Hydrogen-Bonding Residues at the Asymmetric Dimer Site of Trnahis Guanylyltransferase and Their Contributions to Oligomeric State and Activity

Hydrogen-bonding residues at the asymmetric dimer site of tRNAHis guanylyltransferase and their contributions to oligomeric state and activity

Master’s Thesis

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

William Arthur Eberley B.S.

Graduate Program in Biochemistry

The Ohio State University

2011

Thesis Committee

Jane E Jackman, Advisor

James Hopper

Michael Chan

William Arthur Eberley

2011 Abstract tRNAHis guanylyltransferase (Thg1) in eukaryotes plays a critical role in the proper maturation of tRNAHis by catalyzing the 3’-5’ addition of a required non-encoded, guanosine to the 5’ end of RNase P-cleaved tRNAHis. This enzyme’s molecular mechanism is of great interest, and this study evaluates the relationship of the enzyme’s multimeric conformation to enzymatic activity. The crystal structure of Homo sapiens

Thg1 (hThg1) at 2.3Å demonstrates that the enzyme is a homotetramer, in a dimer of dimers arrangement. By close examination of this crystal structure and sequence alignments of Thg1 enzymes from multiple organisms, three amino acid side-chains were thought to be potentially important for monomer-monomer contacts occurring in the asymmetric dimer interface. T98, S102, and S106’s side-chain hydroxyls are seen to be within hydrogen-bonding distance of an adjacent monomer’s main-chain. These residues were investigated for their possible importance through site-directed mutagenesis designed to eliminate or reduce the ability of the three residues to link monomer subunits.

Variant enzymes were analyzed by gel filtration and assays for G-1 addition, both of which confirm a critical role played by these residues. The mutation T98A and a double mutant S102A/S106A in hThg1 showed nearly complete loss of activity while the S102A

32 single variant was less significantly impaired, it at all, for the addition of G-1 to a P labeled tRNAHis. Additionally, replacement of T98 with a different hydrogen-bonding side chain can restore partial activity. Size exclusion chromatography showed the

ii oligomeric state of hThg1 was also altered by several of these mutations and in each case the reduction in G-1 activity can be correlated with a loss of tetrameric enzyme. To further evaluate the altered activity in these variants, filter binding assays were performed to determine whether the disruption of the oligomeric state affected the ability of the variant enzymes to bind to tRNAHis. Enzyme binding was evaluated with 32P labeled tRNA and KD for tRNA was determined for the various mutants, which ranged from KD

= 0.2 µM for wild-type hThg1 to the KD >3 µM exhibited by T98A hThg1. Finally, to extend the analysis of the multimeric structure in Thg1 to an in vivo system, constructs were made for yeast two-hybrid analysis and Thg1 variants from Saccharomyces cerevisiae (sThg1) were created with the same mutations as in the hThg1; this analysis is still ongoing.

iii

Acknowledgments

Sincere thanks to all the members of the Jackman lab for their friendship and help throughout my time in the lab; to the Wu lab for their FPLC, column, and expertise; and to Jane Jackman for her guidance, help, and most especially her patience. Thanks and appreciation also to all the members of my committee as well as members of the Biochemistry staff who have made my time here memorable.

Vita

2006-2010 …………………………………………….B.S. Biochemistry, The Ohio ………………………………………………………….State University

2010-present …...……………………………………….Graduate teaching associate, …………………………………………………………..Department of Biochemistry

Publications

Hyde SJ, Eckenroth BE, Smith BA, Eberley WA, Heintz NH, Jackman JE, Doublie S. 2010. tRNAHis guanylyltransferase (THG1), a unique 3’-5’ nucleotidyl transferase, shares unexpected structural homology with canonical 5’-3’ DNA polymerases. Proc Natl Acad Sci 107: 20305–20310.

Field of Study

Major Field: Biochemistry

Table of Contents

Abstract …………………………………………………………………………… ii

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

Vita …………………….…………………………………………………………. v

List of Figures …………………….………………………………………...……. vii

List of abbreviations ……………………………………………………………... viii

Chapter 1: Introduction. ………….…………………………………………...... 1

Chapter 2: Results ………….……………………………………………..……… 7

Chapter 3: Discussion ………….…………………………………………..…….. 34

Chapter 4: Methods ………….………….………………………………………... 40

References ………………………………………………………………………... 45

List of Figures

1. Modeled view of hThg1 …………………………………………………… 10

2. Sequence alignment of hThg1 and sThg1 .………………………………... 11

3. Chemistry behind the Thg1 mechanism .…………………………...... 14

4. Assay scheme for testing G-1 addition ……………………………...... … 15

5. hThg1 G-1 addition assay with WT and mutants ………………………..… 16

6. Relative activity levels for hThg1 enzymes ………………………………. 17

7. sThg1 G-1 addition assay with WT and mutants …………………...... 21

8. sThg1 G-1 addition assay with GTP only …………………………...... 22

9. Percent activity sThg1 variants with and without ATP …………………… 23

10. Size exclusion chromatography plots ……………………………………... 26

11. Isotherms for hThg1 and mutants showing Kd ..…………………...... 29

12. Isotherms for sThg1 and mutants showing Kd ..…………………...... 30

13. Derivation of the equation used to fit data in the binding assays …………. 31

14. Schematic view of yeast-2-hybrid ………………………………………… 34

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List of Abbreviations hThg1: Homo sapiens tRNAHis guanylyltransferase sThg1: Saccharomyces cerevisiae tRNAHis guanylyltransferase

