title: Translation initiation rate determines the impact of ribosome stalling on bacterial protein synthesis.
abstract: Ribosome stalling during translation can be caused by a number of characterized mechanisms. However, the impact of elongation stalls on protein levels is variable, and the reasons for this are often unclear. To investigate this relationship, we examined the bacterial translation elongation factor P (EF-P), which plays a critical role in rescuing ribosomes stalled at specific amino acid sequences including polyproline motifs. In previous proteomic analyses of both Salmonella and Escherichia coli efp mutants, it was evident that not all proteins containing a polyproline motif were dependent on EF-P for efficient expression in vivo. The α- and β-subunits of ATP synthase, AtpA and AtpD, are translated from the same mRNA transcript, and both contain a PPG motif; however, proteomic analysis revealed that AtpD levels are strongly dependent on EF-P, whereas AtpA levels are independent of EF-P. Using these model proteins, we systematically determined that EF-P dependence is strongly influenced by elements in the 5'-untranslated region of the mRNA. By mutating either the Shine-Dalgarno sequence or the start codon, we find that EF-P dependence correlates directly with the rate of translation initiation where strongly expressed proteins show the greatest dependence on EF-P. Our findings demonstrate that polyproline-induced stalls exert a net effect on protein levels only if they limit translation significantly more than initiation. This model can be generalized to explain why sequences that induce pauses in translation elongation to, for example, facilitate folding do not necessarily exact a penalty on the overall production of the protein.
authors.affiliation: From the Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada and.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Hersch
authors.affiliation: the Department of Microbiology, Ohio State University, Columbus, Ohio 43210.
authors.forename: Sara
authors.initials: S
authors.lastname: Elgamal
authors.affiliation: the Department of Microbiology, Ohio State University, Columbus, Ohio 43210.
authors.forename: Assaf
authors.initials: A
authors.lastname: Katz
authors.affiliation: the Department of Microbiology, Ohio State University, Columbus, Ohio 43210 ibba.1@osu.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Ibba
authors.affiliation: From the Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada and william.navarre@utoronto.ca.
authors.forename: William Wiley
authors.initials: WW
authors.lastname: Navarre
mesh_terms: D020121:5' Untranslated Regions; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010441:Peptide Chain Elongation, Translational / Q000235:genetics*; D010442:Peptide Chain Initiation, Translational / Q000235:genetics*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics* / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics* / Q000378:metabolism
chemical_list: D020121:5' Untranslated Regions; D010455:Peptides; D049452:Green Fluorescent Proteins; C011083:polyproline
keywords: ATP Synthase; Bacterial Pathogenesis; Gene Regulation; Ribosome; Translation Elongation Factor; Translation Initiation; mRNA