Translation initiation rate determines the impact of ribosome stalling on bacterial protein synthesis

J Biol Chem. 2014 Oct 10;289(41):28160-71. doi: 10.1074/jbc.M114.593277. Epub 2014 Aug 22.


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.

Keywords: ATP Synthase; Bacterial Pathogenesis; Gene Regulation; Ribosome; Translation Elongation Factor; Translation Initiation; mRNA.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5' Untranslated Regions
  • Base Sequence
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Genes, Reporter
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Kinetics
  • Models, Genetic
  • Molecular Sequence Data
  • Peptide Chain Elongation, Translational / genetics*
  • Peptide Chain Initiation, Translational / genetics*
  • Peptides / genetics
  • Peptides / metabolism
  • Ribosomes / genetics*
  • Ribosomes / metabolism
  • Salmonella typhimurium / genetics*
  • Salmonella typhimurium / metabolism


  • 5' Untranslated Regions
  • Peptides
  • Green Fluorescent Proteins
  • polyproline