BtThg1: Bacillus thuringiensis tRNAHis guanylyltransferase

FPLC: Fast Protein Liquid Chromatography

TLC: Thin Layer Chromatography

WT: wild-type

AD: activation domain

DBD: DNA binding domain

HisRS: Histidyl tRNA synthetase

Ve: elution volume

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CHAPTER 1: Introduction

Since the introduction of tRNA to the scientific world nearly sixty years ago, labs have been working on deciphering the structure, modifications, identity elements, maturation steps, and sequences for tRNA from organisms in all three domains of life. tRNA is a universally observed adapter molecule used for translation of an mRNA into a protein. In order for translation to be efficient it is necessary to have a continuous pool of tRNA which is charged and able to enter the ribosome; however, the charging of individual tRNA molecules, which all contain the same basic structures, with unique amino acid residues, is a challenge which must be overcome. Since higher order structure of each tRNA is conserved, charging of tRNA by each aminoacyl synthetase must utilize identity elements, which are specific differences in the primary sequence or modification pattern used by synthetases to identify their correct tRNA. These elements are usually only a couple of nucleotides out of the overall sum in the tRNA and are usually concentrated in the aminoacyl acceptor stem and the anticodon (1). The need for primary sequence and post-transcriptional modification thus make it necessary for the cell to contain enzymes capable of modifying tRNA specifically.

It was noted from sequencing work that eukaryotic tRNAHis from multiple organisms contained an additional nucleotide as compared with other eukaryotic tRNAs

(2). Through the use of T2 digestion and chromatography it was determined that the 5’ end of the tRNAHis was predominately pGpGpC opposed to the expected RNase P 1 cleavage product pGpC (2). The additional 5’ guanosine, resulted from a post- transcriptional addition reaction which occurs on nearly all tRNAHis in vitro (2). This additional residue at the 5’ end was later determined to be a very rare type of addition that is unique to tRNAHis; only tRNAHis and one mitochondrial tRNAPhe have this type of additional nucleotide present (3, 4). This residue is highly important due to its role as an identity element in Saccharomyces cerevisiae for aminoacylation by histidyl-tRNA synthetase (7, 8, 9).

These studies have led to a uniquely interesting area of research on the tRNAHis guanylyltransferase (Thg1) which has an interesting job in tRNAHis maturation. All eukaryotic tRNA species are transcribed with additional 5'-leading and 3'-trailing sequences which are cleaved and then matured as required. While some of these maturation events are common to all tRNA species, like the 3’ CCA addition reaction and the 5’-leader cleavage, there are also rare enzymatic events which occur only on specific tRNAs. The G-1 addition reaction catalyzed by Thg1 is one such tRNA-specific processing event. Histidyl-tRNA species in eukaryotes are unique, in that after the pre- tRNA is cleaved by RNase P, a single 5’ guanosine residue, G-1, is added to the tRNA; unlike the capping reaction for eukaryotic mRNA, the additional G-1 residue is linked to the 5'-end of the tRNA via a standard 5'-3' phosphodiester bond (5). Although the presence of G-1 is universally found in all three domains of life, this enzymatic action is necessary in eukaryotes because the additional G-1 nucleotide is not encoded and therefore must be added post-transcriptionally in a non-templated 3’-5’ addition reaction

(6).

In eukaryotes this extra G-1 is added post-transcriptionally across from A73 (10) this is not the case for prokaryotes. In their case the guanosine residue is genetically encoded and the G-1:C73 base pair is recognized by RNase P such that it is retained in 5’- leader cleavage (11). While the methods for acquiring the additional G-1 differ, the role it plays as an identity element is conserved in all domains of life (8). The loss of the G-1 residue was analyzed initially in yeast and E. coli tRNAHis by looking at changes in histidylation by HisRS. Two major identity elements were identified as important for efficient histidylation in each case: guanosine at -1, and the discriminator base at position

73 (7, 8, 12, 13, 14, 15). By eliminating eukaryotes’ ability to effectively add this -1 residue the cellular pool of histidylated tRNAHis decreases and concentrations of uncharged tRNAHis increase (5, 16). This activity was shown to be controlled by the essential gene THG1 in yeast (5) and reductions in the cellular concentrations of Thg1 caused reduced charging in vitro of about 800-fold (16). Interestingly however, the unnatural addition of the identity elements to non-histidyl tRNA leads to greater than

200-fold increase in histidylation of other tRNAs (15). This means Thg1 needs to be

His specific in targeting only tRNA for G-1 addition, to avoid anomalous charging and therefore Thg1 must have identity elements of its own. Investigation of Thg1’s need for recognition elements has identified the GUG anticodon of tRNAHis as a specific recognition element, as evidence showing that incorporation of the GUG anticodon into other tRNA framework retains substrate status for Saccharomyces cerevisiae tRNAHis guanylyltransferase (sThg1) (16).

Thg1 uses three chemical steps in its role of G-1 addition all of which occur within one active site. tRNAHis is initially cleaved by RNase P leaving a monophosphorylated 5’ end which is then adenylylated using ATP to create an AppGp-tRNA and release pyrophosphate. Guanylyltransferase follows this activation with the nucleophilic attack of a GTP via its 3’-OH on the AppGp-tRNA high energy phosphate bond to release AMP and leave pppG-1pGp-tRNA. Finally, pyrophosphate is removed to leave a monophosphorylated pG-1pGp-tRNA. While Thg1 shares many commonalities with more familiar enzymes in that it recognizes tRNA anticodons like aminoacyl-tRNA synthetases (12) and catalyzes the attack of a 3’-hydroxyl on an activated 5’ end much like a standard ligase/nucleotidyltransferase (17); there is no identifiable sequence similarity between Thg1 and other enzymes. Interestingly however even though there is no sequence similarity, there was a distinct homology to DNA polymerases. Based on hThg1 structural data homology was seen with adenylyl and guanylyl cyclases, A family

DNA polymerases, and B family DNA polymerases (18). The fold motif of hThg1,

ßαßßαß, appears to be most similar to the folding of the cyclases; however, overlaying the structure with polymerases suggests a closer relationship between the mechanisms of

Thg1 and A family polymerases based on the location of three conserved carboxylate residues compared to their locations in B family polymerases (18, 19, 20). Based on this homology and mutational data it is clear that a two-metal-ion mechanism is at work in

Thg1similar to T7 DNA polymerase (18).

