Interactions between 23S rRNA and tRNA in the ribosomal E site

Bocchetta, Maurizio; Xiong, Liqun; Shah, Sunita; Mankin, Alexander S.

doi:10.1017/s1355838201001650

Cited by 22 publications

(24 citation statements)

References 34 publications

(31 reference statements)

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“…2). This is the area that had been identified previously as a component of the 50S subunit E site by biochemical studies (Moazed and Noller 1989a;Wower et al 1993Wower et al , 2000Rinke-Appel et al 1995;Joseph and Noller 1996;Bocchetta et al 2001) and by lower resolution structures (Agrawal et al 1996;Stark et al 1997;Yusupov et al 2001). Although CCA is a short sequence, it displays significant specificity and binds only at the A, P, and E sites (data not shown).…”

Section: Resultsmentioning

confidence: 59%

“…These results were corroborated and expanded by later studies. Mankin and colleagues proposed that A76 forms a reverse Hoogsteen base pair with C2431 (2394; Bocchetta et al 2001). Instead, the N3 and N4 of C2431 (2394) hydrogen bonds to the 2Ј hydroxyl and N3 of A76, respectively (Fig.…”

Section: Agreement With Biochemical Resultsmentioning

confidence: 99%

“…Chemical footprinting, protection, and cross-linking studies determined that the portion of the E site on the 30S subunit is close to ribosomal protein S11 and the 3Ј terminus of 16S rRNA (Wower et al 1993), and that the component on the 50S subunit is near the L1 stalk and helix 88 of 23S rRNA (Moazed and Noller 1989a;Wower et al 1993Wower et al , 2000RinkeAppel et al 1995;Joseph and Noller 1996;Bocchetta et al 2001). Using modified tRNAs, it was shown that the terminal adenosine of tRNA contributes significantly to E site binding; removal of A76 decreases tRNA affinity for the E site to a level that is below the experimental detection limit (>100-fold; Lill et al 1988).…”

Section: Introductionmentioning

confidence: 99%

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Structures of deacylated tRNA mimics bound to the E site of the large ribosomal subunit

Schmeing¹,

Moore²,

Steitz³

2003

RNA

104

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During translation, tRNAs cycle through three binding sites on the ribosome: the A, the P, and the E sites. We have determined the structures of complexes between the Haloarcula marismortui large ribosomal subunit and two different E site substrates: a deacylated tRNA acceptor stem minihelix and a CCA-acceptor end. Both of these tRNA mimics contain analogs of adenosine 76, the component responsible for a large proportion of E site binding affinity. They bind in the center of the loop-extension of protein L44e, and make specific contacts with both L44e and 23S rRNA including bases that are conserved in all three kingdoms of life. These contacts are consistent with the footprinting, protection, and cross-linking data that have identified the E site biochemically. These structures explain the specificity of the E site for deacylated tRNAs, as it is too small to accommodate any relevant aminoacyl-tRNA. The orientation of the minihelix suggests that it may mimic the P/E hybrid state. It appears that the E site on the 50S subunit was formed by only RNA in the last common ancestor of the three kingdoms, since the proteins at the E sites of H. marismortui and Deinucoccus radiodurans large subunits are not homologous.

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Section: Resultsmentioning

confidence: 59%

Section: Agreement With Biochemical Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Structures of deacylated tRNA mimics bound to the E site of the large ribosomal subunit

Schmeing¹,

Moore²,

Steitz³

2003

RNA

104

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“…To investigate the role of the P/E hybrid state in translocation, we mutagenized C2394, a key nucleotide of the 50S E site that interacts with the 3Ј-terminal adenosine (A76) of tRNA (1,(18)(19)(20)(21). Plasmids containing rrnB without or with mutation C2394A, C2394G, or C2394U were moved into an Escherichia…”

