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Proline Residues at the C Terminus of Nascent Chains Induce SsrA Tagging during Translation Termination*210

  • Christopher S. Hayes
    Footnotes
    Affiliations
    Department of Biology Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
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  • Baundauna Bose
    Affiliations
    Department of Biology Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
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  • Robert T. Sauer
    Correspondence
    To whom correspondence should be addressed: Dept. of Biology, MIT, Rm. 68-571, 77 Massachusetts Ave., Cambridge, MA 02139. Tel.: 617-253-3163; Fax: 617-258-0673
    Affiliations
    Department of Biology Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
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  • Author Footnotes
    * This work was supported by Grant AI-16892 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    210 The on-line version of this article (available athttp://www.jbc.org) contains Supplemental Table IV).
    ‡ Supported by Fellowship DRG 1686 from the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation.
Open AccessPublished:July 08, 2002DOI:https://doi.org/10.1074/jbc.M205405200
      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 fromEscherichia 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.
      RF
      release factor
      RRF
      ribosome recycling factor
      Ni2+-NTA
      Ni2+-nitrilotriacetic acid
      MALDI
      matrix-assisted laser desorption/ionization
      IPTG
      isopropyl-1-thio-β-d-galactopyranoside
      Aze
      azetidine-2-carboxylic acid
      All eubacteria contain an RNA molecule known as SsrA or tmRNA that functions in the release of stalled ribosomes from mRNAs and in targeting the nascent polypeptides from such ribosomes for proteolysis (
      • Muto A.
      • Ushida C.
      • Himeno H.
      ,
      • Karzai A.W.
      • Roche E.D.
      • Sauer R.T.
      ). SsrA has been shown to act as both a transfer RNA and a messenger RNA (
      • Muto A.
      • Ushida C.
      • Himeno H.
      ,
      • Karzai A.W.
      • Roche E.D.
      • Sauer R.T.
      ,
      • Tu G.F.
      • Reid G.E.
      • Zhang J.G.
      • Moritz R.L.
      • Simpson R.J.
      ,
      • Keiler K.C.
      • Waller P.R.
      • Sauer R.T.
      ). SsrA has a domain that is very similar to a portion of tRNAAla and is aminoacylated with alanine at its 3′ end. SsrA also contains a short open reading frame, which inEscherichia coli encodes the sequence ANDENYALAA (
      • Tu G.F.
      • Reid G.E.
      • Zhang J.G.
      • Moritz R.L.
      • Simpson R.J.
      ). The tmRNA model of SsrA activity postulates that SsrA enters the empty A-site of stalled ribosomes and adds its charged alanine to the nascent polypeptide by transpeptidation. A conformational change then occurs that allows translation to shift from the original mRNA to the ANDENYALAA reading frame within SsrA. As a result, the stalled ribosome is freed from the problematic mRNA, and the SsrA-encoded peptide tag is added to the C terminus of the nascent polypeptide. The SsrA tag is recognized by multiple proteases in E. coli, resulting in rapid degradation of SsrA-tagged proteins (
      • Keiler K.C.
      • Waller P.R.
      • Sauer R.T.
      ,
      • Gottesman S.
      • Roche E.
      • Zhou Y.
      • Sauer R.T.
      ,
      • Herman C.
      • Thevenet D.
      • Bouloc P.
      • Walker G.C.
      • D'Ari R.
      ).
      The determinants of SsrA tagging are well understood in only a few cases. For example, SsrA tagging has been demonstrated for proteins synthesized from truncated messages lacking in-frame stop codons and at protein positions corresponding to rare codons (
      • Keiler K.C.
      • Waller P.R.
      • Sauer R.T.
      ,
      • Williams K.P.
      • Martindale K.A.
      • Bartel D.P.
      ,
      • Roche E.D.
      • Sauer R.T.
      ,
      • Abo T.
      • Inada T.
      • Ogawa K.
      • Aiba H.
      ,
      • Hayes C.S.
      • Bose B.
      • Sauer R.T.
      ). Ribosomes would be expected to stall at the 3′ end of the incomplete mRNA or at the rare codon mRNA position when the cognate charged tRNA was scarce. SsrA tagging of otherwise full-length proteins has also been observed, suggesting that ribosomes stall under some circumstances at stop codons (
      • Roche E.D.
