3 L4 L5 L6 L9 L11 L11 L10 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36 L7/12 C 100 80 60

%SC 40 20 0 S1 S2 S3 S4 S5 S6 S7 S8 S9 S11 S11 S10 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 D 100 80 60

%SC 40 20 0 L1 L2 L3 L4 L5 L6 L9 L11 L11 L10 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36 L7/1

Figure 4.6 Comparing dual protease (trypsin and Asp-N) digestion with and without ion mobility separation. Representative plots of the number of peptides identified for (A) 30S subunit proteins and (B) 50S subunit proteins. Representative plots of protein sequence coverage of (C) 30S subunit proteins and (D) 50S subunit proteins. Ion mobility separation increases the number of peptides detected and sequence coverage of the proteins generated upon dual protease (trypsin and Asp-N) digestion.

Using dual protease digestion and IMS for the three different protease combinations tested,

quantitation of polysome proteins was significantly improved. Figures 4.7 and 4.8 show the amounts

68 of small subunit proteins and large subunit proteins normalized to protein S3 and protein L3,

respectively. Furthermore, there is an almost 50% increase in the number of unique protein

identifications using dual protease digestion and incorporating an additional dimension of gas phase

separation through ion mobility compared to without ion mobility separation (Figure 4.9).

2 Trypsin_Chymotrypsin

1 Amount(pmol)

2 Trypsin_Asp-N

1 Amount(pmol)

2 Trypsin_Glu-C

1 Amount(pmol) S1 S2 S3 S4 S5 S6 S7 S8 S9 S11 S11 S10 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 r-proteins

Figure 4.7 Quantitation of small subunit 30S proteins using ion mobility separation and dual protease digestion. Protein levels are normalized to protein S4.

2 Trypsin_Chymotrypsin

1 Amount(pmol) 2 Trypsin_Asp-N

1 Amount(pmol) 2 Trypsin_Glu-C

1 Amount(pmol) L1 L2 L3 L4 L5 L6 L9 L11 L11 L10 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36 L7/12 r-proteins

Figure 4.8 Quantitation of large subunit proteins using ion mobility separation and dual protease digestion. Protein levels are normalized to protein L3.

69 Tryp_Chym Tryp_Asp-N Tryp_Glu-C Trypsin 150

100

50

0 # of proteins#of identified IMS no IMS

Figure 4.9 The number proteins identified in database searching dramatically increased with ion mobility separation and dual protease digestion.

The increase in the number of peptides detected and sequence coverage afforded more protein

identifications during database searching (Figure 4.9). One of the advantages I observed of using

multiple proteases to digest r-protein is its ability to produce longer peptide digestion products. These

longer peptides exhibit a narrower range of ionization efficiencies, consequently improving r-protein

quantitation. The use of multiple proteases to generate proteolytically unique peptides is particularly

beneficial to small molecular weight r-proteins as they increase the number of peptide detections,

protein sequence coverage and quantitation of low molecular weight r-proteins. Finally, combining

the proteolytic peptides emanating from four different proteases with different cleavage specificities,

the quantitation of r-proteins was dramatically improved (Figure 4.10). All of the low molecular

weight proteins can be quantified using this approach except for proteins S21, L34, L35 and L36.

70 2 Multiple proteases

1 Amount(pmol) S1 S2 S3 S4 S5 S6 S7 S8 S9 S11 S11 S10 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21 r-proteins

2 Multiple proteases

1 Amount(pmol) L1 L2 L3 L4 L5 L6 L9 L11 L11 L10 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36 L7/12 r-proteins

Figure 4.10 Quantitation of small subunit 30S proteins and large subunit proteins using ion mobility separation and multiple endoproteinase digestion using trypsin, chymotrypsin, Asp-N, and Glu-C. A uniform distribution of proteins is observed which is in agreement with their known stoichiometry within the complex. Amounts of small subunit proteins and large subunit proteins are normalized to protein S4 and protein L3 respectively. Error bars represent the standard deviation of 3 multiple measurements.

4.4 Conclusions

The improvements of the LC-MSE method using ion mobility separation and multiple endoproteinase

digestion allow accurate and reproducible quantitation of r-proteins in ribosomes. A uniform

distribution of r-proteins was observed in actively synthesizing ribosomes, in agreement with the

known stoichiometry of these proteins within the complex. Most of r-proteins are reproducibly

quantified by the improved LC-MSE except for a few proteins (S21, L34, L35, and L36), which are

still not quantifiable by this technique even using ion mobility separation and multiple endoproteinase

digestion. Although I obtained good quantitative results for most of the r-proteins, the improved LC-

MSE is still limited to proteins that generate sufficient peptides for quantitation. Some proteins do not

generate many peptides, even using multiple proteases, and therefore cannot be quantified. The

presence of post-translational modifications of r-proteins can also affect the resulting stoichiometry of

the proteins. This is true when dealing with in vivo ribosome assembly complexes wherein some r-

71 proteins are not fully modified during the course of ribosome biogenesis and assembly. For mature

ribosomes, however, r-proteins are fully modified and the errors and variability due to PTMs are less

or negligible. Some proteins might be lost during purification of these complexes as well and could

affect quantitation of the proteins. Although ion mobility separation improved the number of peptides

detected and identified during MS analysis, I believe that the experimental approaches described here

could be used to other LC-MS platforms as well and still obtain good quantitative results without IMS.

72 Chapter 5. Characterization of Ribosomal Proteins Associated with the Different

Functional States of the Ribosomes

5.1 Introduction

The ribosome is a highly dynamic macromolecular machine responsible for synthesizing proteins

within a cell. It is assumed to consist of a defined and homogeneous population of particles as

revealed by the detailed X-ray crystal structures of the complex [1-7]. Several lines of evidence

suggested, however, that ribosomes are heterogeneous particles and this heterogeneity served an

additional regulatory mechanism of the cell to thrive under non-optimal conditions [146,147].

Variations in the r-protein and rRNA complement of the ribosome could lead to the generation of

heterogeneous and specialized ribosomes [146]. Ribosome heterogeneity due to variation in the r-

protein complement of the ribosome was reported 40 years ago. Different classes of ribosomes were

observed with several r-proteins not present in equimolar amounts. Those r-proteins present in less

than one copy per ribosome are subject to variation among the different functional states of the

ribosome [91-94,148-150].

A plethora of biochemical and biophysical techniques have been used to probe ribosome

heterogeneity. Gel-based methods and radiolabeling are commonly used to determine the

stoichiometry of r-proteins associated with ribosomes. These techniques, however, are limited in

measurement accuracy and precision. Here, I used a mass spectrometry approach based on absolute

quantitation of proteins to probe ribosome heterogeneity by evaluating the stoichiometry of r-proteins.

The aim of this chapter is to extend the applicability of the improved LC-MSE to characterize the r-

proteins associated with the different functional states of the ribosome namely free 30S, free 50S,

single ribosomes (70S), and actively synthesizing ribosomes (polysomes).

73 5.2 Experimental

5.2.1 Materials

All material and reagents were obtained as described in Chapter 4.

5.2.2 Bacterial Culture

E. coli wild-type K-12 strain was used to obtain the different functional states of the ribosomes. The

strain was cultured in 2xYT medium following standard growth conditions, harvesting and isolation

procedures previously described with minor modifications as described in Chapter 4 [117]. Two

biological replicates of wild-type K12 cells were prepared for biological reproducibility studies.

5.2.3 Isolation of Free Subunits (30S and 50S), 70S Ribosomes and Polysomes

Free subunits (30S and 50S), 70S ribosomes, and polysomes were purified by sucrose density

gradient ultracentrifugation from the two biological replicates of wild-type K12 cells as described in

Chapter 4.

5.2.4 Dissociation of RNP complex, Protein Digestion and Mass Spectrometry (LC-

MSE)

Dissociation of RNP complexes, protein extraction, and quantitation of total r-proteins from the

different functional states of the ribosomes using the improved LC-MSE was performed as described

in Chapter 4.

5.2.5 Database Searching and Data Analysis

Continuum LC-MSE spectral data were uploaded to PLGS (ProteinLynx Global Server, Version 2.5.2,

Waters, Milford, MA) for protein identification and quantitation as described in Chapetr 4. All

analyses were performed in triplicate for each biological replicate. A total of six independent

74 measurements were done and the values were averaged to obtain the amounts for each protein. To

limit variability between LC-MS runs, protein levels were normalized to L3 and S4 for large subunit

and small subunit proteins, respectively.

