Proline Residues at the C Terminus of Nascent Chains Induce SsrA Tagging during Translation Termination

Abstract

The SsrA or tmRNA quality control system relieves ribosome stalling and directs the addition of a degradation tag to the C terminus of the nascent chain. In some instances, SsrA tagging of otherwise full-length proteins occurs when the ribosome pauses at stop codons during normal translation termination. Here, the identities of the C-terminal residues of the nascent chain are shown to play an important role in full-length protein tagging. Specifically, a subset of C-terminal Xaa-Pro sequences caused SsrA tagging of the full-length YbeL protein from Escherichia coli. This tagging increased when a less efficient stop codon was used, increased in cells lacking protein release factor-3, and decreased when protein release factor-1 was overexpressed. Incorporation of the analog azetidine-2-carboxylic acid in place of proline suppressed tagging, whereas incorporation of 3,4-dehydroproline increased SsrA tagging of full-length YbeL. These results suggest that the detailed chemical or conformational properties of the C-terminal residues of the nascent polypeptide can affect the rate of translation termination, thereby influencing ribosome pausing and SsrA tagging at stop codons.

Stepping Transfer Messenger RNA through the Ribosome

“…The most intense fragment ion signal was obtained from the y 6 ion at m/z 750.38 Ϯ 0. 30. preceding the stop codon were changed to code for Asp and Pro, which were shown to decrease termination efficiency and provoke ribosome pausing (37). In our experiments these mutant tmRNAs were the only tmRNAs present in a strain that contained a thermosensitive RF2 inactivated at 42°C.…”

Making the most of affinity tags

“…Sequence determinants at both the N-and C-termini of proteins can influence their rate of degradation [2][3][4]. Therefore, in some cases affinity tags might improve the yield of recombinant proteins by rendering them more resistant to intracellular proteolysis [5].…”

Modulation of the Rate of Peptidyl Transfer on the Ribosome by the Nature of Substrates

“…The translating ribosome therefore remains attached to the leader transcript, where it blocks Rho factor binding and subsequent transcription termination (65). Furthermore, the presence of the C-terminal Pro in peptides sometimes leads to "full-length tagging" by the SsrA tagging system (25). These effects can be attributed to slow peptide release during termination of protein synthesis, when peptidyl-tRNA in the P site is hydrolyzed in the peptidyltransferase center with the help of termination factors.…”

“…PeptidyltRNA with a C-terminal Pro residue was reported to be exceptionally slow in peptidyl transfer to Pmn on the ribosome (22,23). Furthermore, the C-terminal Pro is essential for the tryptophan-induced ribosome stalling at the end of the tnaC open reading frame (24) or peptide tagging by SsrA (25). However, the effect of different amino acids at the C-terminal position in peptidyl-tRNA on the reaction with aminoacyl-tRNA as A-site substrate has not been studied.…”