      • Sauer R.T.
      ,
      • Hayes C.S.
      • Bose B.
      • Sauer R.T.
      ). The identity of the stop codon can affect SsrA tagging of full-length proteins, with less efficient stop codons leading to higher levels of tagging (
      • Hayes C.S.
      • Bose B.
      • Sauer R.T.
      ). Because termination of translation is a relatively slow process compared with translation elongation (
      • Freistroffer D.V.
      • Kwiatkowski M.
      • Buckingham R.H.
      • Ehrenberg M.
      ), SsrA and protein release factors probably compete for binding to the A-site, while the ribosome idles at an inefficient stop codon. However, SsrA tagging of full-length proteins encoded by genes with efficient stop signals has also been observed (
      • Roche E.D.
      • Sauer R.T.
      ). For example, the intact E. coli YbeL protein is tagged (
      • Roche E.D.
      • Sauer R.T.
      ) even though the ybeL coding region ends with an extended UAAU stop signal which is thought to be the most efficient translation termination signal in E. coli (
      • Poole E.S.
      • Brown C.M.
      • Tate W.P.
      ,
      • Tate W.P.
      • Mannering S.A.
      ).
      In this paper, we dissect the YbeL system to identify the determinants that lead to its efficient tagging by SsrA. Mutational analyses of the last two residues of YbeL demonstrated that the proline residue at the C terminus and the identity of the penultimate residue are major determinants of SsrA tagging. Moreover, substituting proline for the C-terminal residue of thioredoxin, a protein that is not normally tagged, resulted in efficient SsrA-tagging of this variant. Tagging of full-length YbeL was modulated by the identity of the stop codon and by the cellular level of protein release factors. The incorporation of proline analogs into YbeL significantly affected SsrA tagging at the stop codon, suggesting that the structure of the nascent polypeptide is a primary determinant of SsrA tagging of YbeL.

      DISCUSSION

      The results presented here show that several factors contribute to tagging of full-length YbeL by SsrA, including stop codon identity, release factor activity, and the amino acid sequence of the nascent peptide. These observations are consistent with a model in which inefficient translation termination at the ybeL stop codon permits SsrA to compete with RF-1 and RF-2 for entry into the ribosomal A-site, leading to higher levels of full-length protein tagging. The RF-1 and RF-2 proteins are present in ∼10-fold and 50-fold excess, respectively, over SsrA RNA in E. coli during log-phase growth (
      • Adamski F.M.
      • McCaughan K.K.
      • Jorgensen F.
      • Kurland C.G.
      • Tate W.P.
      ). The affinities of these protein release factors for the A-site depends on the identities of the stop codon and following nucleotide (
      • Pavlov M.Y.
      • Freistroffer D.V.
      • Dincbas V.
      • MacDougall J.
      • Buckingham R.H.
      • Ehrenberg M.
      ). Because SsrA does not appear to have an anticodon stem-loop (
      • Felden B.
      • Himeno H.
      • Muto A.
      • McCutcheon J.P.
      • Atkins J.F.
      • Gesteland R.F.
      ,
      • Williams K.P.
      • Bartel D.P.
      ), we assume that its affinity for the A-site is independent of the stop signal. As a result, the competition model predicts the observed dependence of full-length YbeL tagging on stop-codon identity and release factor levels. Moreover, if peptidyl-tRNA hydrolysis is slow for YbeL, then several rounds of release factor association and dissociation may be required before normal termination of translation. This would allow SsrA multiple opportunities to compete for A-site binding.
      In principle, the release factor/SsrA competition model should operate for termination at any stop codon and, by itself, does not explain why full-length YbeL is tagged at such a high level. Several lines of evidence indicate that the last two residues of the nascent YbeL polypeptide also play a critical role in determining the level of tagging. High levels of tagging occurred only when proline occupied the C-terminal position, irrespective of the proline codon and decoding tRNAPro species used. Moreover, the incorporation of certain proline analogs, such as azetidine-2-carboxylic acid, led to dramatic changes in the levels of YbeL tagging. Because the same mRNA codons and tRNAPro molecules direct the incorporation of proline and its analogs, this result provides the strongest evidence that it is the C-terminal proline, rather than mRNA or tRNA determinants, that causes full-length protein tagging. The identity of the penultimate residue of the nascent YbeL chain was also important for full-length tagging, with Asp, Glu, Pro, Ile, and Val leading to the highest tagging levels.
