Translational stalling at polyproline stretches is modulated by the sequence context upstream of the stall site

Abstract

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.

High-Precision Analysis of Translational Pausing by Ribosome Profiling in Bacteria Lacking EFP

“…As a further refinement of our analysis of pausing at PP(X) motifs, we found that the residue just upstream, Z in ZPP(X), has a small effect on the average pause score (Figure 3B). In general, motifs associated with high pausing scores have Lys, Glu, Ile, Val, His, Arg, and Pro at the Z (−3) position and were observed to reduce lacZ expression in a reporter system (Starosta et al, 2014a). We note that the presence of Arg at the −3 position enhances stalling in the RXP(P) motif characterized in our previous study (Woolstenhulme et al, 2013) and at the RPPP pause in recG in Figure 2A.…”

The general mode of translation inhibition by macrolide antibiotics

“…3B) is reminiscent of proline motifs that are poorly translated even by the drug-free ribosomes (22,23). Like with macrolide-dependent arrest, ribosome stalling at the proline motifs is influenced by the nascent chain residues proximal to the C terminus (24,25). On the other hand, some other short motifs, which are known to stall drug-free ribosomes (26), were not enriched in the sites of macrolide-dependent stalling.…”

Distinct tRNA Accommodation Intermediates Observed on the Ribosome with the Antibiotics Hygromycin A and A201A

“…Binding of HygA, A201A and erythromycin (ERY) to an empty E. coli ribosome was monitored using a competition assay with radiolabelled [ 14 C]-erythromycin (170 dpm/pmol) as described before (Karahalios et al, 2006; Petropoulos et al, 2009; Starosta et al, 2010). The position of the ribosome on the mRNA was monitored using a toe-printing assay based on the in vitro coupled transcription-translation system using the PURExpress Δaa ΔtRNA kit (New England Biolabs) as described previously (Starosta et al, 2014). Further details on the in vitro translation and binding assays are provided in the Supplemental Experimental Procedures…”