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Seidelt, Birgit (2010): Structural studies of ribosome stalling and translocation complexes. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

In this study, cryo-electron microscopy (cryo-EM) and single particle reconstruction were used as a main technique to investigate the involvement of bacterial ribosomes in two crucial cellular processes: the regulation of gene expression and the biogenesis of membrane proteins. Whereas most nascent chains are thought to transit passively through the ribosomal exit tunnel during translation, a number of regulatory peptide sequences, such as TnaC and SecM, have been proposed to specifically interact with tunnel components, causing the ribosome to stall which in turn regulates the expression of downstream gene products. In the first part of this study, a 5.8 Å resolution cryo-EM reconstruction of an Escherichia coli 70S ribosome stalled during translation of the TnaC leader peptide could be determined. The high quality of the map allowed the visualization of the TnaC nascent chain within the exit tunnel of the ribosome, making contacts with ribosomal components at distinct sites. At the peptidyl transferase center (PTC), the universally conserved nucleotides A2602 and U2585 adopt conformations that are incompatible with co-habitation of the termination release factors. Moreover, a model could be proposed where interactions within the tunnel are relayed back to the PTC, leading to its inactivation. In addition, a foundation for the elucidation of the SecM-stalling mechanism could also be established. The membrane protein insertase YidC is the prokaryotic member of the conserved YidC/Oxa1/Alb3 protein family. It assists in the assembly and folding of membrane proteins in conjunction with the Sec translocase as well as on its own. E. coli YidC is a hexaspan protein with a large, non-conserved periplasmic domain between the first and second transmembrane (TM) segment. In contrast, YidC2 from the Gram-positive bacterium Streptococcus mutans contains five TM segments and an extended C-terminal region akin to the C-terminal ribosome binding domain of the mitochondrial YidC homolog Oxa1. In the second part of this study, programmed 70S ribosomes carrying the YidC-specific nascent chain MscL could be generated, and visualized in a preliminary low-resolution cryo-EM structure in complex with E. coli YidC. Furthermore, purified S. mutans YidC2 was reconstituted into proteoliposomes and the formation of a ribosome-YidC2-proteoliposome complex could be demonstrated. Thus, the foundations have been laid for the visualization of YidC2 in the membrane environment. Improvement of the preliminary RNC-YidC structure together with determination of an RNC-YidC2 complex are expected to provide insights into the molecular mechanism of YidC mediated membrane protein biogenesis.