      How might the identity of the C-terminal residues of the nascent polypeptide affect translation termination and/or SsrA tagging? Interactions mediated by these amino acids could decrease the affinity of release factors for the A-site, increase the affinity of SsrA for the A-site, or slow hydrolysis of the peptidyl-tRNA bond. The ester bond of prolyl-tRNA hydrolyzes more rapidly than other aminoacyl-tRNAs (
      • Hentzen D.
      • Mandel P.
      • Garel J.-P.
      ), suggesting that the YbeL-Pro-tRNAPro linkage should not be more resistant to hydrolysis from a chemical perspective. Although this observation does not rule out the slow hydrolysis model, it favors versions that focus on conformational effects of the C-terminal residues in positioning the ester bond for catalyzed hydrolysis. Effects mediated via release factors are supported by studies that conclude that the nascent peptide chain can regulate ribosome activity, including translation termination (reviewed in Ref.
      • Lovett P.S.
      • Rogers E.J.
      ). Indeed, Isaksson and co-workers (
      • Mottagui-Tabar S.
      • Bjornsson A.
      • Isaksson L.A.
      ,
      • Bjornsson A.
      • Mottagui-Tabar S.
      • Isaksson L.A.
      ,
      • Mottagui-Tabar S.
      • Isaksson L.A.
      ) have used termination read-through assays to show that the last two residues of proteins influence translation termination, with the Asp-Pro sequence resulting in the most severe inhibition of E. coli translation termination. Although no read-through or frameshift products were observed in the studies reported here, the C-terminal Asp-Pro dipeptide resulted in one of the highest levels of SsrA tagging of full-length YbeL.
      Proline is unique among naturally occurring protein residues in being an imino acid and having its φ dihedral angle constrained to roughly −60° by the pyrrolidine ring. Neither of these properties by themselves explain why proline causes tagging because the azetidine analog (Aze), which does not cause tagging, also lacks an amide proton and has a constrained φ angle similar to proline (
      • Zagari A.
      • Nemethy G.
      • Scheraga H.A.
      ,
      • Zagari A.
      • Nemethy G.
      • Scheraga H.A.
      ). Is tagging related to the fact that the peptide bond of Xaa-Pro dipeptides can isomerize from the trans to the cis conformation at faster rates than dipeptides lacking proline? Cis-transisomerization in solution occurs on a time scale of minutes, whereas translation termination is complete in a few seconds (
      • Freistroffer D.V.
      • Kwiatkowski M.
      • Buckingham R.H.
      • Ehrenberg M.
      ). Moreover, the identity of the residue before proline influences isomerization (
      • Reimer U.
      • Scherer G.
      • Drewello M.
      • Kruber S.
      • Schutkowski M.
      • Fischer G.
      ) in a fashion that is not correlated with the effects of the (−2) residue on YbeL tagging. For example, Tyr-Pro, Phe-Pro, and Trp-Pro peptide bonds are most likely to adopt the cis conformation in solution (
      • Reimer U.
      • Scherer G.
      • Drewello M.
      • Kruber S.
      • Schutkowski M.
      • Fischer G.
      ) but do not result in the highest levels of YbeL tagging. Finally, both the isomerization rate and equilibrium population of the cis isomer is higher for Xaa-Aze than Xaa-Pro peptide bonds (
      • Kern D.
      • Schutkowski M.
      • Drakenberg T.
      ), and yet Xaa-Aze dipeptides at the C terminus of the nascent chain do not cause tagging. These observations argue against isomerization being an important determinant of full-length tagging, but do not rule out this model, becausecis-trans isomerization could be very different in solution than on the ribosome. Energy calculations show that Xaa-Aze and Xaa-Pro have roughly similar conformational preferences but the smaller four-member ring of azetidine is predicted to lead to slightly more flexibility (
      • Zagari A.
