Purine bases at position 37 of tRNA stabilize codon–anticodon interaction in the ribosomal A site by stacking and Mg<sup>2+</sup>-dependent interactions

Konevega, Andrey L.; Soboleva, N. G.; Makhno, V.I.; Semenkov, Yu.P.; Wintermeyer, Wolfgang; Rodnina, Marina V.; Katunin, Vladimir I.

doi:10.1261/rna.5142404

Cited by 113 publications

(110 citation statements)

References 49 publications

(72 reference statements)

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“…27,28 In the interaction of tRNA with the ribosomal A site, such single strand stacking of purines in near neighbourhood to the anticodon stabilizes interaction with the codon. 29 It is tempting to speculate that the single-strand stacking of the ROSE ibpA SD sequence facilitates conformation of hairpin I and II as can be seen by similar T1 cleavage profiles of the corresponding G residues (G22 to G47) in the WT, ΔG74 and C75A RNAs. Consistent with the data presented in Figure 2, hairpin III of the WT RNA was accessible to T1 and S1 (cleaving single-stranded nucleotides) at 45°C but far less at 30°C.…”

Section: Resultsmentioning

confidence: 99%

TheEscherichia coliibpA thermometer is comprised of stable and unstable structural elements

Waldminghaus¹,

Gaubig

Klinkert

et al. 2009

RNA Biology

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Translation of many small heat shock genes in α-and γ-proteobacteria is controlled by the ROSE (Repression Of heat Shock gene Expression) element, a thermo-responsive RNA structure in the 5'-untranslated region. ROSE ibpA regulates translation of the Escherichia coli ibpA gene coding for an inclusion body-associated protein. We present first structural insights into a full-length ROSE element by examining the temperatureinduced conformational changes of ROSE ibpA using detailed enzymatic and lead probing experiments between 20 and 50°C. The initial two hairpins are stable at all temperatures tested and might assist in proper folding of the third temperature-responsive stem-loop structure, which restricts access to the Shine-Dalgarno sequence at temperatures below 35°C. Toeprinting (primer extension inhibition) experiments show that binding of the 30S ribosome to ROSE ibpA is enhanced at high temperatures. In contrast to other ROSE-like elements, the final hairpin is rather short. Single point mutations result in alternative structures with positive or negative effects on translation efficiency. Our study demonstrates how the combination of stable and unstable modules controls translation efficiency in a complete RNA thermometer.

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Section: Resultsmentioning

confidence: 99%

TheEscherichia coliibpA thermometer is comprised of stable and unstable structural elements

Waldminghaus¹,

Gaubig

Klinkert

et al. 2009

RNA Biology

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“…4e, f) were prepared by PCR mutagenesis. Transcription and refolding of the mRNAs were performed as described 54,55 . Complex preparation for cryo-EM.…”

Section: Discussionmentioning

confidence: 99%

The pathway to GTPase activation of elongation factor SelB on the ribosome

Fischer

Neumann

Bock

et al. 2016

Nature

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103

137

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“…The pretranslocation complex with deacylated tRNA fMet in the P site and fMetLeu-tRNA Leu in the A site was prepared by mixing initiation and ternary complexes in buffer A [50 mM Tris⅐HCl (pH 7.5), 70 mM NH 4Cl, 30 mM KCl, 7 mM MgCl2] at 37°C (24). The complex was stabilized by increasing the concentration of MgCl 2 to 21 mM and purified by centrifugation through a sucrose cushion (25). Ribosomes were dissolved in buffer A (21 mM MgCl 2), shock-frozen in liquid nitrogen, and stored at Ϫ80°C.…”

