Abstract

Although the ribosome is a very general catalyst, it cannot synthesize all protein sequences equally well. Certain proteins are capable of stalling the ribosome during their own synthesis. Stalling events are used by both prokaryotic and eukaryotic cells to regulate gene expression. Characterization of natural stalling peptides shows that only a few strategically placed amino acids are needed to inactivate the ribosome. These motifs share little sequence similarity suggesting that there are more stalling motifs yet to be discovered. Here we use two genetic selections in E. coli to discover novel stalling peptides and detail their subsequent characterization. Kinetic studies show that some of these nascent peptides dramatically inhibit rates of peptide release by release factors. We find that residues upstream of the minimal stalling motif can either enhance or suppress this effect. In other stalling motifs, such as polyproline sequences, peptidyl transfer to a subset of aminoacyl-tRNAs is inhibited. Translation factor EF-P alleviates pausing of the polyproline motifs, but has little or no effect on other stalling sequences. The EF-P ortholog eIF5A also alleviates pausing of polyproline sequences in yeast. Our studies show that short peptides sequences are capable of stalling the ribosome during elongation and termination through different mechanisms. These sequences are underrepresented in bacterial proteomes and show evidence of stalling on endogenous E. coli proteins.

Degree

PhD

College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry

Rights

http://lib.byu.edu/about/copyright/

Date Submitted

2014-03-01

Document Type

Dissertation

Handle

http://hdl.lib.byu.edu/1877/etd6795

Keywords

ribosome, translation, stalling, EF-P, eIF5A

Included in

Chemistry Commons

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