Competition between SsrA tagging and translational termination at weak stop codons in <i>Escherichia coli</i>

Collier, Justine; Binet, Emmanuelle; Bouloc, Philippe

doi:10.1046/j.1365-2958.2002.03045.x

Cited by 55 publications

(63 citation statements)

References 45 publications

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“…The 5Ј-truncated secM gene was amplified from the chromosome using primers 5Ј-ATACGCGTCG AC-CTCAGCAA CGCCGCCGAA-3Ј and 5Ј-CGGGATCCAA TAAAATCTCA AACGCC-3Ј. The PCR product digested by SalI and BamHI was cloned into the SalI-BamHI sites of pZA31-M2N (25), giving rise to p⌬ssM2SecM.…”

Section: Methodsmentioning

confidence: 99%

“…Identification of SsrA-H 7 -tagged ⌬ssSecM by Mass SpectrometryTryptic peptides from SsrA-H 7 -tagged ⌬ssSecM were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry according to a described protocol (25). Strain PhB2431 (with a slyD mutation to avoid sample contamination by the histidine-rich peptidyl cis,trans-isomerase SlyD) containing p⌬ssSecM was used for identification of SsrA-H 7 -tagged ⌬ssSecM.…”

Section: Phb2469mentioning

confidence: 99%

“…1A, lane 2) or when SsrA tagging was deficient (lane 5) and is likely the SsrA-tagged ⌬ssM2SecM product. SsrA-tagged proteins can also be stabilized by means of a modified SsrA (SsrA-H 7 ) encoding the peptide tag ANDENYHHHHHHH, which is not recognized by proteases (25). In an ssrAH7 background expressing ⌬ssM2SecM, two translation products were detected using anti-FLAG antibody M2.…”

Section: Elongation-arrested Secm Is Ssra-tagged-elongation Of Secm Tmentioning

confidence: 99%

“…Trans-Translation of secM Occurs at Codons Expressing the Arrest Sequence-SsrA-H 7 allows affinity purification of SsrA-H 7 -tagged proteins (25). To identify the SsrA tagging sites of SecM, we purified SsrA-H 7 -tagged proteins under conditions maximizing SecM elongation arrest.…”

Section: Elongation-arrested Secm Is Ssra-tagged-elongation Of Secm Tmentioning

confidence: 99%

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SsrA Tagging of Escherichia coli SecM at Its Translation Arrest Sequence

Collier

Bohn

Bouloc

2004

Journal of Biological Chemistry

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SecM is expressed from the secM-secA operon and activates the expression of secA in response to secretion defects. The 3 -end of secM encodes an "arrest sequence," which can interact with the ribosomal exit tunnel, preventing complete secM translation under secretion-defective conditions. In a cis-acting manner, ribosome stalling enhances secA translation. Pro 166 is the last residue incorporated when SecM elongation is arrested. We report that secretion deficiencies lead to SsrA tagging of SecM after Pro 166 , Gly 165 , and likely Arg 163 . Northern blot analysis revealed the presence of a truncated secM transcript, likely issued from a secMsecA cleavage. The level of secM transcripts was decreased either when secM translation was totally prevented or when Pro 166 was mutated. However, the accumulation of a truncated secM transcript required secM translation and was prevented when the SecM arrest sequence was inactivated by a point mutation changing Pro 166 to Ala. We suggest that ribosome pausing at the site encoding the arrest sequence is required for formation of the truncated secM mRNA. SsrA tagging affected neither the presence of the secM mRNA nor secA expression, even under translocation-defective conditions. It is therefore likely that SsrA tagging of SecM occurs only after cleavage of secM-secA mRNA within the secM open reading frame encoding the SecM arrest sequence. Accumulation of transcripts expressing arrested SecM generated growth inhibition that was alleviated by the SsrA tagging system. Therefore, SsrA tagging of SecM would rescue ribosomes to avoid excessive jamming of the translation apparatus on stop-less secM mRNA. Nascent proteins emerge from the 50 S ribosomal subunit through an exit tunnel with a length of ϳ100 Å and containing 35-40 amino acids of the elongating polypeptide. Its narrowness prevents peptide folding, and certain sequences from nascent peptides can interact with ribosomal elements of the tunnel to affect protein biosynthesis. The ribosome is therefore sensitive to the composition of the amino acid chain it is synthesizing, and the exit tunnel contributes to the dynamics of protein elongation (1). In Escherichia coli, this is exemplified by a sequence of the periplasmic protein SecM (secretion monitor) at a position close to its C terminus called the arrest sequence, which interacts with the ribosomal exit tunnel and causes elongation arrest within the ribosome (Refs. 2 and 3; for review, see Refs. 4 and 5). Ribosomal arrest is a key element in the regulation of SecA, an ATPase that targets protein precursors to the SecYEG core translocon for secretion. SecA is expressed from the secM-secA-mutT operon; its translation is up-regulated by a block in SecM secretion in a cis-specific manner (2, 6). A secondary structure encompassing the secA Shine-Dalgarno sequence of the secM-secA-mutT RNA can conditionally inhibit secA translation. Due to its arrest sequence, secM translation stalls when its N-terminal signal sequence is not "pulled" by the protein export machinery (7,8). Pau...

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Section: Methodsmentioning

confidence: 99%

Section: Phb2469mentioning

confidence: 99%

Section: Elongation-arrested Secm Is Ssra-tagged-elongation Of Secm Tmentioning

confidence: 99%

Section: Elongation-arrested Secm Is Ssra-tagged-elongation Of Secm Tmentioning

confidence: 99%

See 2 more Smart Citations

SsrA Tagging of Escherichia coli SecM at Its Translation Arrest Sequence

Collier

Bohn

Bouloc

2004

Journal of Biological Chemistry

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“…These sites can be (1) clusters of rare codons, 27 (2) weak termination codons. [28][29][30] They can also be the result of a flawed co-translational process, such as wrong protein folding or secretion; then, trans-translation is necessary to relieve the subsequent translational arrests, whatever the codon context. 3 The mechanism by which trans-translation is activated on these full-length mRNAs has been elusive until the discovery of the role played by the bacterial toxin RelE in inhibiting protein synthesis under nutrient deprivation conditions.…”

Section: Smpb As the Handyman Of Tmrna During Trans-translationmentioning

confidence: 99%