Prolyl-tRNAPro in the A-site of SecM-arrested Ribosomes Inhibits the Recruitment of Transfer-messenger RNA

Garza-Sánchez, Fernando; Janssen, Brian D.; Hayes, Christopher S.

doi:10.1074/jbc.m608052200

Cited by 90 publications

(105 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…5a). These could either represent species with tRNA still bound to the nascent chain or, possibly, covalently attached tmRNA added by the SsrA rescue system (19). When introduced into the LepB[10D, L ϭ 42] construct, the Ser Ϫ2 3 Pro mutation yielded f FL Ϸ 0.3 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Exploration of the Arrest Peptide Sequence Space Reveals Arrest-enhanced Variants

Cymer

Hedman

Ismail

et al. 2015

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background:The stalling efficiency of translational arrest peptides (APs) is sensitive to mechanical pulling forces on the nascent chain. Results: We identify new APs with enhanced stalling efficiency. Conclusion: Mechanical pulling forces reduce stalling induced by diproline stretches in efp Ϫ cells. Significance:Our results provide new insights into AP-induced translational stalling and offer new in vivo force sensors.Translational arrest peptides (APs) are short stretches of polypeptides that induce translational stalling when synthesized on a ribosome. Mechanical pulling forces acting on the nascent chain can weaken or even abolish stalling. APs can therefore be used as in vivo force sensors, making it possible to measure the forces that act on a nascent chain during translation with single-residue resolution. It is also possible to score the relative strengths of APs by subjecting them to a given pulling force and ranking them according to stalling efficiency. Using the latter approach, we now report an extensive mutagenesis scan of a strong mutant variant of the Mannheimia succiniciproducens SecM AP and identify mutations that further increase the stalling efficiency. Combining three such mutations, we designed an AP that withstands the strongest pulling force we are able to generate at present. We further show that diproline stretches in a nascent protein act as very strong APs when translation is carried out in the absence of elongation factor P. Our findings highlight critical residues in APs, show that certain amino acid sequences induce very strong translational arrest and provide a toolbox of APs of varying strengths that can be used for in vivo force measurements.During protein synthesis, the nascent polypeptide chain moves through the ϳ100-Å-long exit tunnel in the large ribosomal subunit (1). Strong interactions between the nascent chain and the tunnel might adversely affect protein synthesis and can even lead to complete blockage of the ribosome, a mechanism exploited by many antibiotics (2). It has therefore been postulated that the ribosome exit tunnel has a "Teflonlike" surface that minimizes interactions with the nascent chain to avoid adverse effects on translation (1, 3).Nevertheless, some nascent chain segments are able to interact with the ribosomal exit tunnel in ways that block or slow down translation (4, 5). In bacteria, such translational arrest peptides (APs) 2 are often used to regulate the translation of downstream open reading frames in polycistronic mRNAs (6, 7). APs interact with distinct ribosomal RNA and protein components within the ribosomal exit tunnel (8), inducing conformations at the ribosome active site that can block the peptidyl transfer reaction (9 -11).In the case of the AP-containing SecM protein in Escherichia coli, the arrest of nascent chain elongation can be overcome by the activity of the motor protein SecA, which presumably breaks the AP-tunnel interactions by mechanically pulling on the nascent chain (12). On the basis of this notion, we hypothesized that...

show abstract

Section: Resultsmentioning

confidence: 99%

Exploration of the Arrest Peptide Sequence Space Reveals Arrest-enhanced Variants

Cymer

Hedman

Ismail

et al. 2015

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Competition cocultures were harvested into an equal volume of ice-cold methanol, and the cells were collected by centrifugation and frozen at -80°C. Total RNA was extracted with guanidinium isothiocyanate (GITC)-phenol as described (40,41) and resuspended in 10 mM sodium acetate (pH 5.2), which stabilizes the aminoacyl linkage (42). Total RNA (5 μg) was analyzed by Northern blot hybridization using [ 32 P]-labeled oligonucleotides 3894, CH452, CH800, CH801, and CH1417 as probes (16).…”

Section: Methodsmentioning

confidence: 99%

Activation of contact-dependent antibacterial tRNase toxins by translation elongation factors