While similarities are being recognized with the folding and the mechanism it is also important to remember the oligomeric structure which was seen in the

4 crystallographic data of hThg1 (18), which crystallized as a homotetramer. Substrate recognition by Thg1 is clearly important, as misincorperation of G-1 could lead to incorrect aminoacylation, but the tRNA bound structure has not yet been solved. Based on known data however some interesting hypotheses can be tested. It is known that the site of recognition for Thg1 is the GUG anticodon (16) and that this recognition and activity are required; however it is also known that Thg1 acts at the 5’ end of tRNA which is 70Å removed from this site (6). Based on the crystal structure the folding of the monomer unit seems to be conserved and the length of a single monomer unit seems to be unlikely to span across 70Å to interact with both the GUG and the 5’ end. Combined with the data seen in initial yeast two-hybrid experiments which demonstrate strong self- interaction (21), the cogent extension is that monomeric forms of this protein, which have been observed in the crystal structure to form a tetramer (18), are likely to be fully functional only as a multimer.

Thus understanding the basis for Thg1multimerization, including both the asymmetric and symmetric interfaces which are seen in the homotetramer, is an important step in understanding this required function in eukaryotes. This homotetramer with two interfaces, asymmetric and symmetric, cause the burying of 4,200 Å2 and 1,800

Å2 respectively and form the most stable oligomeric state according to stability calculations (6). The crystal structure also shows some likely hydrogen-bonding interactions between monomers at the asymmetric interface however no such bonds can be readily identified for the symmetric interface. The malfunction of this protein leads to severely deleterious effects on eukaryotic organisms so understanding the ability of this

5 enzyme to form an active tetramer is important for maintaining a healthy organism.

Therefore the goal of this project was to investigate the interactions on the asymmetric interface and lay the ground work for continued investigation of oligomeric formation in eukaryotic Thg1.

CHAPTER 2: Results

Creation of substitution variants for the three residues of interest: T98, S102, and S106

In the hThg1 crystal structure, the three residues T98, S102 and S106 reside on a long helix that forms the asymmetric interface between two monomer subunits, and are within hydrogen-bonding distance of backbone residues on the adjacent monomer (Fig

1). Thus, variants were constructed to eliminate the hydrogen-bonding capability of these side-chains by replacement of T98, S102, and S102/S106 with an alanine in human tRNAHis guanylyltransferase. Both T98A and S102A hThg1 variants had been previously constructed in a plasmid background allowing overexpression in E. coli under the control of an IPTG-inducible promoter and purified using affinity chromatography against the N- terminal His6-tag. For this work, the double mutant S102A/S106A was constructed from the DNA of the S102A mutant strain by Quick Change and T98C, T98S, and T98Y were constructed from T98A variant plasmid DNA template. The purpose of creating these variants was to probe the ability of this site to tolerate small changes in the potential hydrogen-bonding capability between the sidechains of the helix and the backbone of the adjacent ß-stand. Variants were also constructed using the yeast wild-type Thg1 plasmid as a template, yielding T99A, S103A, and S107A which are the equivalent residues to those varied in the human enzyme (Fig 2). As with the hThg1 variants, each of these enzymes were designed with an N-terminal His6-tag to allow for metal ion-affinity

7 purification on a cobalt column. For all of the Thg1 variant proteins, the final purified preparation (prepared as described in Chapter 4) was judged to be >90% by visual inspection on Coomassie blue stained denaturing polyacrylamide gels, and each protein migrated to the appropriate position for the wild-type enzyme, around 32 kDa for hThg1(18) and 28 kDa for sThg1(5).

Figure 1: a) Ribbon view of hThg1 tetramer based on crystallographic data (18). b) Close-up of the asymetric dimer interface which contains the three residues of interest T98, S102, and S106. c) Asymetric interface with potential hydrogen-bond distances measured 9

Figure 2: CLUSTAL 2.1 multiple sequence alignment of hThg1 and sThg1, with bold red text marking T98, S102, and S106 (human Thg1 numbering; corresponding to residues T99, S103 and S107 in sThg1).

His In vitro activity assays for G-1 addition to tRNA catalyzed by hThg1 variants

Using each of the enzymes which were previously purified, assays were performed which tested the relative ability of each enzyme to catalyze the physiologically

His relevant reaction of these enzymes, addition of G-1 to wild-type (A73-containing) tRNA . tRNA substrates were first 5’ monophosphorylated with 32P, and then in a reaction containing ATP and GTP, the enzymes were allowed to react for 2 hours before being treated by RNase A, which cleaves tRNA 3’ of pyrimidines and EDTA, which inactivates the enzyme through metal chelation. Samples were then treated with calf intestinal phosphatase, which removes any phosphate not protected by participation in a phosphodiester bond, and run on a silica TLC plate to identify different products. The mechanism for Thg1 addition reactions are known to involve three steps including: activation by ATP, nucleotidyltransfer, and pyrophosphate removal (Fig. 3) (18). This means that for 5’ monophosphorylated tRNA, the reaction in vitro should proceed by first forming an activated adenylylated-tRNA, followed by the pppG-1-tRNA, and finally the pG-1-tRNA; in fact both of these reaction products can be observed by performing reactions at limited Thg1 concentration. Reaction products resolved by thin-layer chromatography (TLC) as shown in Figure 4 can be differentiated based on their

32 migration: the farthest migrating product is the G-1-addition reaction product (G pGpC) while the Pi derived from unreacted substrate migrates lower on the TLC plates, as seen in (Fig. 5). Under certain reaction conditions (not shown in Fig 4), the activated intermediate species (visualized as Ap32pGpC) can also be observed.