      • Nemethy G.
      • Scheraga H.A.
      ,
      • Zagari A.
      • Nemethy G.
      • Scheraga H.A.
      ). This additional flexibility may permit the Xaa-Aze nascent chains to avoid steric clashes with release factors or help position the ester bond for hydrolysis. The role of the (−2) residue in Pro-dependent tagging may also result from conformational effects on the C-terminal residue and/or from direct interactions between the (−2) side chain and the termination machinery.
      In our studies, Asp-Pro and Pro-Pro resulted in the highest relative levels of tagged to untagged YbeL protein. For the Pro-Pro sequence, for example, roughly 40% of the total YbeL protein was tagged (Fig.4A). Asp-Pro and Pro-Pro are highly underrepresented at the C terminus of proteins in most bacteria (Table IV; Supplemental Material). In E. coli, only one of 4355 proteins ends with Asp-Pro (10 expected) and none end with Pro-Pro (7 expected). Among 23,558 proteins in the Bacillus/Clostridium family, just one protein ends with Asp-Pro (43 expected) and four end with Pro-Pro (20 expected). Some bacteria, however, show only a slight bias against these C-terminal dipeptides. In the gamma proteobacterium Xylella fastidiosa, for example, six proteins end with Asp-Pro (8 expected) and six terminate with Pro-Pro (8 expected). In fact, Xaa-Pro dipeptides are found more frequently at the C terminus (5.7%) than at internal positions (5.0%) in X. fastidiosa. By contrast, significant biases against C-terminal Xaa-Pro sequences are observed for many other gamma proteobacteria, including E. coli(2.9% terminal/4.5% internal), Buchnera sp. APS (0.7% terminal/3.0% internal), Haemophilus influenzae(1.7% terminal/3.7% internal), and Pasteurella multicoda(1.9% terminal/3.9% internal). The translation-termination machinery or SsrA system of X. fastidiosa may have evolved to avoid interactions with C-terminal prolines in nascent polypeptides. Interestingly, archaeabacteria, which have no SsrA system and have protein release factors that are unrelated to the bacterial RF-1 and RF-2 proteins, show no systematic biases against C-terminal prolines.
      In previous work (
      • Hayes C.S.
      • Bose B.
      • Sauer R.T.
      ), we showed that tagging of E. coliribokinase (RbsK) at or near the C terminus depended on the presence of a rare arginine codon at the mRNA position encoding the C-terminal residue, as well as the levels of the cognate charged tRNAArg, and the efficiency of the terminal signal. In unpublished work,
      C. S. Hayes, B. Bose, and R. T. Sauer, unpublished results.
      we have found that RbsK tagging increases in a RF-3 deletion strain. Thus, tagging of both YbeL and RbsK has the same dependence on the stop codon and the levels of release factors, as would be expected if SsrA competes with release factors. However, RbsK tagging depends in large part on the scarcity of the rare tRNA whereas full-length YbeL tagging is largely dependent on the chemical nature of the C-terminal residues of the nascent chain. Full-length tagging of the λ cI repressor (
      • Roche E.D.
      • Sauer R.T.
      ) and UDP-galactose-4-epimerase2 have also been observed but neither of these systems involves a C-terminal proline or a rare C-terminal codon. Perhaps other C-terminal amino acid sequences can also influence tagging via interactions with release factors or SsrA.
      Full-length protein tagging seems inherently wasteful. Why should some fraction of protein synthesis result in SsrA-tagged proteins that are rapidly degraded shortly after release from the ribosome? In a few cases, this may be an inevitable consequence of a functional requirement for a specific C-terminal sequence like Asp-Pro or Pro-Pro. In other instances, such as regulatory leader peptides, the full-length protein/peptide may have no function other than influencing downstream gene expression and thus recycling of its amino acids would be beneficial. Three of the ten leader peptides in E. coli end with Xaa-Pro sequences, and another leader coding sequence ends with two consecutive rare arginine codons. Still other proteins may use inefficient translation termination as a final opportunity to regulate gene expression at the level of protein synthesis and degradation.

      Acknowledgments

      We thank Drew Endy, Sean Moore, and Peter Chivers for helpful discussions.

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