Genetic identification of nascent peptides that induce ribosome stalling.
Several nascent peptides stall ribosomes during their own translation in both prokaryotes and eukaryotes. Leader peptides that induce stalling can regulate downstream gene expression. Interestingly, stalling peptides show little sequence similarity and interact with the ribosome through distinct mechanisms. To explore the scope of regulation by stalling peptides and to better understand the mechanism of stalling, we identified and characterized new examples from random libraries. We created a genetic selection that ties the life of Escherichia coli cells to stalling at a specific site. This selection relies on the natural bacterial system that rescues arrested ribosomes. We altered transfer-messenger RNA, a key component of this rescue system, to direct the completion of a necessary protein if and only if stalling occurs. We identified three classes of stalling peptides: C-terminal Pro residues, SecM-like peptides, and the novel stalling sequence FXXYXIWPP. Like the leader peptides SecM and TnaC, the FXXYXIWPP peptide induces stalling efficiently by inhibiting peptidyl transfer. The nascent peptide exit tunnel and peptidyltransferase center are implicated in this stalling event, although mutations in the ribosome affect stalling on SecM and FXXYXIWPP differently. We conclude that ribosome stalling can be caused by numerous sequences and is more common than previously believed.
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, USA.
Douglas R
DR
Tanner
Daniel A
DA
Cariello
Christopher J
CJ
Woolstenhulme
Mark A
MA
Broadbent
Allen R
AR
Buskirk
D000595:Amino Acid Sequence; D000818:Animals; D015964:Gene Expression Regulation, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010441:Peptide Chain Elongation, Translational; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D012641:Selection, Genetic
D019151:Peptide Library; D010455:Peptides; D011993:Recombinant Fusion Proteins