By using quantification software it is possible to measure the amount of radioactivity contained in each spot on the image in Figure 5 and compare relative activities of wild-type Thg1 to those of Thg1 variants. For quantification of relative

His activities, the amount of product (G-1 containing tRNA , represented by Gp*GpC as described above) was determined for each enzyme at a single equivalent enzyme concentration, and then used to calculate the percent remaining activity for each variant enzyme relative to wild-type Thg1. Compared to the activity of wild-type enzyme, the mutations of T98A (16%), T98Y (11%), and S102A/S106A (6%) exhibit significantly decreased activity. The S102A (60%), T98C (24%), and T98S (61%) variants each exhibit activity levels which are reduced from the wild-type but are still detectable using the in vitro activity assay (Fig4). These results suggest that the enzyme does rely on hydrogen-bonding interactions to remain in an active tetrameric form based both on the fact that these residues are highly conserved and that the enzyme activity of the variant enzymes correlates with the hydrogen-bonding potential of these three residues.

The in vitro assays were performed under standard reaction conditions (described in Chapter 4), which include 125 mM NaCl in the assay buffer; however, to directly compare the activities of the variants under conditions used for gel-filtration analysis of the hThg1 variants (see below), enzymatic activities were also tested in several additional buffer systems. It was determined that the same activities (relative to the wild-type control) were observed even at NaCl concentrations as low as 50 mM, as well as in buffer containing 20 mM Tris-HCl,50 mM NH4Cl, pH 8.

Figure 3: Three step chemistry behind the Thg1 enzymatic addition of a 5’ G-1 onto tRNAHis.

Figure 4: Reaction scheme for testing Thg1 activity where addition of the guanosine protects the 32P from phosphatase cleavage and the products are separated by silica TLC.

Figure 5: G-1 addition assay TLC silica plate visualized by PhosphorImager. Plate shows wild-type Thg1 in 5-fold dilutions from 2.7µg, a representative spot for each mutant with enzyme concentrations of 2.7 µg, and a control with no enzyme

Figure 6: Six mutants of hThg1 with their relative G-1 activity levels to wild-type enzyme. This graph shows activities based on ~100 ng of each enzyme run concurrently, with a wild-type substrate turnover of 7%.

His In vitro activity assays for G-1 addition onto tRNA with sThg1

In terms of mechanistic and kinetic characterization, the Thg1 enzyme from

Saccharomyces cerevisiae (sThg1) has been extensively studied; however, no structure of this enzyme is currently available. Nonetheless, based on the >52% sequence similarity between Homo sapiens Thg1 and sThg1 (Fig. 2) (6), we predicted that a similar structure would be observed with sThg1, including a similar multimeric organization. Indeed, preliminary measurements using sedimentation velocity analytical ultracentrifugation indicated a molecular weight that agreed well with a tetramer for sThg1, although the results were not entirely conclusive due to evidence for non-specific aggregation in the samples (J. Jackman, unpublished). To test the hypothesis that sThg1 adopts a similar oligomeric structure to hThg1, enzyme mutations were also made in yeast to test the role of the predicted asymmetric dimer interface residues in catalysis. The T99, S103, and

S107 residues of sThg1 are analogous to the previously described T98, S102, and S106 residues of hThg1, so sThg1 variants were constructure in which each of these residues was changed to alanine.

The results of enzymatic activity assays for G-1 addition with the sThg1 alanine variants differed in some ways from the results obtained with the analogous variants in hThg1. If anything, the amount of product formed with either the S103A and S107A single mutations was increased relative to the amount of product formed by wild-type sThg1. This result was not entirely surprising, since the single S102A alteration in the context of hThg1 did not significantly disrupt enzymatic activity (the single S106A variation was not tested in hThg1). Combinations of these alterations may be required to

17 see any effects on enzymatic activity, as with hThg1. However, somewhat unexpectedly, while the T99A mutant did show a loss of G-1 addition function it was not as deficient as the T98A from hThg1 (Fig 5) with >20% activity (Fig7). Further assessment of the nature of the inter-subunit interactions between monomers of sThg1 may require additional biochemical and structural characterization.

Interestingly, we observed evidence for additional products being formed other than the expected G-1 addition product in reactions with the S103A and S107A variants.

The migration of these spots is consistent with the expected migration of a reaction product identified previously to correspond to an activated intermediate form of the tRNA in which a 5'-GMP has been added in 5'-5' linkage. Although wild-type sThg1 prefers to use ATP for the activation step of the reaction (Fig. 3), when GTP is provided as the only nucleotide in the assays, it can also be used to create the activated intermediate for attack of the incoming G-1 residue. To confirm the identity of this

His reaction product as the guanylylated reaction intermediate (5'-Gpp-tRNA ) G-1 addition assays were performed in the presence of GTP as the sole nucleotide in the assays (Fig

8). From this test it was clear that wild-type sThg1, S103A, and S107A mutants were all able to catalyze G-1 addition without ATP, which can only occur through the use of GTP for the activation step (Fig 9), but the activity of the variant enzymes was demonstrably more robust than that observed for wild-type sThg1. The molecular basis for the apparently relaxed nucleotide specificity for the activation step of the reaction caused by either of the single serine variants is not understood at this time, but interestingly,

18 similarly relaxed activation nucleotide specificity has been observed with bacterial Thg1 from Bacillus thuringiensis, and remains to be investigated further.

Figure 7: This shows WT sThg1 and the 3 mutants all in the typical G-1addition assay described in the methods as containing ATP and 1 mM GTP. Image has been visualized by a PhosphorImager.

Figure 8: This shows WT sThg1 and the 3 mutants all in the typical G-1 addition assay as described in the Methods except this only contains 1 mM GTP and no ATP. Image has been visualized by a PhosphorImager.