5.3 Results and Discussion

The presence and stoichiometry of r-proteins associated with the different functional states of the

ribosome namely free 30S, free 50S, 70S ribosomes, and polysomes were evaluated in a wild-type K-

12 strain. Chloramphenicol was added three minutes before harvesting to stop translational elongation

and prevent ribosomes from running off the mRNA so polysomes can be maintained. Sucrose

gradient fractionation of the crude cell lysate under non-dissociating conditions yielded the different

functional states of the ribosomes, which can be detected and purified. Figure 5.1 shows the 10-50%

sucrose gradient profile of the crude cell lysate from E. coli cells. The free subunits (30S and 50S),

70S ribosomes, and the actively synthesizing ribosomes (polysomes) are shown. Previous studies

using gel-based techniques showed that the r-protein complement of the ribosomes is not present in

equimolar amounts leading to the generation of different classes of ribosomes [91-94,148-150]. One

of the reasons for this apparent heterogeneity is the variation of the r-protein component of the

ribosome. To gain insights into the chemical heterogeneity of the ribosome and its various functional

states, I used the improved LC-MSE approach described in Chapter 4 to determine the composition

and stoichiometry of r-proteins associated with these complexes.

75 70 70S 60

50

40 cytoplasmic free fraction 50S 30 free 30S Relative Abs. at 260at Abs. nm Relative 20 polysome

10

0 0 5 10 15 20 25 30 35 40 Fraction No.

Figure 5.1 10-50% sucrose gradient profile of crude cell lysate from wild-type E. coli K-12 cells in non-dissociating conditions. The different functional states of the ribosomes including the free subunits (30S and 50S), single ribosomes (70S), and polysomes are shown.

5.3.1 Stoichiometric measurements of r-proteins in actively translating polysomes

To evaluate the composition and stoichiometry of r-proteins associated with the different functional

classes of the ribosome, r-proteins from these complexes were extracted and analyzed by the

improved LC-MSE approach. Figure 5.2 shows the protein amounts of the large subunit and small

subunit proteins in wild-type polysomes. The protein levels are normalized to primary binding

proteins L3 and S4 for the large subunit and small subunit, respectively. These proteins are expected

to be present in every ribosome as they are important for ribosome assembly and function. R-proteins

are uniformly distributed in polysomes, which is in agreement that actively translating ribosomes

contain one copy of each r-protein per ribosome. Proteins L30, L34, L35, L36 and S21 were present

in reduced amounts as these proteins do not generate sufficient peptides for quantitation. The

ribosome population in polysomes is homogeneous with respect to its r-protein complement.

Although the exact copy number of the stalk proteins L7/L12 is not reflected in the quantitative result,

it is probably due to the highly flexible nature of these proteins and loss of these proteins during

fractionation. Likewise, the small ribosomal subunit proteins are also uniformly distributed in

76 polysomes. Interestingly, protein S1 is present in equimolar amounts, which is consistent that one

copy of S1 is present per ribosome in actively translating ribosomes [151]. S1 acts by allowing

ribosomes to bind to the mRNA during protein synthesis and is therefore essential in actively

translating ribosomes. Protein S1, however, is barely present in free 30S subunit, which supports

previous biochemical data that this protein is only required transiently during protein synthesis [14,

151,152].

2 ) 1 pmol

0 L1 L2 L3 L4 L5 L6 L9 L11 L11 L10 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36

2 L7/12

1 NormalizedProtein Level (

0 S1 S2 S3 S4 S5 S6 S7 S8 S9 S11 S11 S10 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21

Figure 5.2 Quantitation of large 50S subunit and small 30S subunit r-proteins associated with wild- type polysomes. Protein amounts were normalized to protein L3 and protein S4 for the large subunit and small subunit, respectively. Data represent the mean of the normalized protein amounts of two biological preparations of polysomes with three technical replicates each. Error bars represent the standard deviation of the 6 trials.

5.3.2 Stoichiometric measurements of r-proteins in 70S ribosomes

In the case of single or 70S ribosomes, one would expect that the stoichiometry of r-proteins in 70S

would be similar to that of the polysomes as most of the 70S ribosomes are derived from polysomes.

However, some of the 70S ribosomes probably are 70S-like or translation initiation complexes

lacking some proteins, similar to what is observed in 80S-like complexes in yeast [153]. Figure 5.3

shows the amounts of proteins in 70S. Although most of the proteins are stoichiometric, several

77 proteins are present in reduced amounts. Proteins L5, L20, and L29 are present in reduced amounts in

70S compared to polysomes. Because maturation of the subunit occurs in polysomes [37], some

proteins are probably not incorporated yet into the 70S complex resulting in reduced amounts for

some of these proteins. In addition, r-proteins can rapidly exchange as well and could possibly

explain the apparent degree of heterogeneity of the 70S ribosomes analyzed in this work. Figure 5.4

shows the log2 fold change of proteins in 70S over the proteins in polysomes. Protein S1, and tertiary

binding proteins S3, S10, and S13 are relatively present in lower amounts in 70S than in polysomes,

suggesting that the 70S is probably composed of immature 30S complexes.

2 ) 1 pmol

0 L1 L2 L3 L4 L5 L6 L9 L11 L11 L10 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36 L7/12 2

1 NormalizedProtein Level (

0 S1 S2 S3 S4 S5 S6 S7 S8 S9 S11 S11 S10 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21

Figure 5.3 Quantitation of large 50S subunit and small 30S subunit r-proteins associated with wild- type 70S ribosomes. Protein amounts were normalized to protein L3 and protein S4 for the large subunit and small subunit, respectively. Data represent the mean of the normalized protein amounts of two biological preparations of 70S ribosomes with three technical replicates each. Error bars represent the standard deviation of the 6 trials.

78 2

0

-2 L1 L2 L3 L4 L5 L6 L9 L11 L11 L10 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36

2 L7/12 (fold change) 2 log

0

-2 S1 S2 S3 S4 S5 S6 S7 S8 S9 S11 S11 S10 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21

Figure 5.4 Plot of log2 fold change of r-proteins in K12 70S ribosomes over K12 polysomes. Protein amounts were normalized to protein L3 and protein S4 for the large subunit and small subunit, respectively. Positive values indicate that the amounts of proteins are reduced in 70S ribosomes compared to K12 polysomes while negative values indicate otherwise. Data represent the mean of the normalized protein amounts of two biological preparations of 70S ribosomes with three technical replicates each. Error bars represent the standard deviation of the 6 trials.

5.3.3 Stoichiometric measurements of r-proteins in free 50S and 30S subunits

The free subunits (30S and 50S) are derived from dissociated 70S ribosomes and premature subunits.

Therefore one would expect that these particles are heterogeneous with respect to their protein

complement. Figure 5.5 shows the amounts of r-proteins associated with wild-type free subunits. The

distribution of the large subunit proteins is very similar to that from the 70S ribosomes. For the small

subunit proteins, the non-uniform distribution is dramatic as several of the proteins are present in

significantly reduced amounts. The 30S subunit has been suggested as a hotspot for ribosome

heterogeneity [146]. Protein S1 is barely present in the free 30S subunit. Van Duin et al. (1970)

suggested that S1 is required for maximum capacity of ribosomes to bind mRNA and is present in

actively translating ribosomes [151]. As this protein is only present in a fraction of ribosomes at any

instant, they further suggested that S1 is rapidly exchanging from one ribosome to another during

protein synthesis. This could explain why S1 is present in reduced amounts in the free 30S subunit.

79 Likewise, the free subunits contain premature assembly particles devoid of late binding proteins. For

instance, the tertiary binding proteins S2, S3, and S10 are present in reduced amounts probably due to

these proteins not being incorporated yet into the complex. In addition, r-proteins could also exchange

from the free subunits to those in actively translating complexes or they can also perform extra-

ribosomal protein functions such as in S10. Protein S10 is significantly reduced in free 30S subunit

and has been reported to perform other functions than ribosome assembly and protein translation

[154]. S10 also acts as a transcriptional elongation factor by binding to the RNA polymerase and

probably exchanges between the free-ribosomal pool and extra ribosomal protein S10 resulting in a

relatively reduced amounts in free 30S subunit [154].

2

1 ) pmol 0 L1 L2 L3 L4 L5 L6 L9 L11 L11 L10 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36 L7/12

2

1 NormalizedProtein Level (

0 S1 S2 S3 S4 S5 S6 S7 S8 S9 S11 S11 S10 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21

Figure 5.5 Quantitation of large 50S subunit and small 30S subunit r-proteins associated with wild- type free subunits. Protein amounts were normalized to protein L3 and protein S4 for the large subunit and small subunit, respectively. Data represent the mean of the normalized protein amounts of two biological preparations of 70S ribosomes with three technical replicates each. Error bars represent the standard deviation of the 6 trials.

