Mechanisms of programmed ribosomal -1 frameshifting in bacteria

by Neva Caliskan
Doctoral thesis
Date of Examination:2013-05-29
Date of issue:2014-07-15
Advisor:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Holger Stark
Referee:Prof. Dr. Ralf Ficner
Persistent Address: http://dx.doi.org/10.53846/goediss-4534

 

 

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Abstract

English

Translation of proteins must be accurate to synthesize functional proteins. However, in some cases gene information is reprogrammed which overwrites the normal decoding rules of translation. One of these cases is programmed ribosomal frameshifting, in which the ribosome moves to an alternative, the overlapping reading frame upon encountering specific signals embedded in the mRNAs. A variety of models has been proposed to explain -1 frameshifting mechanistically. However, it is unclear at which step of the elongation cycle -1 frameshifting takes place. Here we have examined -1 ribosomal frameshifting in real-time by rapid kinetics using a minimal IBV 1a/1b frameshifting system which leads to ~70% frameshifting in vivo measured by a dual luciferase assay. We analyzed the formation of the zero- and -1-frame peptides and compared the efficiency of translation of the frameshifting and control mRNAs. We showed that frameshifting takes place following the incorporation of Lys corresponding to the second codon of the slippery sequence, immediately preceding the recoding site. We show that the efficiency of frameshifting does not depend on the competition or availability of tRNAs binding in zero or -1 frames. The presence of the slippery site alone enhances the rapid decoding from the -1 reading frame. The presence of the pseudoknot alone strongly delays decoding of both zero- and -1 frames following the Lys codon. The analysis of 50S translocation by a time-resolved puromycin assay and by a fluorescence based tRNA translocation assay suggests that the translocation on the 50S subunit is independent of the presence of frameshifting elements, leaving 30S translocation as the only step for frameshifting. Using FRET between the ribosome and EF-G we showed that EF-G remains bound longer on the ribosomes during frameshifting. Together, these results suggest the mechanism and exact timing of -1 frameshifting.
Keywords: Ribosome; Translation; -1 frameshifting; Pseudoknot; IBV
 
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