Figure 9: This chart shows percent activity relative to WT of substrate to G*pGpC under conditions with 1 mM GTP and 0.1 mM ATP (standard conditions as in Methods) in blue and 1 mM GTP in red.

Size exclusion chromatography results for hThg1enzymes

To directly probe the oligomeric state of Thg1, the purified wild-type and variant

Thg1 proteins were analyzed using size exclusion chromatography by FPLC. In this way, we hoped to correlate the changes to G-1 addition activity we had observed upon alteration of the hydrogen-bonding residues with changes in quaternary interactions between Thg1 subunits. Wild-type hThg1 is eluted from the size exclusion column in a major peak at ~13 mL ve (elution volume), and a secondary peak of ~15 mL ve. By comparison to the elution of commercially available molecular weight standards run under identical conditions, the former peak corresponds to a species with an apparent molecular weight of ~160 kDa, while the latter peak elutes with an apparent MW ~ 50 kDa. The calculated molecular weight of the hThg1 monomer is ~32 kDa, and thus the larger molecular weight species is consistent with formation of the tetrameric structure, but the exact quaternary structure of the smaller peak cannot be precisely defined (ie, whether this peak likely corresponds to monomeric or dimeric hThg1).

Using the same conditions, the T98A and S102A/S106A mutations in hThg1 have repeatedly shown increases in the amount of protein eluting in the lower multimeric order peak in comparison to wild-type hThg1, while the single S102A variant does not exhibit measureable differences from wild-type. This observed loss of higher order structure is consistent with the effects on functionality we observed in the in vitro assays. We note that the buffer conditions used for the data in Figure 10 were a 50 mM sodium phosphate buffer, pH 7.2 containing 0.5 M NaCl. This relatively high salt concentration was used to match the conditions previously determined to be optimal for long term storage of sThg1, 23 but we note that these high salt conditions may also induce some loss of multimeric structure (although differences between the wild-type and variant enzymes are still likely to represent differences in structural stability). Due to these concerns about high NaCl conditions contributing to decreased tetramer formation, other buffers have been evaluated including a 20 mM Tris-HCl, 50 mM NH4Cl, pH 7.5 buffer in which the predominant peak of enzyme is eluted as the high molecular weight (tetramer) species.

In each case, fractions from the peaks of eluted enzyme have been collected and characterized by SDS-PAGE to confirm that both peaks in absorbance correspond to our protein of interest, and moreover are not the result of degradation. By using size exclusion chromatography it is possible to draw a correlative relationship between the hydrogen-bonding residues in hThg1 and its ability to form the conserved tetramer structure. Furthermore, the ability to form this tetramer structure is directly related to the

His ability to add a G-1 on tRNA .

Figure 10: Each of these samples was run on a Superdex-200 column using BioRad gel- filtration standards (1,350–670,000 Da) to evaluate molecular weight. All samples were run in a buffered sodium phosphate system with 0.5M NaCl at pH7.2. Curves are based on absorbance at 280nm in arbitrary units (AU) and elution volume (ve) in ml. Major peaks correspond with MWs of 165 kDa and 50 kDa respectively a) WT hThg1 b) T98A c) S102A d) S102A/S106A. 25

The effects of alteration of interface residues on Thg1 binding to tRNAHis

Activity for G-1 addition has been shown to be correlated with each enzyme’s ability to form a tetramer, but the molecular basis for these alterations in activity has not been addressed. Based on their distant locations in the structure, the alterations that we have made in dimer interface residues would not be predicted to have direct effects on the previously identified active-site residues, thus leading to a breakdown in the chemical steps by which G-1 is added. The hypothesis would be instead that enzymes that are unable to form a tetramer might also fail to bind tRNA substrate properly, particularly given the relatively equivalent size of each tRNA substrate in relation to a Thg1 monomer. To test this, each enzyme was incubated with a labeled tRNAHis and then subjected to a double-filter binding assay, first through nitrocellulose (which binds to enzyme, and thus will trap any labeled tRNA that is tightly associated with Thg1) followed by Hybond N+ , which traps any remaining labeled tRNA that is not associated with Thg1. By using eight dilutions of each enzyme and incubating them with a small amount of substrate ([E] >>[ tRNA]), and plotting the resulting percentage of bound tRNA as a function of [Thg1], a dissociation curve could be constructed using the

His equation derived in Figure 13, yielding the apparent KD for Thg1:tRNA (Fig 11 and

12) (24).

The overall results for the various hThg1 variants in the binding assays were again consistent with the data which has been previously shown. Those enzymes which

His showed limited activity in the G-1 addition assay showed reduced binding to tRNA relative to the wild-type enzyme, and those variants that exhibited wild-type activity 26 yielded similarly low KD values, when binding was measured at pH 6. Of the hThg1 mutants the binding for T98A occurred with the highest KD, with nearly a 20-fold increase from wild-type (3.4 µM). Again, however, the results for sThg1 were not as conclusive regarding the roles of the hydrogen-bonding residues. Where binding to the full length tRNA was tested, the observed KD values were unchanged for S103A and

S107A sThg1 variants, although binding to tRNAHis was reduced by as much as 5-fold in the T99A case. However, the tRNA binding affinities observed with sThg1 are weaker than affinities measured for hThg1, even for the wild-type enzymes, tested at two different pH values, and thus KD values can not be determined as precisely for sThg1 using this type of filter-binding assay due to the relatively higher amounts of protein that must be added to saturate the binding reactions. Nonetheless, as with the in vitro activity data, the ability of sThg1 mutants to retain binding and activity even with these mutations suggests that perhaps they are not as easily perturbed as their hThg1 counterparts.