80 4

2

0

-2

-4 L1 L2 L3 L4 L5 L6 L9 L11 L11 L10 L13 L14 L15 L16 L17 L18 L19 L20 L21 L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33 L34 L35 L36 L7/12

(fold change) 4 2

log 2

0

-2

-4 S1 S2 S3 S4 S5 S6 S7 S8 S9 S11 S11 S10 S12 S13 S14 S15 S16 S17 S18 S19 S20 S21

Figure 5.6 Plot of log2 fold change of r-proteins in free subunits over K12 polysomes. Protein amounts were normalized to protein L3 and protein S4 for the large subunit and small subunit, respectively. Positive values indicate that the amounts of proteins are reduced in free subunits compared to K12 polysomes while negative values indicate otherwise. Data represent the mean of the normalized protein amounts of two biological preparations of 70S ribosomes with three technical replicates each. Error bars represent the standard deviation of the 6 trials.

Protein S6 is present in one copy per 70S ribosome [155]. This protein is posttranslationally modified

with glutamic acid residues and is reduced under stress conditions [49,154]. Because the free 30S

subunit is composed of recycled 30S subunits (dissociated from 70S) and immature 30S, S6 is

probably incorporated into polysomes once it is fully modified so one copy of this protein is found in

polysomes while it is significantly reduced in the free 30S subunit. Stoichiometric measurement of

protein S6 is consistent with these observations (Figure 5.5 and Figure 5.6). Williamson and co-

workers showed that S6 has a relatively higher pool size compared to the primary binding proteins

and suggested that the dynamic modification of this protein probably account for its higher pool size

[154].

81 5.3.4 Reproducibility studies of r-protein quantitation using the improved LC-MSE

The stoichiometric measurements of the amounts of proteins were performed using two biological

replicates of E. coli wild-type cells. For each biological replicate, the different functional states of the

ribosomes were purified and analyzed by the improved LC-MSE. Tables 5.1 and 5.2 summarize the

run-to-run reproducibility of the stoichiometric measurements of the small subunit and large subunit

r-proteins in wild-type polysomes, respectively. Quantitation using the improved LC-MSE approach

showed very good run-to-run reproducibility with CV less than 15% for both large subunit and small

subunit proteins. Likewise, the biological reproducibility was also examined using two biological

preparations of wild-type cells. Tables 5.3 and 5.4 represent the biological reproducibility of the

stoichiometric measurements. Although the %CV between biological replicates can reach up to 35%

for some proteins, this is expected because of the highly dynamic nature of the ribosome.

82 Table 5.1 Run-to-run reproducibility of the stoichiometric measurements of small 30S subunit proteins in wild-type polysomes (POLY). r-protein POLY 1 001 POLY 1 002 POLY 1 003 AVG STDEV %CV S1 0.76 0.88 0.73 0.79 0.08 9.67 S2 0.92 0.89 0.85 0.89 0.03 3.94 S3 1.47 1.30 1.43 1.40 0.09 6.19 S4 1.00 1.00 1.00 1.00 0.00 0.00 S5 0.85 0.77 0.73 0.79 0.06 7.74 S6 - 0.99 1.03 1.01 0.03 3.34 S7 1.09 1.06 1.20 1.12 0.08 6.75 S8 0.71 0.66 0.68 0.68 0.03 3.69 S9 - 0.83 0.80 0.81 0.03 3.20 S10 0.99 0.98 1.00 0.99 0.01 1.13 S11 0.69 0.63 0.67 0.67 0.03 4.52 S12 0.72 0.73 0.77 0.74 0.03 3.53 S13 1.13 0.87 1.02 1.01 0.13 13.1 S14 0.96 0.77 0.82 0.85 0.10 11.5 S15 0.89 - 0.91 0.90 0.01 0.87 S16 1.07 0.95 1.19 1.07 0.12 11.1 S17 0.95 0.95 0.96 0.95 0.01 0.68 S18 0.51 0.49 0.48 0.49 0.02 3.11 S19 1.02 1.03 1.03 1.03 0.01 0.67 S20 0.86 0.87 0.91 0.88 0.03 3.27 S21 0.42 0.38 0.42 0.41 0.02 5.61

83 Table 5.2 Run-to-run reproducibility of the stoichiometric measurements of large 50S subunit proteins in wild-type polysomes (POLY). r-protein POLY 1 001 POLY 1 002 POLY 1 003 AVG STDEV %CV L1 0.72 0.76 0.72 0.74 0.02 3.38 L2 1.05 1.12 1.01 1.06 0.06 5.34 L3 1.00 1.00 1.00 1.00 0.00 0.00 L4 0.73 0.84 0.85 0.81 0.07 8.18 L5 1.05 1.01 1.08 1.05 0.03 3.28 L6 0.87 0.82 0.78 0.82 0.04 5.35 L7/12 1.74 1.89 1.91 1.85 0.09 4.97 L9 0.67 0.76 0.76 0.73 0.05 7.38 L10 0.91 0.89 0.82 0.87 0.05 5.50 L11 0.73 0.76 0.76 0.75 0.02 2.91 L13 0.99 1.08 - 1.04 0.06 5.58 L14 0.84 1.00 0.85 0.89 0.09 10.3 L15 0.87 0.92 0.87 0.88 0.03 3.13 L16 1.02 1.12 0.89 1.01 0.12 11.6 L17 0.95 0.92 0.83 0.90 0.06 6.53 L18 0.97 0.93 0.87 0.92 0.05 5.44 L19 0.89 0.99 0.99 0.96 0.06 5.85 L20 0.66 0.80 0.65 0.70 0.08 11.4 L21 0.96 1.00 0.90 0.95 0.05 5.41 L22 0.81 0.82 0.76 0.79 0.03 3.90 L23 0.79 0.80 0.71 0.76 0.05 6.64 L24 1.09 1.16 1.01 1.09 0.08 6.97 L25 0.68 0.68 0.60 0.65 0.04 6.71 L27 0.78 0.81 0.61 0.73 0.11 14.7 L28 0.65 0.58 0.61 0.62 0.04 5.97 L29 0.80 0.76 0.70 0.75 0.05 6.19 L30 0.46 - 0.55 0.51 0.06 12.5 L31 0.96 1.01 0.84 0.94 0.09 9.35 L32 0.82 0.84 0.80 0.82 0.02 2.64 L33 1.10 1.15 1.00 1.08 0.07 6.70 L34 0.00 0.00 0.00 0.00 0.00 - L35 0.32 0.29 0.30 0.30 0.02 5.07 L36 0.27 0.29 0.28 0.02 5.89

84 Table 5.3 Biological reproducibility of the stoichiometric measurements of small 30S subunit proteins in wild-type polysomes (POLY). r-protein POLY 1 POLY 2 AVG STDEV %CV S1 0.50 0.79 0.64 0.21 32.0 S2 0.83 0.89 0.86 0.04 4.71 S3 1.28 1.40 1.34 0.09 6.35 S4 1.00 1.00 1.00 0.00 0.00 S5 0.82 0.79 0.80 0.02 2.96 S6 0.98 1.01 1.00 0.02 1.95 S7 1.32 1.12 1.22 0.15 12.0 S8 0.96 0.68 0.82 0.20 23.9 S9 1.03 0.81 0.92 0.15 16.5 S10 0.86 0.99 0.93 0.09 10.1 S11 0.89 0.67 0.78 0.16 20.1 S12 0.79 0.74 0.77 0.04 4.83 S13 1.06 1.01 1.03 0.04 3.63 S14 1.07 0.85 0.96 0.16 16.5 S15 1.07 0.90 0.99 0.12 12.3 S16 1.23 1.07 1.15 0.11 9.90 S17 1.27 0.95 1.11 0.22 20.0 S18 0.82 0.49 0.66 0.23 35.3 S19 0.99 1.03 1.01 0.02 2.36 S20 0.88 0.88 0.88 0.00 0.12 S21 0.55 0.41 0.48 0.11 21.9