Jones

Garza-Sánchez

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Contact-dependent growth inhibition (CDI) is a mechanism by which bacteria exchange toxins via direct cell-to-cell contact. CDI systems are distributed widely among Gram-negative pathogens and are thought to mediate interstrain competition. Here, we describe tsf mutations that alter the coiled-coil domain of elongation factor Ts (EF-Ts) and confer resistance to the CdiA-CT EC869 tRNase toxin from enterohemorrhagic Escherichia coli EC869. Although EF-Ts is required for toxicity in vivo, our results indicate that it is dispensable for tRNase activity in vitro. We find that CdiA-CT EC869 binds to elongation factor Tu (EF-Tu) with high affinity and this interaction is critical for nuclease activity. Moreover, in vitro tRNase activity is GTP-dependent, suggesting that CdiA-CT EC869 only cleaves tRNA in the context of translationally active GTP·EF-Tu·tRNA ternary complexes. We propose that EF-Ts promotes the formation of GTP·EF-Tu·tRNA ternary complexes, thereby accelerating substrate turnover for rapid depletion of target-cell tRNA.cell communication | protein synthesis | toxin-immunity proteins | ribonuclease | bacterial competition

show abstract

“…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. Another example where Pro-tRNA appears to bind to the A site of the ribosome (66,67). The presence of Pro-tRNA Pro in the peptidyltransferase center may predispose the ribosome to stall upon synthesis of SecM (68).…”

mentioning

confidence: 99%

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

Wohlgemuth

Brenner

Beringer

et al. 2008

Journal of Biological Chemistry

164

194

View full text Add to dashboard Cite

The ribosome catalyzes peptide bond formation between peptidyl-tRNA in the P site and aminoacyl-tRNA in the A site. Here, we show that the nature of the C-terminal amino acid residue in the P-site peptidyl-tRNA strongly affects the rate of peptidyl transfer. Depending on the C-terminal amino acid of the peptidyl-tRNA, the rate of reaction with the small A-site substrate puromycin varied between 100 and 0.14 s ؊1 , regardless of the tRNA identity. The reactivity decreased in the order Lys ‫؍‬ Arg > Ala > Ser > Phe ‫؍‬ Val > Asp Ͼ Ͼ Pro, with Pro being by far the slowest. However, when Phe-tRNA Phe was used as A-site substrate, the rate of peptide bond formation with any peptidyl-tRNA was ϳ7 s ؊1 , which corresponds to the rate of binding of Phe-tRNA Phe to the A site (accommodation). Because accommodation is rate-limiting for peptide bond formation, the reaction rate is uniform for all peptidyltRNAs, regardless of the variations of the intrinsic chemical reactivities. On the other hand, the 50-fold increase in the reaction rate for peptidyl-tRNA ending with Pro suggests that full-length aminoacyl-tRNA in the A site greatly accelerates peptide bond formation.The enzymatic activity of the ribosome is to catalyze peptide bond formation. During the peptidyl transfer reaction, the ␣-amino group of aminoacyl-tRNA bound to the A site of the ribosome attacks the ester bond of peptidyl-tRNA in the P site, which results in peptidyl-tRNA extended by one amino acid in the A site and deacylated tRNA in the P site. The tRNA substrates are aligned in the active center of the ribosome by interactions of their CCA ends with 23 S rRNA bases (1-3). The ribosome lowers the activation entropy of the reaction (4, 5) by orienting the reacting groups precisely relative to each other (2, 3), providing an electrostatic environment that reduces the free energy of forming the transition state, shielding the reaction against bulk water (6, 7), or a combination of these effects (8).The peptidyl transfer reaction is modulated by conformational changes at the active site (3, 8 -10) as well as by the nature of the substrates. Rapid peptide bond formation requires fulllength tRNA in both A and P sites, and the reaction rate is influenced by the length of the tRNA fragments when model substrates are used (8, 10 -14). The reaction rate is also influenced by the nature of the amino acid side chain of the A-site substrate (13,(15)(16)(17), but is independent of the nucleophilicity of the attacking amino group in model substrates (18). Moreover, the length of the peptidyl chain and the nature of the C-terminal amino acid of the peptidyl-tRNA in the P site seem to have an effect (10,12,13,19). Early studies with 50 S ribosomal subunits indicated that efficient peptidyl transfer was observed with 3Ј-terminal RNase T1 fragments of N-acetylArg-tRNA Arg and fMet-tRNA fMet as model P-site substrates and an analog of aminoacyl-tRNA, puromycin (Pmn 4 ; O-methyltyrosine linked to N 6 -dimethyladenosine via an amide bond), as A-site substrate (20). In contrast,...

show abstract

Prolyl-tRNAPro in the A-site of SecM-arrested Ribosomes Inhibits the Recruitment of Transfer-messenger RNA

Cited by 90 publications

References 44 publications

Exploration of the Arrest Peptide Sequence Space Reveals Arrest-enhanced Variants

Exploration of the Arrest Peptide Sequence Space Reveals Arrest-enhanced Variants

Activation of contact-dependent antibacterial tRNase toxins by translation elongation factors

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

Contact Info

Product

Resources

About