Ribosomes are the protein synthesizing factories of the cell, polymerizing polypeptide chains from their constituent amino acids. However, distinct combinations of amino acids, such as polyproline stretches, cannot be efficiently polymerized by ribosomes, leading to translational stalling. The stalled ribosomes are rescued by the translational elongation factor P (EF-P), which by stimulating peptide-bond formation allows translation to resume. Using metabolic stable isotope labeling and mass spectrometry, we demonstrate in vivo that EF-P is important for expression of not only polyproline-containing proteins, but also for specific subsets of proteins containing diprolyl motifs (XPP/PPX). Together with a systematic in vitro and in vivo analysis, we provide a distinct hierarchy of stalling triplets, ranging from strong stallers, such as PPP, DPP, and PPN to weak stallers, such as CPP, PPR, and PPH, all of which are substrates for EF-P. These findings provide mechanistic insight into how the characteristics of the specific amino acid substrates influence the fundamentals of peptide bond formation.

The polymerization of amino acids into proteins occurs on ribosomes, with the rate influenced by the amino acids being polymerized. The imino acid proline is a poor donor and acceptor for peptide-bond formation, such that translational stalling occurs when three or more consecutive prolines (PPP) are encountered by the ribosome. In bacteria, stalling at PPP motifs is rescued by the elongation factor P (EF-P). Using SILAC mass spectrometry of Escherichia coli strains, we identified a subset of PPP-containing proteins for which the expression patterns remained unchanged or even appeared up-regulated in the absence of EF-P. Subsequent analysis using in vitro and in vivo reporter assays revealed that stalling at PPP motifs is influenced by the sequence context upstream of the stall site. Specifically, the presence of amino acids such as Cys and Thr preceding the stall site suppressed stalling at PPP motifs, whereas amino acids like Arg and His promoted stalling. In addition to providing fundamental insight into the mechanism of peptide-bond formation, our findings suggest how the sequence context of polyproline-containing proteins can be modulated to maximize the efficiency and yield of protein production.

No abstract

The ribosome is a large, complex and dynamic ribonu-cleoprotein particle consisting of a large and a small subunit. In Escherichia coli, the large (50S) subunit contains two rRNA molecules (23S and 5S rRNA) and 33 ribosomal proteins (r-proteins), whereas the small (30S) subunit contains one rRNA molecule (16S rRNA) and 21 r-proteins [1]. Assembly of ribosomes is a complex and highly coordinated process, which is initiated during rRNA transcription [2] and involves processing, modification and folding of rRNA and r-proteins, as well as their association to form functional ribosomal subunits. Many extraribosomal factors are involved in the ribosome assembly process, especially in eukaryotes [3]. In bacteria, the number of extraribosomal components so far identified as being involved in ribosome assembly is smaller [4,5]. The absence of the RNA modification enzymes RluD [6] and RlmJ [7], the RNA helicases DeaD ⁄ CsdA [8] and SrmB [9], the cold-shock protein RbfA [10,11] and the heat-shock proteins DnaK [12-14] and GroEL [15] leads to the accumulation of ribosome assembly precursor particles, suggesting involvement of these proteins in ribosome assembly. A group of proteins known as 'small GTPases' have also been shown to participate in the assembly of the small subunit (Era [10,11] and RsgA [16]) or the large subunit (CgtA E ⁄ Obg [17,18] and EngA ⁄ Der [19]). The E. coli genome contains five genes for DEAD-box RNA helicases: deaD, dbpA, rhlB, rhlE and srmB [20]. RNA helicase DeaD ⁄ CsdA is involved in translation [21], RNA degradation [22] and ribosome assembly [8]. It has two names [23], but because CsdA (cold shock DEAD-box protein; named by Brandi et al. 1999 [23]) also designates the E. coli gene encoding cysteine sulfinate desulfinase (previously ygdJ-ygdK) [24], we prefer to use the original name DeaD (DEAD-box protein) [26]. Disruption of the deaD gene leads to no growth defect at 37 °C, whereas prominent growth defects, with long adaptation periods, are observed at temperatures below 30 °C [27]. Over-expression of RNA helicase RhlE suppresses the cold-sensitive growth defect of a strain lacking DeaD Ribosome subunit assembly in bacteria is a fast and efficient process. Among the nonribosomal proteins involved in ribosome biogenesis are RNA helicases. We describe ribosome biogenesis in Escherichia coli strains lacking RNA helicase DeaD (CsdA) or DbpA. Ribosome large subunit assembly intermediate particles (40S) accumulate at 25 °C and at 37 °C in the absence of DeaD but not without DbpA. 23S rRNA is incompletely processed in the 40S and 50S particles of the DeaD) strain. Pulse labeling showed that the 40S particles are converted nearly completely into functional ribosomes. The rate of large ribosomal subunit assembly was reduced about four times in DeaD-deficient cells. Functional activity tests of the ribosomal particles demonstrated that the final step of 50S assembly, the activation step, was affected when DeaD was not present. The results are compatible with the model that predicts multiple DeaD-catalyz...

Bacterial ribosomes stall on polyproline stretches and require the elongation factor P (EF-P) to relieve the arrest. Yet it remains unclear why evolution has favored the development of EF-P, rather than selecting against the occurrence of polyproline stretches in proteins. We have discovered that only a single polyproline stretch is invariant across all domains of life, namely, a proline triplet in ValS, the tRNA synthetase that charges tRNAVal with valine. Here we show that expression of ValS in vivo and in vitro requires EF-P and demonstrate that the proline triplet located in the active site of ValS is important for efficient charging of tRNAVal with valine, preventing formation of mischarged Thr-tRNAVal, as well as for efficient growth of E. coli in vivo. We suggest that the critical role of the proline triplet for ValS activity may explain why bacterial cells co-evolved the EF-P rescue system.