85 Table 5.4 Biological reproducibility of the stoichiometric measurements of large 50S subunit proteins in wild-type polysomes (POLY). r-protein POLY 1 POLY 2 AVG STDEV %CV L1 0.87 0.74 0.80 0.09 11.5 L2 1.01 1.06 1.03 0.04 3.58 L3 1.00 1.00 1.00 0.00 0.00 L4 0.84 0.81 0.82 0.02 2.48 L5 1.07 1.05 1.06 0.01 1.22 L6 0.78 0.82 0.80 0.03 3.85 L7/12 1.36 1.85 1.60 0.34 21.3 L9 0.78 0.73 0.75 0.04 5.06 L10 0.78 0.87 0.83 0.06 7.82 L11 0.73 0.75 0.74 0.01 1.79 L13 0.93 1.04 0.98 0.07 7.58 L14 0.72 0.89 0.81 0.12 15.2 L15 0.83 0.88 0.86 0.04 4.58 L16 0.78 1.01 0.90 0.16 17.9 L17 0.77 0.90 0.83 0.09 11.2 L18 0.76 0.92 0.84 0.12 13.8 L19 0.84 0.96 0.90 0.08 9.41 L20 0.59 0.70 0.65 0.08 11.9 L21 0.89 0.95 0.92 0.04 4.84 L22 0.69 0.79 0.74 0.08 10.4 L23 0.70 0.76 0.73 0.04 5.99 L24 0.76 1.09 0.92 0.23 24.5 L25 0.51 0.65 0.58 0.10 17.0 L27 0.62 0.73 0.68 0.08 11.5 L28 0.71 0.62 0.66 0.07 10.4 L29 0.78 0.75 0.77 0.02 2.28 L30 0.33 0.51 0.42 0.12 29.3 L31 0.93 0.94 0.93 0.01 0.69 L32 0.75 0.82 0.79 0.05 5.83 L33 0.80 1.08 0.94 0.20 21.4 L34 0.00 0.00 0.00 0.00 - L35 0.35 0.30 0.33 0.03 10.7 L36 0.37 0.28 0.32 0.06 19.3

86 5.4 Conclusions

In this Chapter, I extended the applicability of the improved LC-MSE approach to probe the

heterogeneity of ribosomes by determining the stoichiometry of r-proteins associated with the

different functional states of the ribosomes isolated in E. coli wild-type cells. The actively translating

ribosomes (polysomes) contain stoichiometric amounts of proteins consistent that these proteins play

roles in fine-tuning ribosome functions in protein synthesis. The 70S contains stoichiometric amounts

of proteins as well but significant heterogeneity were found with free subunits as they are composed

of immature complexes and dissociated subunits from 70S. The stoichiometric measurements among

the different classes of ribosomes showed very good run-to-run reproducibility with %CV less than

15%. In addition, the biological reproducibility of the amounts of proteins, although exhibiting %CV

as high as 35%, most proteins showed consistent biological reproducibility. Because ribosomes are

highly dynamic complexes within a cell, the composition of the ribosomes varies depending on its

functional state, which could explain for the apparent heterogeneity of the particles observed in vivo.

87 Chapter 6. Mass Spectrometry-Based Characterization of the Erythromycin-Induced

Ribosome Assembly Particles in Escherichia coli

6.1 Introduction

Detailed X-ray crystal structures of the bacterial ribosome have revealed a wealth of information that

increased our understanding of the structure and function of the ribosome as well as antibiotic action

[1-7,16,21,22]. Although much is known how antibiotics inhibit important ribosomal functions such

as mRNA decoding, peptidyl transfer and elongation, less is understood how certain antibiotics

interfere in the biogenesis and assembly of the ribosome, which is an emerging and important new

target of antimicrobial agents. Thus, insights gained into the interplay between antibiotics and

ribosomal subunit formation can facilitate rational drug design and development of small molecule

drugs or ligands specific to bacterial ribosome assembly. The aim of this Chapter is to characterize

antibiotic-induced ribosome assembly particles in vivo using mass spectrometry, in particular those

particles resulting from misfolding during ribosome assembly. The use of a temperature-sensitive

ribonuclease E mutant SK5665 strain allows isolation and characterization of improperly folded

ribosome assembly particles.

6.2 Experimental

6.2.1 Materials

Tryptone and yeast extract were obtained from Fisher Scientific (Fairlawn, NJ). Trypsin (sequencing

grade) and RNase-free DNase were purchased from Promega (Madison, WI). Lysozyme from

chicken egg white was purchased from Worthington (Lakewood, NJ). Erythromycin, thymidine,

dithiothreitol (DTT), iodoacetamide (IA) and molecular-grade sucrose were obtained from Sigma (St.

Louis, MO). Acids and organic solvents were HPLC grade or better.

88 6.2.2 Bacterial culture

E. coli wild-type K-12 was cultured as described in Chapter 4. E. coli strain SK5665 (thyA715, rne-l)

was obtained from the E. coli Genetic Stock Center (Yale University, New Haven, CT). The strains

were cultured following standard growth conditions, harvesting and isolation procedures described

previously 1 with minor modifications. For SK5665, prior optimization of growth conditions was

performed to observe and detect the 50S assembly particles.

A single colony of SK5665 was inoculated in 50 mL 2xYT media (1.6% Bacto Tryptone, 1% Bacto

Yeast Extract, 0.5% NaCl) and incubated overnight at 25 °C and 250 rpm. One (1) ml of cell

suspension was then transferred into 1-liter culture media supplemented with 5 µg/mL thymidine and

18 µg/mL erythromycin for SK5665. At early mid-log phase (A600: 0.3-0.4), cells were harvested and

refrigerated for 30 min at 4 °C. The cells were then collected by centrifugation and resuspended in

lysis buffer (60 mM KCl, 60 mM NH4Cl, 50 mM Tris-HCl, 6 mM MgCl2, 6 mM 2-mercaptoethanol,

16% sucrose, pH 7.5-7.7) containing lysozyme. After incubation with RNase-free DNase, the lysed

cells were clarified by centrifugation and the ribosome-rich supernatant was retained. The crude cell

supernatant was further centrifuged for 2 h at 48,000 rpm, 4 °C in a 50 Ti rotor with a Beckman-

Coulter ultracentrifuge (Fullerton, CA). The polysome-depleted ribosome was resuspended in buffer

D (60 mM KCl, 60 mM NH4Cl, 50 mM Tris-HCl, 6 mM MgCl2, 6 mM beta-mercaptoethanol, pH

7.5-7.7) and stored at -80 °C until further use.

6.2.3 Isolation of the 50S assembly particles (Δ50S) and mature 50S ribosomal subunits

from SK5665

Following culturing, the Δ50S particles and mature 50S subunits were purified by sucrose density

gradient ultracentrifugation. A 0-45% sucrose gradient in dissociation buffer (60 mM NH4Cl, 10 mM

Tris-HCl, 1.1 mM MgCl2, 0.1 mM EDTA, 2 mM beta-mercaptoethanol, pH 7.5-7.7) was prepared by

89 the freeze-thaw method. Briefly, 34 mL of 22.5% sucrose in dissociation buffer were dispensed into

polyallomer tubes and frozen overnight at -80 °C. The gradients were allowed to thaw and

approximately 80-100 ODU of crude ribosome were gently layered on top of the gradients. The

gradients were then centrifuged for 15 h at 23,000 rpm at 4 °C using a Beckman SW32 rotor/ SW28

swinging bucket rotor. The fractions were then collected using an ISCO automated fraction collector

with continuous monitoring at 254 nm. Fractions corresponding to the assembly particle and mature

subunits were pooled and pelleted by centrifugation for 20 h at 48,000 rpm at 4 °C. The ribosome

pellets were resuspended in Buffer D. A second sucrose density gradient fractionation using a 0-45%

sucrose gradient in non-dissociating conditions was used to purify the assembly particle. A 1% non-

denaturing agarose gel was performed to assess purity of the assembly particle and mature subunits.

6.2.4 Liquid Chromatography- Tandem Mass Spectrometry (LC-MS/MS)

R-proteins from Δ50S and mature 50S subunits were obtained by acetone precipitation after rRNA

extraction with acetic acid as described in Chapter 4. Fifteen micrograms (15 µg) of total protein

were digested with trypsin after reduction and alkylation with dithiothreitol (DTT) and iodoacetic

acid (IA), respectively. Tryptic digests were analyzed by LC-MS/MS on a Thermo Scientific

(Waltham, MA) LTQ XL linear ion trap mass spectrometer and Surveyor HPLC equipped with a

Finnigan Micro AS autosampler for automated injections. Reversed-phase chromatography on an

XBridgeTM C18 column (1.0 mm, 2.1 mm x 100 mm, Waters, Milford, MA) was performed at a flow

rate of 70 µL min-1 using 5% acetonitrile (0.1% formic acid) as mobile phase A and 95% acetonitrile

(0.1% formic acid) as mobile phase B. Gradient elution was carried out from 10 % B to 50 % B for 55

min, followed by 2 % B min-1 to 80 % B. Mass spectra were recorded in positive ion mode with a

capillary temperature of 275 °C, spray voltage of 5 kV and 35, 10, 10 arbitrary flow units of sheath,

auxiliary and sweep gas, respectively. All LC-MS/MS were performed in triplicate.

90 6.2.5 Database Searching

LTQ-acquired MS/MS spectral data were converted from raw instrument output into Mascot Generic

Format using Mascot Daemon (Version 2.2.2, Matrix Science) prior to launching Mascot searches.

Mascot (version 2.0, Matrix Science) searches were performed on a 16-processor system Mascot

server at the University of Cincinnati, Genome Research Center

(http://research.cchmc.org/mascot/home.html). The SwissProt 2010_08 protein sequence database

consisting of 518,415 sequences and 182,829,264 residues was used in all searches. Searches were

limited to E. coli (22708 sequences) and allowed only tryptic peptides. Parameters set for LTQ

searches included use of monoisotopic masses with peptide mass tolerance of 1.2 Da and fragment

mass tolerance of 0.6 Da. Two internal missed cleavages were allowed with cysteine residues being

carbamidomethylated (+57.0215 Da) and methionine being oxidized (+15.9949 Da). A composite

decoy search was performed and specific cut-off scores were determined allowing <1% false

discovery rate. A minimum of two peptide matches was considered for positive protein identification.

Manual inspection of MS/MS data was performed for proteins having only two peptide matches to

verify sequence assignments.

6.2.6 Quantitation of r-proteins and assembly factors associated with the erythromycin-

induced 50S particles

To obtain quantitative information of the proteins associated with mature 50S and Δ50S assembly

particles, the LC-MSE approach described in Chapter 4 was used. Instead of using multiple enzymes,

only the tryptic peptides from total proteins were used for quantitation. The low yield of the Δ50S

particles did not permit multiple digestions using different proteases. The protein amounts for each

trial were normalized to protein L3. Plots of the log10 fold change of proteins over wild-type K12 50S

were generated for both SK5665 Δ50S and SK5665 50S to allow comparison between mature 50S

subunits (K12 and SK5665) and erythromycin-induced Δ50S particles.

91 6.3 Results and Discussion

Although the r-protein composition of the assembly particles has been identified from both studies

using 2D gel and mass spectrometry [31,32,107], the presence of extra ribosome assembly factors

associated with the particles has not been reported. In addition, absolute quantification of proteins in

these complexes has not been implemented which could give information relating to complex

stoichiometry and biogenesis. Because one cannot pinpoint whether or not the heterogeneity of the

particles reported in previous studies is due to the real intermediates observed in vivo or just mere

contamination during the purification process, I devised a strategy based on the differences in subunit

packing under dissociating and non-dissociating conditions during sucrose gradients to isolate the 50S

particle. In addition, absolute quantification of proteins associated with the particles was performed to

determine the stoichiometry of the proteins associated with the complex.

6.3.1 Isolation and purification of the Δ50S assembly particle

The components of the erythromycin-induced Δ50S ribosomal subunit assembly particles in E. coli

SK5665 cells were characterized using mass spectrometry-based methods. These data were then

compared with the components of the mature wild-type K12 50S and SK5565 50S subunit in

erythromycin treated and untreated cells. These comparisons reveal differences between the mature

50S subunit and Δ50S particle, which could be used to time stamp what stage the antibiotic inhibits

formation of ribosomal subunit. To identify the components of the Δ50S particle, SK5665 cells were

grown with and without erythromycin (final concentration 18 µg/mL) at 25°C. At this non-permissive

temperature, RNase E is inactive allowing detection and isolation of misassembled Δ50S particles.

The growth of SK5665 cells was significantly slower in the presence of the antibiotic compared to

without the antibiotic (Figure 6.1).

92 0.8

0.7

0.6

0.5

0.4

0.3

Absorbance600at nm 0.2

0.1

0 0 100 200 300 400 500 600 700 800 Time (min) SK 5665 with ery-1 SK 5665 with ery-2 SK 5665 with ery-3 SK 5665 w/out ery

Figure 6.1 Growth curves of SK5665 cells grown in 2xYT media (25°C) with and without erythromycin. Final erythromycin concentration used was 18 µg/mL.

Figure 6.2 shows the 0-45% sucrose gradient profile of the crude ribosome in non-dissociating

conditions. An assembly particle was observed which sediments between the 30S and 50S (Figure

6.2A). This particle is absent in cells grown without the antibiotic (Figure 6.2B). 1% non-denaturing

gel electrophoresis of the fractions corresponding to the assembly particle revealed the presence of

23S rRNA, which indicates that is a 50S assembly particle. No assembly particle related to the 30S

subunit was detected in the presence of erythromycin. This suggests that erythromycin probably

interferes specifically the formation of the large subunit 50S as previously suggested by Champney

and co-workers.

93 A (+) Ery 70S B (-) Ery 70S

50S 30S

254 254 30S Δ50S A A 50S

Fraction no. Fraction no.

Figure 6.2 0-45% sucrose gradient profile of crude ribosome from SK5665 cells (A) with erythromycin (Ery) and (B) without erythromycin. Cells grown with erythromycin showed an abnormal particle that sediments ~35-40S in sucrose gradients. This particle is absent in cells grown without the antibiotic.

To define the composition of the Δ50S particle, a strategy was devised to isolate the particle that is

devoid of contamination from mature 50S and 30S subunits. Scheme 6.1 shows the experimental

strategy used to purify the Δ50S assembly particle. The crude ribosome was first fractionated in

sucrose gradients under dissociating conditions (1.1 mM Mg2+). The 70S ribosome dissociates into its

corresponding 30S and 50S subunits. Following fractionation of the subunits, agarose gel

electrophoresis of the fractions revealed the presence of 23S rRNA which co-sediments in the 30S

subunit region containing 16S rRNA as well. Fractions (26-28) were pooled and collected for further

purification. To maintain the intact 70S subunit, stoichiometric amounts of a divalent ion (Mg2+) must

be present. At low Mg2+ ion concentration, the assembly particle Δ50S assumes ribosomal subunit

packing similar to that of the 30S subunit sediments at an almost similar rate with that of the 30S

during ultracentrifugation [156]. During the first sucrose fractionation, the mature 50S subunit can be

fully isolated from the Δ50S. A second gradient fractionation under non-dissociating conditions was

used to finally isolate the Δ50S particle. At higher Mg2+ ion concentration (12 mM Mg2+), Δ50S

assumes a different shape and conformation than that of the mature 30S subunit and sediments at

different rates during centrifugation. The agarose gel of fractions after fractionation in non-

94 dissociating conditions shows a 23S rRNA that can be separated from the 16S rRNA. Fractions (34-

37) were pooled to yield the pure Δ50S.

Crude ribosome (+) Ery

dissociating conditions

50S 30S A254

Fraction no. 23S 16S

non-dissociating conditions 30S A254 Δ50S

Fraction no. 23S 16S

Scheme 6.1 Isolation scheme used to purify the assembly particle in cells grown with the antibiotic. The differences between the sedimentation profiles of the subunits and Δ50S particle under dissociating and non-dissociating conditions were exploited as means to isolate and purify the assembly particle.

95 6.3.2 Components of the erythromycin-induced 50S ribosomal subunit assembly

particle

After the Δ50S and the mature 50S subunits have been purified, the protein components were isolated

as described previously [117]. Tryptic peptides of total proteins were analyzed and identified by LC-

MS/MS and database searching. Figure 6.3A shows a representative total ion chromatogram (TIC) of

the tryptic digests from wild-type K-12 50S proteins showing only the first 60 minutes of the LC-

MS/MS run. Figure 6.3B shows the mass spectrum of the peptide peak eluting at 36.7 minutes

showing a base peak at m/z 840.8. Collision-induced dissociation (CID) of this precursor ion yielded

a fragmentation pattern matched to a peptide fragment from ribosomal protein L6 (Figure 6.3C).

Database searching identified the r-proteins, ribosome assembly factors, and other proteins with

known and unknown functions in ribosome assembly. Table 6.1 summarizes the large subunit

proteins identified in wild-type K-12 50S, SK5665 50S with and without erythromycin and the Δ50S

particle. For the K-12 50S and SK5665 50S subunits, 33 out of 34 (97%) r-proteins were identified,

while only 20 out of 34 (59%) proteins were identified in Δ50S particle. Proteins L6, L16, L18, L27,

L30 and L31 vary in the 3 replicate analyses while proteins L7/L12, L10, L28, L33, L34 and L35

were consistently absent. These results suggest that the Δ50S particles are heterogeneous with respect

to its protein content. The proteins (L7/L12, L10, L28, L33, L34 and L35) not detected in the Δ50S

are late binding proteins suggesting that erythromycin affects later stages of 50S formation. Likewise,

proteins that are important during initial 50S subunit assembly in vitro (L4, L13, L20, L22, L24) were

present in the mature 50S subunit from K-12 and S5665 with and without erythromycin [52,160].

These proteins are also present in the Δ50S particle.