Figure 11: Kd (uM) determination for the binding of tRNAHis with hThg1, T98A, and S102 by fitting the points generated by graphing percent bound vs. concentration to the equation y=1/(1+Kd/[Thg1])

Figure 12: Kd (µM) determination for the binding of tRNAHis with sThg1, T99A, S103A, and S107A by fitting the points generated by graphing percent bound vs. concentration to the equation y=1/(1+Kd/[Thg1])

Figure 13: Derivation of the equation used to fit the data points and determine KD. Equations above all make the assumption that because [Thg1]>>[tRNA] the [Thg1]free=[Thg1]total.

Preliminary work to establish a yeast two-hybrid assay to evaluate protein-protein interactions between Thg1 enzymes

In an effort to understand the in vivo interaction between monomeric subunits in

Thg1 mutants, reagents necessary to perform a yeast two-hybrid assay were constructed.

The concept of this process is to establish strains in which a desired enzyme (in this case,

Thg1) is attached to separated activation domain (AD) and DNA binding domain (DBD) of a transcription factor that, when brought together by interactions of the enzyme to be tested will become active, and will stimulate the expression of reporter genes that have been placed under control of a galactose-inducible promoter. In constructing the plasmids necessary for performing the two-hybrid assay, restriction enzyme cut sites were added to Thg1 which could be used for ligation into a similarly restriction-digested vector. The vectors used were VP16 (AD) and GBT9 (DBD) (kindly provided by Dr.

Jim Hopper) and the Thg1 enzyme to be tested was integrated N-terminally in the DBD and C-terminally in the case of the AD through the use of restriction sites BamHI/NotI in

AD and BamHI/PstNI in DBD. Additional restriction digests were used to verify the successful ligation before each was confirmed by sequencing. Once the strains were constructed the selectable markers LEU2 for the DBD and TRP1 for the AD could be used to introduce them into a yeast two-hybrid strain by transformation and plating on selective media. Finally reporters could be used to identify interaction by testing the activity of the genes HIS3, URA3, and lacZ (which are all under control of galactose- responsive promoters in the strain) (Fig 14). Testing of these constructs is still in progress. This method of testing will allow for expansion from simply looking at the in

31 vitro assay results with single mutants, which have been individually constructed at the asymmetric interface, to a reverse yeast two-hybrid which can evaluate not only the asymmetric but also the symmetric interface through the use of a mutagenized cassette which gives a library of different mutants where only those that disrupt the Thg1 inter- subunit interactions will be identified and studied.

Figure 14: Images represent the general scheme of the yeast two-hybrid setup from incorporating the Thg1 sequence to testing interaction and activating reporters.

CHAPTER 3: Discussion

Thg1 has many interesting and unusual characteristics. This enzyme catalyzes three distinct chemical steps within a single active site during its unusual 3'-5' nucleotide addition reaction where it selectively binds tRNAHis and modifies it by adding a guanosine onto the 5’ end via a standard phosphodiester bond. Through this work, we have used site-directed mutagenesis, in vitro enzymatic assays and physical methods for characterization of multimeric structure to provide independent support for the proposed homotetrameric structure of eukaryotic Thg1 enzymes. Moreover, our data suggest that the multimeric structure of eukaryotic Thg1 is at least partially stabilized by three residues which, based on their positions in the hThg1 crystal structure, appear to bridge monomer subunits through hydrogen-bonds to help create the asymmetric interface which appears to be important for correct enzymatic function.

Thg1 enzymes are found in organisms from all three domains of life. In addition

His to its essential role in tRNA maturation in eukaryotes where the recognition element G-

1 is non-encoded, Thg1 homologs have been implicated in other various reactions that take advantage of the enzymes' ability to catalyze 3'-5' nucleotide addition reactions, including tRNA 5'-end repair and mitochondrial 5'-tRNA editing. Notably, the residues investigated in this study (T98, S102, and S106 in hThg1) are similarly conserved in

Thg1 enzymes from all domains of life, suggesting that similar roles for these residues

34 may be observed for enzymes from other domains of life. Consistent with this hypothesis, previous characterization of the bacterial Thg1 homolog from Bacillus thuringiensis (BtThg1) by sedimentation velocity using analytical ultracentrifugation has confirmed a molecular weight for this enzyme consistent with formation of a homotetramer (data not shown), as is observed for hThg1.

The small hydrogen-bonding residues investigated in this study are demonstrably important for function, and we propose that they each play a role in stabilizing one of the dimer interfaces observed in the hThg1 crystal structure. By site-directed mutagenesis and in vitro analysis it is clear that in hThg1, loss of the side-chain hydroxyl groups at positions 98, 102, and 106 can cause a decrease in G-1 addition activity. In fact the single mutation of T98A can cause almost a complete loss of activity in the enzyme and also causes a dramatic reduction in the amount of the tetrameric form of the enzyme, as judged by size exclusion analysis. The effects of the single alanine alterations at S102 or

S106 are less dramatic; however the combination of mutations in both S102 and S106 similarly leads to dramatic loss of G-1 addition activity and overall tetrameric structure of the variant enzyme. This suggests that the interaction between the monomer units at the asymmetric interface of the homotetramer hThg1 is highly important, a fact supported additionally by these residues’ highly conserved nature.

Interestingly, the precise nature of the oligomeric state of the defective hThg1 variants is not clear from our results. We note that the species that migrates on size- exclusion with an apparently lower molecular weight than the tetramer species is calculated to have a molecular mass of ~45-65 kD by comparison to the commercial 35 standards run on the same column under identical conditions, while the predicted molecular mass of each hThg1 monomer is 32 kD. We envision two likely alternatives; first, that the lower apparent molecular weight species corresponds to the migration of monomeric hThg1, and the migration of this species does not correlate exactly with the molecular weight due to the shape of the molecule, which can affect migration on gel filtration. Second, it is possible that the perturbations caused by the alterations investigated here do not completely disrupt all intermolecular interactions, and the species observed on gel filtration represents the dimeric form of the enzyme. To further evaluate these possibilities will require the use of techniques that allow more precise measurement of molecular weight, such as analytical ultracentrifugation.