Section: Methodsmentioning

confidence: 99%

Structure of ratcheted ribosomes with tRNAs in hybrid states

Julián

Konevega

Scheres³

et al. 2008

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

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167

176

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During protein synthesis, tRNAs and mRNA move through the ribosome between aminoacyl (A), peptidyl (P), and exit (E) sites of the ribosome in a process called translocation. Translocation is accompanied by the displacement of the tRNAs on the large ribosomal subunit toward the hybrid A/P and P/E states and by a rotational movement (ratchet) of the ribosomal subunits relative to one another. So far, the structure of the ratcheted state has been observed only when translation factors were bound to the ribosome. Using cryo-electron microscopy and classification, we show here that ribosomes can spontaneously adopt a ratcheted conformation with tRNAs in their hybrid states. The peptidyl-tRNA molecule in the A/P state, which is visualized here, is not distorted compared with the A/A state except for slight adjustments of its acceptor end, suggesting that the displacement of the A-site tRNA on the 50S subunit is passive and is induced by the 30S subunit rotation. Simultaneous subunit ratchet and formation of the tRNA hybrid states precede and may promote the subsequent rapid and coordinated tRNA translocation on the 30S subunit catalyzed by elongation factor G.translocation ͉ elongation factor G ͉ cryo-electron microscopy D uring the elongation cycle, the tRNAs⅐mRNA complex is translocated to allow reading of the following codon on mRNA. Translocation is promoted by elongation factor G (EF-G) and accompanied by GTP hydrolysis. During translocation, the ribosome changes from the pretranslocation state with deacylated tRNA in the peptidyl site (P site) and peptidyltRNA in the aminoacyl site (A site) to the posttranslocation state where A-and P-site tRNAs have moved to P and exit (E) sites, respectively. The universal design of ribosomes from two subunits inspired early suggestions that translocation may involve a movement of the subunits relative to each other (1, 2). Such models imply the existence of intermediate states for the tRNAs that differ from the classic A, P, and E states. Chemical probing experiments with pretranslocation ribosomes indicated that after peptidyl transfer the acceptor ends of the tRNAs spontaneously moved on the large 50S subunit toward their posttranslocation positions (3), indicating that peptidyl-tRNA and deacylated tRNA entered hybrid A/P and P/E states, respectively. The transition was observed in the absence of EF-G, and the driving force for the movement was attributed to different affinities of the A, P, and E sites on the 50S subunit (4) for the chemically different acceptor ends of the tRNAs (5, 6). Nevertheless, cryo-electron microscopy (cryoEM) of ribosomes in the pretranslocation state showed the tRNAs in their classic A, P, and E states (7). Although this result was difficult to reconcile with the hybrid-state model, it is important to note that in that complex the occupancy of the E site by deacylated tRNA may have prevented the P-site tRNA to enter the P/E-site hybrid state.CryoEM of ribosomes in complex with EF-G revealed a relative rotation between subunits referred to as ratc...

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Purine bases at position 37 of tRNA stabilize codon–anticodon interaction in the ribosomal A site by stacking and Mg²⁺-dependent interactions

Abstract: The anticodon loop of tRNA contains a number of conserved or semiconserved nucleotides. In most tRNAs, a highly modified purine is found at position 37 immediately 3 to the anticodon. Here, we examined the role of the base at position 37 for tRNA Phe

Cited by 113 publications

References 49 publications

TheEscherichia coliibpA thermometer is comprised of stable and unstable structural elements

TheEscherichia coliibpA thermometer is comprised of stable and unstable structural elements

The pathway to GTPase activation of elongation factor SelB on the ribosome

Structure of ratcheted ribosomes with tRNAs in hybrid states

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Purine bases at position 37 of tRNA stabilize codon–anticodon interaction in the ribosomal A site by stacking and Mg2+-dependent interactions

Abstract: The anticodon loop of tRNA contains a number of conserved or semiconserved nucleotides. In most tRNAs, a highly modified purine is found at position 37 immediately 3 to the anticodon. Here, we examined the role of the base at position 37 for tRNA Phe

Cited by 113 publications

References 49 publications

TheEscherichia coliibpA thermometer is comprised of stable and unstable structural elements

TheEscherichia coliibpA thermometer is comprised of stable and unstable structural elements

The pathway to GTPase activation of elongation factor SelB on the ribosome

Structure of ratcheted ribosomes with tRNAs in hybrid states

Contact Info

Product

Resources

About

Purine bases at position 37 of tRNA stabilize codon–anticodon interaction in the ribosomal A site by stacking and Mg²⁺-dependent interactions