Translation Initiation Rate Determines the Impact of Ribosome Stalling on Bacterial Protein Synthesis

Steven J Hersch,Sara Elgamal,Assaf Katz,Michael Ibba,William Wiley Navarre

doi:10.1074/jbc.m114.593277

Steven J Hersch, Sara Elgamal + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m114.593277

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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.

Highlights

Elongation factor P (EF-P) rescues ribosomes stalled at consecutive prolines; not all proteins with polyprolines show elongation factor P (EF-P)-dependent expression
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
We generated serial swaps of increasing length from the 5Ј end of the mRNA transcript to the PPG motif or in the reverse direction (Fig. 2). Results from this analysis demonstrated that the coding region beyond 12 codons upstream of the PPG motif had no additional reversal of EF-P dependence, even when the entire N terminus from the ATG start codon to the polyproline motif was swapped

Summary

Background

Elongation factor P (EF-P) rescues ribosomes stalled at consecutive prolines; not all proteins with polyprolines show EF-P-dependent expression. 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. Proteomic analysis by amino acid isotope labeling (SILAC) in both of these species has demonstrated that a large percentage of polyproline-containing proteins are unaffected by the absence of EF-P [29, 30] This divergent impact of specific stall sequences on protein levels establishes polyproline motifs as an excellent model for examining the interplay between elongation stalls and cellular protein synthesis. Our data indicate that not all polyproline motifs induce stalling to equal degrees and that EF-P dependence is only observed when a given polyproline sequence impacts the rate of synthesis more than translation initiation

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION