Recoding of bacteriophage T4 gene 60 mRNA by programmed translational bypassing
by Mariia Klimova
Date of Examination:2020-02-10
Date of issue:2020-07-28
Advisor:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Kai Tittmann
Referee:Prof. Dr. Ralf Ficner
Referee:Prof. Dr. Heike Krebber
Referee:Prof. Dr. Holger Stark
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EnglishDuring synthesis of a topoisomerase subunit encoded by the gene 60 of bacteriophage T4, the ribosome takes off from a certain codon, slides over a 50-nt non-coding segment of mRNA and resumes translation on a specific landing codon, which together is denoted as translational bypassing. The signals required for bypassing are programmed within the gene 60 mRNA and include the take-off and the landing codon, the stop codon adjacent to the take-off codon, several mRNA stem-loops and the nascent peptide that interacts with the exit tunnel of the ribosome. Gene 60 mRNA structures orchestrate ribosome movements during bypassing, but it is unclear which event initiates the take-off from the mRNA and what defines the directionality of sliding. We have investigated bypassing in a reconstituted in vitro translation system from E. coli and have probed the ribosome dynamics during bypassing using single-molecule FRET technique. We show that the nascent peptide interactions within the exit tunnel of the ribosome together with a short mRNA stem-loop formed in the A site induce a non-canonical hyper-rotated state of the ribosome during pausing and before the take-off. Elongation factor G (EF-G) interacts with the pausing ribosome and facilitates pseudo-translocation, using the mRNA stem-loop as an A-site tRNA mimic. This disrupts the codon-anticodon interaction in the P site and initiates the take-off. During forward sliding about two guanosine 5′-triphosphate (GTP) molecules are hydrolyzed per nucleotide of the noncoding gap, which we suggest is important for efficient sliding and landing. Our data suggest that EF-G plays an important, previously unanticipated role in translational bypassing initiation. Previous in-vivo data suggested that the ribosomal protein L9 may act as a regulator of bypassing, in particular in the context of polysomes. We show that polysome formation reduces the bypassing efficiency, whereas the deletion of the L9 does not increase bypassing in vitro. Our work reveals unforeseen details of the bypassing mechanism and regulation and opens up new perspectives for studies of recoding events.
Keywords: Translation; Ribosome; Recoding; Bypassing mechanism; messenger RNA; elongation factor G