The hThg1 crystal structure reveals that the enzyme is arranged in a "dimer of dimers" architecture, with two non-identical interfaces for inter-subunit interactions. The first is the asymmetric dimer interface investigated in this work, for which the long alpha helices containing T98, S102 and S107 appear to make substantial contributions to overall stability. The second interface can be thought of as a symmetric dimer interface in which two of these hThg1 dimers come together in a surface burying 1,800 Å2 of surface. While the area buried on this interface is considerably smaller than the 4,200 Å2 buried on the asymmetric dimer interface it is still contributing significantly to the overall stability of the enzyme (18). The stabilizing influences on this interface however are not discernable from the crystal structure. In the case of the asymmetric interface hydrogen- bonding opportunities presented themselves along with other stabilizing influences such as two salt-bridges; however, the symmetric interface presents no obvious salt-bridges,

36 hydrophobic patches, hydrogen-bonding, or covalent links. Though not addressed specifically in this research project the interactions at this interface will be of further interest in future, and to that end the yeast two-hybrid system which has been started will be the first way to begin looking at this interface. By using a reverse two-hybrid system in vivo testing of mutagenized cassettes which span this interface should allow a greater understanding of which residues or sections of protein are playing vital roles in the formation of this interface. The tRNAHis substrate for Thg1 activity is comparable in size to that of an individual Thg1 monomer. This, combined with the demonstrated need for eukaryotic Thg1 to simultaneously recognize the tRNAHis anticodon while acting 70

Å away at the 5'-end of the tRNA, suggests that a single Thg1 monomer would not be catalytically active. In fact, measurements of the stoichiometry of tRNA binding by hThg1 have suggested a stoichiometry of only 1-2 tRNA molecules bound per Thg1 tetramer (B. Smith and J. Jackman, unpublished). The correlation demonstrated in this work between decreased G-1 addition activity and decreased stability of the tetrameric enzyme is suggestive of a changing ability of the variant enzymes to bind substrate.

Binding assays show that in each case where a decrease in tetrameric enzyme formation and activity occurred, there was also an increased dissociation constant for the interaction between Thg1 and tRNA. While wild-type hThg1 is able to bind tRNAHis relatively tightly, the tRNA binding affinity of an S102A mutant is somewhat reduced, and the

T98A mutant binds tRNA nearly 20-fold weaker than the wild-type enzyme. Together these data make a strong argument for the importance of the three hydrogen-bonding residues located at this dimer interface.

The results we have observed for sThg1 alterations are not as conclusive as what was observed for the hThg1 case. The analogous T99A sThg1 variant showed a reduction in G-1 addition activity of nearly 80%, consistent with a potential role for this residue in participating in similar inter-subunit hydrogen-bonding interactions, but no defects in enzymatic activity were observed with S103A and S107A variant enzymes.

Another interesting observation was made for the S103A and S107A mutants in that they are activatable by GTP alone, to a greater extent than is observed with the wild-type sThg1 enzyme. Taken together, these results may suggest a possible difference in the oligomeric state of sThg1 as compared with its crystallized human counterpart. Also the ability to utilize the GTP in activation could be indicative of some global protein flexibility induced by these alterations which allow for the use of additional nucleotides in the activation steps. One interesting note on the sequence of sThg1 is the presence of an additional hydrogen-bond-capable residue nearby the other residues that were tested,

T106, mutational analysis of this position might reveal a reliance on this residue for hydrogen-bonding interactions at this interface, and thus that the structure is slightly different in yeast.

If indeed some changes in inter-subunit associations have occurred between orthologous Thg1 proteins, then teasing out the interactions between monomers via a two-hybrid system will become advantageous. The ability to show in vivo an interaction between monomers of Thg1 allows a greater ability to understand how these interfaces are constructed. While the focus of this research as been on the asymmetric site, there is also the symmetric interface which has no easily perceivable interactions holding it

38 together—though data suggests that it must be held somehow. By continuing with individual site-directed mutations understanding the asymmetric site would likely be possible, however dealing with an interaction which does not contain a clear starting point for mutation could be tedious and benefit from the in vivo ability of a cell to pick out mutants for monomer interaction from a library of carefully mutagenized protein.

Understanding the interactions between the individual subunits in a functional homotetrameric Thg1 could give additional insights to the field of enzymology and protein dynamics based on its unusual activity and the similarities in architecture which are seen between Thg1 and other enzymes, such as canonical 5'-3' DNA polymerases

(12).

CHAPTER 4: Methods

Construction and purification of mutant enzymes

Site-directed mutagenesis to create both single and double alanine variants of hThg1 (without 29 mitochondrial targeting N-terminal amino acids) and sThg1 were done by QuickChange mutagenesis (Stratagene) with reactions containing 50 ng of template plasmid DNA encoding yeast His6-Thg1 and 125 ng of each oligonucleotide (5).

Once the variants were sequenced to confirm mutagenesis each construct was transformed into E. coli XL-1 Blue competent cells. Using the His6 tag, each protein was purified by cobalt based metal-ion affinity chromatography TALON resin (Clontech) detailed in (6,5). Final pure protein was dialyzed against 0.5 M NaCl, 4 mM MgCl2, 1

μM EDTA, 1 mM DTT, 50% glycerol, pH 7.5 (except in specified cases where NaCl concentrations have been exchanged for: 250 mM NaCl, 50 mM NaCl, or 50 mM NH4Cl and 20 mM Tris-HCl). Concentration was determined by BioRad protein assay using an

IgG standard. Pure proteins were stored at -20 °C.