96 RT: 0.10 - 59.95 SM: 7B A 21.82 NL: 100 1.67E7 TIC F: ITMS + 90 p ESI Z ms 21.08 [400.00- 2000.00] MS 80 18.13 29.70 K12_50S_15ug _2 70

33.13 60 14.27 23.56 24.94 50

36.72 40 13.38 RelativeAbundance 30 9.30 39.27 20 1.77 8.58

2.53 40.95 45.47 50.71 57.58 58.34 10

0 5 10 15 20 25 30 35 40 45 50 55 Time (min)

K12_50S_15ug_2 #2744-2818 RT: 36.17-37.13 AV: 15 SM: 7B NL: 9.17E3 F: ITMS + p ESI Z ms [400.00-2000.00] 840.82 B 100

90

80

70

60 700.28

50

40 560.46 RelativeAbundance 30 681.66 1361.46 634.20 20 951.42 453.30 10 807.18 1050.46 1067.34 1190.40 1419.52 1679.86 1770.56 1939.10 0 400 600 800 1000 1200 1400 1600 1800 2000 m/z

K12_50S_15ug_2 #2744 RT: 36.68 AV: 1 NL: 1.51E4 F: ITMS + c ESI d Full ms2 [email protected] [220.00-1695.00] 682.29 C 100 + y6 951.40 y + b + 90 9 10 * + 998.28 b 8 A L L N S M V I G V T E G F T K 80 825.30 + 50S ribosomal subunit protein L6 70 y10 1050.39 60

50 * + b 7 + y5 712.21 40 + y + + + y 7 + + b13 b

RelativeAbundance 581.26 4 b9 b11 14 452.23 781.38 1285.27 30 + 899.28 1099.29 + 1432.41 + b7 b12 + + b6 b15 y3 1228.31 + 20 y13 395.20 630.06 1533.39 y + 573.20 1382.47 10 2 248.18 499.12 1618.89 0 400 600 800 1000 1200 1400 1600 m/z Figure 6.3 Representative LC-MS/MS spectral data of the tryptic digests from K12 50S proteins at 15 µg starting total protein concentration. (A) Total ion chromatogram (TIC) of the tryptic digests (B) MS spectrum of the peak eluting at 37 min showing a base beak at m/z 840.8. (C) MS/MS spectrum obtained by collision-induced dissociation (CID) of m/z 840.8. The fragmentation pattern corresponds to a peptide fragment from the E. coli large ribosomal subunit protein L6.

97 Table 6.1 Large 50S ribosomal subunit proteins detected in wild-type K12 50S and SK5665 50S with and without erythromycin and Δ50S particle. r-protein wt K12 SK5665 SK5665 SK5665 50S (-) Ery 50S (-) Ery 50S (+) Ery Δ50S (+) Ery L1 + + + + L2 + + + + L3 + + + + L4 + + + + L5 + + + + L6 + + + ± L7/L12 + + + − L9 + + + + L10 + + + − L11 + + + + L13 + + + + L14 + + + + L15 + + + + L16 + + + ± L17 + + + + L18 + + + ± L19 + + + + L20 + + + + L21 + + + + L22 + + + + L23 + + + + L24 + + + + L25 + + + + L27 + + + ± L28 + + + − L29 + + + + L30 + + + ± L31 + + + ± L32 + + + + L33 + + + − L34 + + + − L35 + + + − L36 − − − − +, proteins detected −, proteins not detected ±, proteins variably detected in 3 replicate analyses

Aside from the r-proteins detected in the Δ50S particle and mature 50S, non-ribosomal proteins

factors were also identified. Table 6.2 summarizes the assembly factors associated with the Δ50S

particles and mature 50S subunit. The ATP-dependent RNA helicases SrmB and DbpA are

specifically known to facilitate proper assembly 50S subunit in vivo [106,157-159]. SrmB, a putative

DEAD-box RNA helicase associates with a pre-50S particle and promotes binding of protein L13 at

98 early stages of ribosome assembly [157]. DbpA, DEAD-box protein A is another RNA helicase

implicated in the assembly and maturation of the 50S subunit in vivo. DbpA acts at later stages of

ribosome assembly than SrmB [106,158]. The presence of these assembly factors in Δ50S particle

indicates that it is indeed immature and not a fully assembled 50S particle. Other proteins with

unknown function in ribosome biogenesis and assembly such as the RNA binding protein yhbY and

protein hfq were also detected.

Table 6.2 Non-ribosomal proteins detected in mature 50S of wild-type K12 and SK5665 with and without erythromycin and Δ50S particle. Accession Non-ribosomal K12 SK5665 50S SK5665 SK5665 number Proteins 50S (-) Ery (-) Ery 50S (+) Ery Δ50S (+) Ery P0AGK4 RNA binding protein − − − + yhbY

C4ZR49 Protein hfq − − − +

P0A9P6 Cold-shock DEAD box + − − + protein A

P21507 ATP-dependent RNA − − − + helicase srmB

B1XET7 Betaine aldehyde + + + − dehydrogenase

C4ZXI2 L-seryl-tRNA (Sec) − + + − selenium transferase

P0ABD3 Bacterioferritin − − − ±

P0A910 Outer membrane − − + + protein A

P04128 Type-1 fimbrial − − − ± protein, A chain +, proteins detected −, proteins not detected ±, proteins variably detected in 3 replicate analyses

To determine whether the Δ50S particle exhibits similarity to the in vitro and in vivo 50S assembly

intermediates reported previously [52-54], I compared the protein complement of Δ50S with those

50S intermediates. Table 6.3 shows the protein complement of in vitro and in vivo 50S intermediates

99 and reveals close resemblance of the Δ50S with that of the in vivo p250S (43S) with respect to its

protein composition.

Table 6.3 Comparison of the protein complement of in vitro and in vivo 50S intermediates with that of the erythromycin-induced Δ50S particle. r-protein In vitro In vivo p150S In vivo p250S SK5665 (33/41S) (32S) (43S) Δ50S (+) Ery L1 + + + + L2 + − − + L3 + − + + L4 + + + + L5 + + + + L6 − − − ± L7/L12 + − + − L9 + + + + L10 + + + − L11 + − + + L13 + + + + L14 − − + + L15 + − + + L16 − − − ± L17 + + + + L18 − + + ± L19 + − + + L20 + + + + L21 + + + + L22 + + + + L23 + − + + L24 + + + + L25 − + + + L27 − + + ± L28 − − − − L29 + + + + L30 − + + ± L31 − − − ± L32 − − − + L33 + − + − L34 + U U − L35 U U U − L36 U U U − +, proteins detected −, proteins not detected U, unknown

100 6.3.2 Quantitation of r-proteins and assembly factors associated with the erythromycin-

induced 50S particles

To obtain quantitative information for the proteins associated with the Δ50S particle mature 50S, the

LC-MSE approach was used. Figure 6.4A shows the plot of log10 (fold change) of proteins from

mature SK5665 50S over wild K12 50S subunit. Although some variations in the amounts of proteins

between mature SK5665 50S and wild-type K12 50S can be seen, these differences are not dramatic

and probably due to strain-dependent variations. Figure 6.4B shows the plot of log10 (fold change) of

proteins from Δ50S particle over wild K12 50S. Several proteins including L4, L6, L9, L10, L14, L15,

L16, and L28 are significantly reduced in the Δ50S particle. All of these proteins except, L4 and L9,

are late binding proteins suggesting that erythromycin acts at late stages of ribosome assembly.

1 A Early binders Moderate binders Late binders

0

-1

1

(fold change) B 10 log

0

-1 L4 L3 L1 L2 L9 L5 L6 L11 L11 L24 L13 L22 L21 L17 L23 L25 L29 L34 L33 L15 L18 L19 L14 L20 L10 L16 L28 L30 L32 L27 L31 L35 L36 L7/12 r-proteins

Figure 6.4 Quantitation of r-proteins in Δ50S particle and mature 50S subunit. (A) Plot of log10 (fold change) of proteins from mature SK5665 50S over wild K12 50S subunit. (B) Plot of log10 (fold change) of proteins from Δ50S particle over wild K12 50S subunit. R-proteins are grouped into early binders, moderate binders, and late binders as previously described [62]. Protein amounts were normalized to protein L3. Measurements were done in triplicate and error bars represent standard deviations of the three trials.

101 Siibak and co-workers showed that proteins L2, L6, L9, L15, L16, L22, L27, L28, L29, L30, L32,

L34, L35, and L36 are significantly reduced in 35S particles in erythromycin and chloramphenicol

treated cells [60,107]. The Δ50S particles isolated here shows reduced levels of similar proteins.