Section: Mutations Of C2394 Confer Moderate Defects In Cell Growthmentioning

confidence: 99%

Role of hybrid tRNA-binding states in ribosomal translocation

Walker

Shoji

Pan

et al. 2008

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

108

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During translation, tRNAs must move rapidly to their adjacent sites in the ribosome while maintaining precise pairing with mRNA. This movement (translocation) occurs in a stepwise manner with hybrid-state intermediates, but it is unclear how these hybrid states relate to kinetically defined events of translocation. Here we analyze mutations at position 2394 of 23S rRNA in a pre-steadystate kinetic analysis of translocation. These mutations target the 50S E site and are predicted to inhibit P/E state formation. Each mutation decreases growth rate, the maximal rate of translocation (k trans), and the apparent affinity of EF-G for the pretranslocation complex (i.e., increases K 1/2). The magnitude of these defects follows the trend A > G > U. Because the C2394A mutation did not decrease the rate of single-turnover GTP hydrolysis, the >20-fold increase in K 1/2 conferred by C2394A can be attributed to neither the initial binding of EF-G nor the subsequent GTP hydrolysis step. We propose that C2394A inhibits a later step, P/E state formation, to confer its effects on translocation. Replacement of the peptidyl group with an aminoacyl group, which is predicted to inhibit A/P state formation, decreases k trans without increasing K1/2. These data suggest that movements of tRNA into the P/E and A/P sites are separable events. This mutational study allows tRNA movements with respect to both subunits to be integrated into a kinetic model for translocation.elongation factor G ͉ translation ͉ exit site ͉ GTPase M ovement of tRNAs through the ribosome is believed to occur in a stepwise manner (1). Chemical protection experiments showed that, after peptidyl transfer, the acceptor ends of the tRNAs can move spontaneously with respect to the 50S subunit to form the hybrid state. In the hybrid state, the deacylated tRNA occupies the 30S P site and 50S E site (P/E site) while the peptidyl-tRNA occupies the 30S A site and 50S P site (A/P site). It was proposed that hybrid state formation precedes codon-anticodon movement within the 30S subunit, and only the latter event requires catalysis by elongation factor G (EF-G). Support for the hybrid-state model came from Förster resonance energy transfer (FRET) experiments in which an Ϸ20-Å movement of the 5Ј end of the newly deacylated tRNA toward ribosomal protein L1 was inferred after peptide bond formation, whereas the position of the peptidyl group changed little (2). Because L1 lies near the 50S E site, these data are consistent with movement of tRNA into the P/E site. Further evidence came from single-molecule FRET studies that monitored the distance between probes attached to the elbows of tRNAs in the A and P sites. Fluctuations between two distinct configurations were observed that were structurally consistent with the classical and hybrid states (3). More recently, a third state of the pretranslocation (PRE) complex was observed by single-molecule FRET, consistent with one tRNA bound to the P/E site and the other bound to the A/A site (4). Experiments that monitored mRNA in ribos...

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“…The removal or substitution of A 76 decreases the affinity of the ribosomal E-site for tRNA at least 100-fold (15,32,35). Failure of the G 76 mutant of tRNA Val to translate poly(GUA) may be due to the inability of the G 76 mutant to bind correctly to the E-site, thus blocking movement of tRNA from the P-to the E-site.…”

Section: Trna 3ј-terminal Adenosine In Ribosomal Translationmentioning

confidence: 99%

Functional Importance of the 3′-Terminal Adenosine of tRNA in Ribosomal Translation

Virumäe¹,

Saarma²,

Horowitz³

et al. 2002

Journal of Biological Chemistry

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The universally conserved 3-terminal CCA sequence of tRNA interacts with large ribosomal subunit RNA during translation. The functional importance of the interaction between the 3-terminal nucleotide of tRNA and the ribosome was studied in vitro using mutant in vitro transcribed tRNA Val A76G. Val-tRNA CCG does not support polypeptide synthesis on poly(GUA) as a message. However, in a co-translation system, where ValtRNA CCG represented only a small fraction of total ValtRNA, the mutant tRNA is able to transfer valine into a polypeptide chain, albeit at a reduced level. The A76G mutation does not affect binding of Val-or NAcValtRNA CCG to the A-or P-sites as shown by efficient peptide bond formation, although the donor activity of the mutant NAcVal-tRNA CCG in the peptidyl transfer reaction is slightly reduced compared with wild-type NAcVal-tRNA. Translocation of 3-CCG-tRNA from the Pto the E-site is not significantly influenced. However, the A76G mutation drastically inhibits translocation of peptidyl-tRNA G 76 from the ribosomal A-site to the Psite, which apparently explains its failure to support cell-free protein synthesis. Our results indicate that the identity of the 3-terminal nucleotide of tRNA is critical for tRNA movement in the ribosome.

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Interactions between 23S rRNA and tRNA in the ribosomal E site

Cited by 22 publications

References 34 publications

Structures of deacylated tRNA mimics bound to the E site of the large ribosomal subunit

Structures of deacylated tRNA mimics bound to the E site of the large ribosomal subunit

Role of hybrid tRNA-binding states in ribosomal translocation

Functional Importance of the 3′-Terminal Adenosine of tRNA in Ribosomal Translation

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