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.
Highlights
Ribosomes translate message encoded within mRNA into an amino acid sequence
Translation of LepA in the absence of EF-P led to appearance of two prominent bands; a ∼40 kDa peptidyl-tRNA band reflecting the mass with sites of stalling observed in the absence of EF-P or in the absence of the amino acid glutamine. (B,C) Autoradiograms of SDS-PAGE of in vitro translation reactions of (B) wild-type NlpD, 5:1NlpD and 10:1NlpD chimeras and (C) wild-type LepA, 5:1LepA and 10:1LepA chimeras
Using a series of in vitro and in vivo translation reporters, we revealed that the context upstream of the polyproline motif can influence the efficiency of stalling and modulate the dependence of the protein expression on EF-P
Summary
Ribosomes translate message encoded within mRNA into an amino acid sequence. The rate of amino acid polymerization varies for each amino acid, being significantly slower for proline (Pro). The imino rather than amino group determines Pro as a poor A-site acceptor of peptidyl moiety during peptide-bond formation [1,2], as well as poor donor when present in the P-site [3,4,5]. The translational stalling occurs when the peptidyl-Pro-Pro-tRNA is located in the P-site [3,7] and results from slow peptide-bond formation with the Pro-tRNA located in the A-site [3]. Translational stalling is observed at diprolyl motifs (XPPZ), with the strength of stalling influenced by the nature of X and Z amino acids flanking the proline residues [3,7,8]. While polyproline stretches produce the strongest translational stalling, ribosome stalling is observed with Asp and Ala preceding and/or with Trp, Asp, Asn and Gly following the diprolyl motif [3,7,8]
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