When the proteins are grouped based on their binding rates to the 50S subunit as previously described

[62], moderate and late binding proteins are significantly reduced in the Δ50S particles suggesting

that these particles are mostly devoid of tertiary binding proteins (Figure 6.4). In addition, based on

the in vitro assembly map of the 50S subunit, proteins that are variable in the Δ50S particle have high

dependence on binding to protein L15. Protein L15 is significantly reduced in the Δ50S particle.

Proteins L10 and L28 are highly dependent on L15 for binding and are present in reduced amounts as

well. If these two proteins cannot bind, L7/L12 and L33 cannot bind. Quantitative analysis showed

that L7/L12 and L33 were not detected in the assembly particle while present in both wild-type K12

and SK5665 mature 50S subunits.

In vitro studies showed the erythromycin binds to protein L15 8. Bacterial protein L15 is a 15 kDa

protein of the large ribosomal subunit that interacts with more than ten other proteins during 50S

assembly in vitro [11,160]. It has an unusual N-terminal tail structure that extends into the proximity

of the exit tunnel of the 50S subunit where proteins L22 and L4 are also located [13]. One possibility

is that, during assembly of the 50S subunit, erythromycin binds to L15 and prevents proper

conformational changes that allow new binding sites for other proteins to form. When these binding

sites are not formed, proteins dependent on L15 will not be incorporated to the 50S subunit. This

leads to formation of misfolded 50S ribosome assembly particle that accumulates in erythromycin-

treated cells. To test whether L15 is responsible for the formation of the assembly particle, truncation

mutants of the protein could be created to define the important and functional regions of L15

responsible for erythromycin binding. If misfolded particles are not detected in erythromycin-treated

102 cells, then the region of the protein where the mutation is incorporated probably is responsible for

erythromycin binding.

The amounts of assembly factors and other proteins associated with the Δ50S particle were also

evaluated. DbpA is present ~14 fold higher than that of SrmB, which is consistent with the role of

SrmB at earlier stages of assembly than DbpA (Figure 6.5). This result further supports that the Δ50S

particle is formed as a result of 50S inhibition at later stages of subunit assembly.

120 100 80 60 40 Amount(fmol) 20 0 DbpA SrmB OMPA

Figure 6.5 Quantitation of ribosome assembly factors DbpA and SrmB detected in Δ50S particle.

Although I was able to isolate and characterize the Δ50S particle that accumulates in the presence of

erythromycin, almost all of the small 30S subunit proteins were also identified in the Δ50S fraction

(Table 6.4). Proteins S1, S2, S14 and S15 were not detected. All proteins detected except S15 are late

binding proteins.

103 Table 6.4 Small 30S ribosomal subunit proteins detected in wt K12 30S and SK5665 30S with and without erythromycin and Δ50S particle.

r-protein wt K12 SK5665 SK5665 SK5665 30S (-) Ery 30S (-) Ery 30S (+) Ery Δ50S (+) Ery S1 + + + − S2 + + + − S3 + + + + S4 + + + + S5 + + + + S6 + + + + S7 + + + + S8 + + + + S9 + + + + S10 + + + + S11 + + + + S12 + + + + S13 + + + + S14 + + + − S15 + + + − S16 + + + + S17 + + + + S18 + + + + S19 + + + + S20 + + + + S21 + + + + +, proteins detected −, proteins not detected ±, proteins variably detected in 3 replicate analyses

6.4 Conclusions

A strategy was devised to isolate and purify the erythromycin-induced 50S assembly particle in

SK5665 cells grown in the presence of the antibiotic erythromycin. Quantitative analysis of the

proteins associated with the Δ50S particles revealed reduced amounts of proteins L4, L6, L9, L10,

L14, L15, L16, and L28. This result suggests a heterogeneous collection of 50S intermediates with

different subsets and varying amounts of proteins. Proteins dependent on protein L15 are significantly

reduced in Δ50S particles. One possibility is that during 50S subunit assembly, binding of

erythromycin to L15 inhibits conformational changes that allow new binding sites for other proteins.

Hence, protein dependent on L15 will not be incorporated to the 50S subunit. This leads to formation

of misfolded 50S ribosome assembly particle that accumulates in erythromycin-treated cells devoid of

104 late assembly proteins. Likewise, the ribosome assembly factors SrmB and DbpA are also detected in

the Δ50S. These assembly factors facilitate proper folding and assembly of the 50S subunit at later

stages in vivo. These results indicate that erythromycin affects later stages of 50S subunit ribosome

assembly.

105 Chapter 7. Conclusions and Future Directions

7.1 Summary and Conclusions

The goal of this dissertation was to use mass spectrometry-based approaches to gain insights into the

composition and structural organization of ribosomes and ribosome assembly particles in vivo,

particularly those particles that result from perturbations (e.g. deletion of assembly factors,

antibiotics).

In Chapter 3, a combination of MS-based approaches including 15N-labeling and data-dependent LC-

MS/MS, MALDI-TOF MS and LC-MSE were used to define the composition, relative amounts and

posttranslational modifications of proteins associated with pre-30S complexes in rimM and rbfA

deletion mutants. Quantitative analysis of the proteins is in agreement with the Nomura assembly map

wherein tertiary binding proteins are under-represented in the complexes suggesting their roles in late

stages of 30S assembly. The lack of RimM has a more profound effect than the lack of RbfA on the

amount of proteins in the 30S particles, which is in agreement that RimM is needed in a step prior to

RbfA during maturation of 16S rRNA. In addition, the modification status of protein S5 and S18 was

established demonstrating the extent of protein modification correlates with the formation of specific

rRNA interactions during 30S assembly.

Relative quantitation of proteins by 15N-labeling and LC-MS/MS provides information on the

differential expression of proteins in normal and perturbed samples. This approach, however, is

limited to comparing two samples at a time, labeling can be expensive and laborious and not

amenable to other multicellular organisms. In Chapter 4, the applicability of a label-free approach,

LC-MSE, for absolute “ribosome-centric” quantification of r-proteins was explored. Because the

information obtained in this experiment is related to the number of peptides identified per protein,

106 experimental conditions were optimized that allow accurate and reproducible quantitation of r-

proteins associated with mature ribosomes from Escherichia coli. Using the additional dimension of

gas phase separation through ion mobility and the use of multiple endoproteinase digestion allow

reproducible and accurate quantitation of proteins associated with mature ribosomes. A uniform

distribution of r-proteins was observed in actively synthesizing ribosomes, in agreement with the

known stoichiometry of these proteins within the complex. Most of the r-proteins are reproducibly

quantified by the improved LC-MSE except for several proteins (S21, L34, L35, and L36), which are

not quantifiable even using ion mobility separation and multiple endoproteinase digestion because

these proteins generate too few peptides.

In Chapter 5, the improved LC-MSE approach was then extended to probe the heterogeneity of

ribosomes by determining the stoichiometry of r-proteins associated with the different functional

states of the ribosomes isolated in E. coli wild-type cells. The actively translating ribosomes

(polysomes) contain stoichiometric amounts of proteins consistent that these proteins play roles in

fine-tuning ribosome functions in protein synthesis. The 70S contains stoichiometric amounts of

proteins as well, but significant heterogeneity were found with free subunits as they are composed of

immature complexes and dissociated subunits from 70S. The stoichiometric measurements among the

different classes of ribosomes showed very good run-to-run reproducibility with %CV less than 15%.

In addition, the biological reproducibility of the amounts of proteins, although exhibiting %CV as

high as 35%, were consistent. Because ribosomes are highly dynamic complexes within a cell, the

composition of ribosomes varies depending on its functional state, which could explain the apparent

heterogeneity of the particles observed in vivo.

Finally, Chapter 6 was devoted towards characterizing in vivo assembly complexes formed in the

presence of the antibiotic erythromycin. A strategy was devised to isolate and purify the

erythromycin-induced 50S assembly particle in SK5665 cells grown in the presence of the antibiotic

107 erythromycin. Quantitative analysis of the proteins associated with the Δ50S particles revealed

reduced amounts of proteins L4, L6, L9, L10, L14, L15, L16, and L28. This result suggests a

heterogeneous collection of 50S intermediates with different subsets and varying amounts of proteins.

Late assembly proteins were not detected in the Δ50S particle as well, which indicates that

erythromycin affects late stages of 50S ribosome assembly. Ribosome assembly factors, SrmB and

DbpA are detected in the Δ50S. These assembly factors facilitate proper folding and assembly of the

50S subunit in vivo.