G-1 Addition Assay

32 Activity of the enzymes for the addition of a G-1 was evaluated with a 5’- P- labeled yeast tRNAHis as previously described (12). Substrate tRNA, with a specific activity of 5000-10,000 Ci/mmol, was then incubated for 2 hrs at room temperature with

1 mM GTP, 0.1 mM ATP (unless GTP only), 25 mM Hepes pH 7.5, 10 mM MgCl2, 3 mM DTT, 125 mM NaCl, 0.8 µg BSA, and five dilutions of a specific purified Thg1 enzymes (concentrations of 2-5 mg/ml) in fivefold dilutions. After 2 hrs each reaction was digested with 5 µg ribonuclease A and enzyme inactivated with 0.5 µl of 0.5 M

EDTA at 50 ˚C for 10 min before being treated with 0.5 units of calf intestinal phosphatase (Roche) at 37 ˚C for 30 min. This results in label being associated with either a G32pGpC fragment which is the extended product or 32P alone from unreacted substrate. These products are then resolved on a silica TLC plate with an n- propanol:NH4OH:H2O (55:35:10) solvent,visualized by using a PhosphorImager, and analyzed with Image Quant software.

Size exclusion chromatography

Analysis was done using a Superdex 200 10/300 GL column and AKTA FPLC

(GE Healthsciences). Samples were run in a 50 mM sodium phosphate and 0.5 M NaCl buffer at pH 7.2, except where other conditions are noted. Sample sizes ranged from

200-500 µl each with concentrations of 2.7-10 mg/ml pure protein. BioRad gel-filtration standards were used under same buffer conditions to evaluate molecular weights.

Filter binding assay

Binding of the various Thg1 enzymes were evaluated against a body-labeled tRNAHis which was created through T7 runoff transcription with [α-32P]-ATP. A 30 µl reaction volume was made up of: 22 µl of a common mix (50.8 mM Tris-HCl pH 8, 1 mM spermidine, 30.5 mM MgCl2, 7.5 mM DTT, 4.1 mM UTP, 4.1 mM CTP, 4.1 mM

32 GTP, 0.5 mM ATP and H2O), 5 µl [α- P]-ATP, 2 µl of NsiI digested DNA

(concentrations ~2 mg/ml), and 1 µl of purified T7 RNA polymerase. This reaction was initiated by the addition of T7 and was allowed to run for 2 hrs at 37 ˚C. 3 µl of DNaseI was then added and incubation was continued for 30 min to cleave the DNA. A 10% polyacrylamide gel with 4 M urea was then used to purify the RNA. The gel is exposed to film and the band of tRNA is excised and eluted in 0.5 M ammonium acetate, 0.1%

SDS, 5 mM EDTA, and H2O overnight at 37 ˚C. tRNA is then isolated from the gel with a Spin-X column (Fisher Scientific), phenol extracted, and ethanol precipitated.

Filter binding assays are setup as described (24) using 50 µl reaction volume containing 5 µl binding buffer (50 mM bis-Tris pH 6.0 (Tris pH 7.5 was used in high pH binding trials), 10 mM MgCl2, 3 mM DTT, 125 mM NaCl, and H2O), ~5000cpm labeled tRNAHis(<0.5 µl), 5 µl purified enzyme Thg1 (eight twofold dilutions from 100-0.39

µM), and H2O. This reaction was allowed to stand at room temperature for 1 hr.

Nitrocellulose and Hybond filters were soaked in binding buffer and then clamped into a dot-blot apparatus (Nitrocellulose on top of Hybond). 35 µl of the reaction mixture was then added to each well and vacuum-pumped through the filters. Each well was then washed thoroughly by running >200 µl of binding buffer through before allowing each filter to dry and be visualized by using a PhosphorImager and evaluated with ImageQuant software. Percent bound was then determined and plotted against protein concentration with Kd determined from fitting the points with the equation y = 1/(1+Kd/[Thg1]).

Preparation of yeast two-hybrid constructs

Constructs for a yeast two-hybrid assay were made from vectors pVP16 (AD) and pGBT9 (DBD) (Provided by James Hopper) with hThg1 and sThg1 inserts. These strains were constructed by ligation after restriction digestion. For the AD-inserts 5’-PstNI and

3’-NotI cut sites were cloned into the Thg1 sequence and 5’-BamHI and 3’-PstNI sites were created for the DBD-inserts.

Cut sites were added by PCR using oligonucleotides (Sigma-Aldrich) (33-35 nucleotides Tm= 75-80°C) under conditions provided by iProof (200 ng template DNA,

250 ng of each oligonucleotide, 10 µl of iProof HF buffer, 1µl dNTP mix, and H2O in a total volume of 49.6 µl). Reactions were initiated by adding 0.4 µl iProof DNA polymerase and running the suggested PCR cycle (98 °C for 30 sec – 1 cycle; 98°C for

10 sec,72°C for 30 sec –30 cycles; 72°C for 10 min –1 cycle). PCR products were then gel purified on a Tris-acetate-EDTA (TAE) 1.5% agarose gel using a QIAquick Gel

Extraction Kit (Qiagen).

Vectors and inserts were both digested with the appropriate restriction enzymes

(NEB) for 2 hrs at 37°C. Digested products were purified by a QIAquick PCR

Purification Kit (Qiagen) to prepare for ligation. Ligation was done at 16°C for 16 hr with a 10 µl reaction containing 10-fold excess insert to vector with USB T4 ligase. 5 µl of ligation reaction was then transformed into XL-1 Blue competent cells and grown on

Lysogeny broth agarose plates containing ampicillin overnight at 37°C. Colonies from these plates were then grown in liquid media and DNA was isolated by QIAprep

Miniprep (Qiagen). To confirm ligation success, an additional restriction digest was done and the products were run out on a TAE 1% agarose gel. DNA which was confirmed to be ligated was then sequenced to confirm.

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