Release of a Natural mRNA During Recycling of the Prokaryotic Ribosome
by Moritz Willy Rudolf Willmer
Date of Examination:2023-02-02
Date of issue:2023-06-20
Advisor:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Kai Tittmann
Referee:Prof. Dr. Holger Stark
Referee:Dr. Alex Caspar Faesen
Referee:Dr. Sonja, Lorenz
Referee:Prof. Dr. Hauke Hillen
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EnglishRibosome recycling is the last step of the bacterial translation cycle. The release of the peptide leaves a 70S ribosome with a deacylated tRNA in the P site and the stop codon of the mRNA in the A site. The combined action of the ribosome recycling factor (RRF) and elongation factor G (EF-G) cause the splitting of the ribosomal subunits. The mechanism of tRNA and mRNA release and the order of events are less well understood and are controversially discussed in the literature. Based on studies using various non-native model mRNAs different orders of events were proposed. In this thesis the release of a natural mRNA and subunit splitting in a native termination context are investigated. A system had to me established to measure the recycling reaction at the stop codon of a previously fully translated ORF of an mRNA featuring a natural 3’-UTR. Using a bioinformatic transcriptome analysis, the lpp mRNA (coding for the major outer membrane prolipoprotein Lpp) was chosen as a representative natural mRNA. The lpp mRNA features a structured 3’-UTR with a transcription terminator hairpin, which is a predominant feature of terminal OFRs. To observe mRNA release from the 30S subunit, a FRET-assay was developed. For that purpose, a bioconjugation method utilizing the lipoic acid ligase LplAW37V was utilized and adapted for site-specific introduction of a fluorophore to the 30S subunit. Subunit splitting and mRNA release were measured in bulk kinetic experiments using the stopped-flow technique. The results show that mRNA release of the natural lpp mRNA happens almost simultaneously to subunit splitting, with subunit splitting being necessary for the fast release of the structured natural mRNA. mRNAs with truncated or no 3’-UTR, i.e. without the native mRNA hairpin, can dissociate from the ribosome without requiring subunit splitting. Single molecule TIRF microscopy was used as an additional technique and supported the results obtained with the bulk kinetic experiments. In conclusion, the results demonstrate the context dependence of ribosome function, the importance of choosing a physiological relevant model system and explain the partially contradicting models of previous studies. This study contributes towards integrating mRNA release into a comprehensive model of prokaryotic ribosome recycling and adds to understanding this vital step in bacterial translation.
Keywords: Ribosome; Recycling; mRNA