The mass spectrometry-based approaches presented here can be extended for the rapid

characterization of RNA-protein complexes and ribosome assembly complexes resulting from various

perturbations. Because of the improved LC-MSE method’s robustness, high-throughput capability and

amenability to both relative and absolute quantitation, this approach would be applicable for rapid

“ribosome-centric” characterization of proteins in more complex systems such as in eukaryotes,

mammalian cells and human tissues. Insights gained on how the ribosome is being formed from its

constituent RNA and proteins, and how antibiotics and perturbations affect this process will be

valuable in understanding the dynamics and detailed molecular mechanisms involved in ribosome

formation. Deciphering these complex processes will enable the development and rational drug

design of small molecules or ligands specific to bacterial ribosome assembly.

7.2 Future Directions

In Chapter 6, the composition of the erythromycin-induced 50S assembly particle (Δ50S) was

evaluated. Several small 30S ribosomal subunit proteins were also detected in Δ50S. The possibility

that both the assembly of the 30S and 50S subunits is affected indirectly by erythromycin or just mere

contamination during purification cannot be ruled out. Therefore, another model system was used to

characterize the events and effects of antibiotics in the formation of the large 50S ribosomal subunit

108 in vivo. The E. coli AD2291 strain has a plasmid containing the bacterial rrnB operon under the

control of a temperature-inducible phage λ promoter. A streptavidin binding aptamer is incorporated

into the functionally neutral helix 25 of the 23S rRNA, which allows purification of pure 23S rRNA

complexes (Figure 7.1) [161-162]. Expression of the tagged 23S rRNA is induced by raising the

temperature to 42°C (Figure 7.2). As the transcription and assembly of the tagged rRNA proceeds,

23S rRNA-protein complexes are captured and isolated. Affinity purification using streptavidin

agarose resin was performed to purify the 23S rRNA complexes captured at different time points

(Figure 7.3).

G A G A C G C G G U G U U G U G C G C G A G A G G A G A U G U G G C 540 G C 540 U A U A G C G C 550 C G 550 C G G C G C G U G U A U A U 23S rRNA C G Helix 25 U G C G A U U A G C G C C G C G G C G U C G C G G C G C C G C A G C A G U G C U C G C G A G A A A G G C G U U A A U G C A A A C G U Streptavidin binding aptamer

Figure 7.1 E. coli model system used to characterize the events during the biogenesis and assembly of the 50S subunit. A streptavidin binding aptamer (red segment) is incorporated into the functionally neutral helix 25 of the 23S rRNA allowing purification of pure ribosomal 23S rRNA complexes.

109 Induction of mutant 23S rRNA transcription

23S No transcription 16S 25°C X P1 P2 T1 T2 λ phage promoter 5S tRNA

23S Transcription proceeds 16S 42°C P1 P2 T1 T2 λ phage promoter 5S tRNA

Figure 7.2. Expression of tagged rRNA from the phage λ promoter is induced by raising the temperature from 25°C to 42°C. As the transcription and assembly of the tagged rRNA proceeds, 23S rRNA complexes are captured at different time points by addition of rifampicin.

Streptavidin-agarose beads

Incubate with streptavidin agarose beads crude ribosomes

streptavidin-biotin complex wash

biotin

unbound subunits

Incubate with biotin tagged 50S subunit

Figure 7.3 Streptavidin affinity purification of tagged 23S rRNA complexes. The aptamer-tagged complexes were captured at different time points after induction. Streptavidin affinity purification allows isolation of pure ribosomal particles. Elution of the mutant ribosomes from streptavidin beads was carried out by incubation of the mixture with 25 mM biotin.

110 To evaluate the applicability of this model system in gaining insights into the biogenesis and

assembly of the ribosome in vivo, experimental conditions for successful purification of tagged 23S

complexes were established. The presence of the aptamer tag in induced and uninduced cells was

confirmed by reverse-transcription PCR. The expected RT-PCR product of the wt rRNA (~359 bp)

coincides well with the DNA ladder. The tagged rRNA having the streptavidin aptamer is also in

accordance with the expected size of the product (~436 bp). Likewise, when the plasmid was from

AD2291 and PCR was performed, the product also exhibited similar size with that of the mutant

rRNA containing the aptamer. No bands were detected in samples without reverse transcriptase

except for sample at 25°C (Lane 3).

25°C 25°C 42°C 42°C 42°C 42°C DNA ladder 70S 70S 70S 70S 50S 50S Reverse + − + − + − Transcriptase

bp

500 ~436 bp ! 400 ~359 bp ! 350

Figure 7.4 Reverse transcription-PCR analysis to confirm the presence of streptavidin binding aptamer. 2.5% agarose gel profile of PCR products. The wild-type and tagged 23S rRNAs were reverse transcribed into their corresponding cDNA sequences. PCR of the resulting cDNA using appropriate primers confirmed the presence of the aptamer in the mutant rRNA (~436 bp product). The aptamer was absent in uninduced cells or wild-type 23S rRNA (~359 bp product).

The composition of the tagged 23S rRNA complexes was then determined by mass spectromtery. As

the transcription and assembly of the tagged rRNA proceeds, complexes were captured and isolated at

different time points (0 min, 2 min, 5 min, 10 min) by addition of rifampicin. The proteins associated

111 with the complexes at various time points were analyzed by LC-MSE. Table 7.1 summarizes the

proteins detected in these samples. After two minutes after induction, proteins are already bound to

the 23S complexes. After 10 minutes induction, most of the proteins are already present in the

complex.

Table 7.1 Large 50S ribosomal subunit proteins detected in AD2291 tagged 23S rRNA complexes harvested at different time points

r-protein 0 min 2 min 5 min 10 min L1 − + + + L2 − + + + L3 − + + + L4 − + + + L5 − + + + L6 − − + + L7/L12 − − + + L9 − + + + L10 − + + + L11 − − + + L13 − + + + L14 − + + + L15 − + + + L16 − + + + L17 − + + + L18 − − + + L19 − + + + L20 − − + + L21 − + + + L22 − + + + L23 − − − − L24 − + + + L25 − − + + L27 − − − − L28 − + + + L29 − + + + L30 − − + + L31 − − − − L32 − − + + L33 − + + + L34 − + + + L35 − − − − L36 − − − − +, proteins detected −, proteins not detected

112 To determine the effects of antibiotics on ribosome formation, the minimum inhibitory concentrations

(MICs) of various antibiotics were determined. The 50S inhibitors erythromycin and chloramphenicol

and the 30S inhibitors neomycin and paromomycin were used. Figure 7.5 shows the MICs of the

different antibiotics determined using the graphical approach [163]. These concentrations of

antibiotics will be used to evaluate the their effects on ribosome formation using the AD2291 model

system.

Ery Cam

1.6 1.8 50 ug/mL 1.75 ug/mL 1.4 1.6

1.2 1.4 1.2 1 1 0.8 0.8 0.6 0.6 Abs at 600Absat nm Abs at 600Absat nm 0.4 0.4 0.2 0.2 0 0 0 20 40 60 80 100 120 0 1 2 3 4 5 6 Concentration (ug/mL) Concentration (ug/mL)

Neo Paro

0.7 0.7

0.6 0.6 1.50 ug/mL 1.50 ug/mL 0.5 0.5

0.4 0.4

0.3 0.3 Abs at 600Absat nm 0.2 600Absat nm 0.2

0.1 0.1

0 0 0 1 2 3 4 5 6 0 2 4 6 8 10 Concentration (ug/mL) Concentration (ug/mL)

Figure 7.5 Determination of the minimum inhibitory concentration (MIC) determination of different antibiotics against AD2291 cells. The 50S inhibitors, erythromycin and chloramphenicol, and the 30S inhibitors, neomycin and paromomycin were used.

To gain insight into the mechanism how antibiotics affect ribosome assembly in vivo, r-protein

binding kinetics will be analyzed. Rates of protein binding to the assembling 23S rRNA complexes

113 produced at different time points will be determined from progress curves generated for each r-

protein (Figure 7.6). Kinetic data and binding rates will be evaluated as previously described by

Williamson and co-workers [61]. These experiments will be performed on AD2291 cells in the

presence and absence of antibiotics. The hypothesis is that if the antibiotic inhibits a specific stage

during ribosome formation, the rate constant of proteins implicated in antibiotic binding/interference

will be affected (decrease kobs) compared to the control without the antibiotic. The rate constants of

the primary binding proteins will not be affected compared to that of the late binding proteins. On the

other hand, if the antibiotic affects protein synthesis in general, the rate constants of all proteins

including the primary binding proteins will be affected when compared to without the antibiotic. I

envisioned that these experimental strategies could distinguish the mechanisms how various

antibiotics affect ribosome formation or protein synthesis in vivo.

1.0

0.5 L1 L2 ScaledProtein amount

0.0 0 10 20 30 40 50 60 Time (min)

Figure 7.6 Example of protein binding progress curve generated by plotting the scaled protein level at various time points. Protein levels will be obtained by quantifying the proteins using the LC-MSE approach.

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