Functional and structural studies of a C-terminally extended YidC Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) Fakultät Naturwissenschaften Universität Hohenheim Institut für Mikrobiologie vorgelegt von Ines Seitl aus Esslingen a.N. 2015 Dekan: Prof. Dr. Heinz Breer 1. berichtende Person: Prof. Dr. Andreas Kuhn 2. berichtende Person: Prof. Dr. Heinz Breer Eingereicht am: 30.09.2015 Mündliche Prüfung am: 18.02.2016 Die vorliegende Arbeit wurde am 26.11.2015 von der Fakultät Naturwissenschaften der Universität Hohenheim als „Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften“ angenommen. Parts of this thesis have been published in Seitl I., Wickles S., Beckmann R., Kuhn A. and Kiefer D. (2014) The C-terminal regions of YidC from Rhodopirellula baltica and Oceanicaulis alexandrii bind to ribosomes and partially substitute for SRP receptor function in Escherichia coli. Mol Microbiol 91(2):408- 421 Parts of this thesis have been presented at national and international conferences Symposium Goethe University Frankfurt, 2010: Evolution of protein translocation systems Poster presentation “The positively charged C-terminal extensions of marine YidC- homologs are crucial for interaction with E. coli ribosomes” Gordon Conference, Galveston USA 2012: Protein transport across cell membranes Poster presentation incl. young investigator talk “Functional analysis of the C-terminal extensions of marine YidC homologs in the SRP-dependent membrane insertion” EMBO Conference, Dubrovnik 2013: From structure to function of translocation machines Poster presentation “Characterization of the interaction between the C-terminal domain of a planctomycete YidC homolog and the bacterial ribosome” TABLE OF CONTENT 1 INTRODUCTION .......................................................................................................................... 1 1.1 The prokaryotic ribosome .................................................................................................. 1 1.1.1 The duty cycle of the bacterial ribosome ................................................................... 2 1.1.2 The ribosomal exit tunnel: a flexible binding platform .............................................. 4 1.1.3 Ribosome nascent chain complexes .......................................................................... 8 1.2 Protein targeting in bacteria ............................................................................................ 11 1.2.1 The Signal Recognition Particle and its RNA ............................................................ 15 1.2.2 Signal sequence recognition by SRP ......................................................................... 18 1.2.3 Co-translational targeting: SRP-FtsY assembly......................................................... 20 1.3 The YidC/Oxa1/Alb3 insertase family ............................................................................... 23 1.3.1 Functional specialization in bacteria, mitochondria and chloroplasts ..................... 23 1.3.2 The bacterial membrane insertase YidC .................................................................. 28 1.3.3 YidC and the ribosome ............................................................................................. 34 1.4 Aims of the study ............................................................................................................. 36 2 MATERIALS AND METHODS .................................................................................................... 37 2.1 Culture media and additives ............................................................................................ 37 2.2 Bacterial strains ................................................................................................................ 37 2.3 Plasmids ............................................................................................................................ 39 2.4 PCR primer........................................................................................................................ 43 2.5 Peptide synthesis ............................................................................................................. 46 2.6 Methods in molecular biology ......................................................................................... 47 2.6.1 Polymerase chain reaction (PCR) ............................................................................. 47 2.6.2 Agarose gel electrophoresis ..................................................................................... 48 2.6.3 Restriction digest of DNA ......................................................................................... 48 2.6.4 Ligation of DNA fragments ....................................................................................... 48 2.6.5 Transformation of chemically competent E. coli cells ............................................. 49 2.6.6 Preparation of chemically (RbCl) competent E. coli cells ......................................... 49 2.6.7 Isolation of plasmid DNA from bacterial cultures .................................................... 49 2.6.8 DNA sequencing (Sanger et al., 1977) ...................................................................... 50 2.6.9 Site-directed mutagenesis (Quikchange) ................................................................. 50 2.6.10 EtOH precipitation of plasmid DNA .......................................................................... 52 2.6.11 Homologous recombination to generate the E. coli strain MC-Ffh ......................... 52 2.6.12 In vitro transcription ................................................................................................. 57 2.7 in vivo complementation assay ........................................................................................ 58 2.8 Fluorescence microscopy – GFP localization ................................................................... 58 2.9 AMS derivatization – pulse chase & immuno-precipitation ............................................. 59 2.10 Protein purification .......................................................................................................... 61 2.10.1 Purification of E. coli YidCNHis10 ................................................................................. 61 2.10.2 Purification of YidC-Rb NHis10...................................................................................... 62 2.10.3 Purification of E. coli Ffh & Ffh C406S / M423C ....................................................... 63 2.10.4 Purification of ribosomal proteins L24 and L29 ....................................................... 65 2.10.5 Purification of the C-terminal insertase domains RbCT, OaCT, O1CT and A3CT ...... 66 2.11 Protein concentration determination according to Scopes ............................................. 68 2.12 in vitro ribosome binding assay ........................................................................................ 68 2.13 Reconstitution of an RNC-YidC complex .......................................................................... 69 2.14 Electron microscopy and image processing ..................................................................... 69 2.15 in vitro pull down assays .................................................................................................. 70 2.16 Circular dichroism (CD) - spectroscopy ............................................................................ 70 2.17 Microscale thermophoresis (MST) measurements .......................................................... 71 3 RESULTS.................................................................................................................................... 75 3.1 Chimera of the membrane insertase YidC with altered C-terminal regions .................... 75 3.1.1 Complementation of E. coli YidC .............................................................................. 76 3.1.2 Purification of the YidC proteins .............................................................................. 77 3.1.3 Ribosome binding of YidC is improved by the extended C-terminal tails ................ 81 3.2 Structural studies of the YidCRb - ribosome complex ....................................................... 83 3.2.1 Cryo-EM structure of the YidCRb – RNC complex ...................................................... 83 3.2.2 The ribosomal protein L29 binds to the C-terminal tail of YidC-Rb ......................... 88 3.2.3 Structure of the YidC/Oxa1/Alb3 C-terminal domains (CTDs) ................................. 91 3.3 In vivo studies: function of the C-terminal YidC regions .................................................. 98 3.3.1 Insertion of MscL via YidC-Rb in the absence of a functional SRP system ............... 98 3.3.2 Localization of MscL-GFP in the absence of Ffh or FtsY ......................................... 102 3.4 Binding studies of SRP and the SRP signal sequence of KdpD ....................................... 104 3.4.1 Purification and labeling of Ffh from E. coli ........................................................... 106 3.4.2 Reconstitution of a functional SRP protein ............................................................ 109 3.4.3 Binding studies of SRP signal sequences to SRP using microscale thermophoresis 112 4 DISCUSSION ........................................................................................................................... 119 4.1 C-terminally