A Renewed Focus on Transfer RNA

Daviter, Tina; Frank, Vered; Ramakrishnan, V.

doi:10.1126/science.1113415

Cited by 15 publications

(14 citation statements)

References 18 publications

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“…Precise kinetic experiments revealed that the Hirsh suppressor misreads near-cognate codons by accelerating the forward rate constants of GTPase activation and accommodation into the peptidyltransferase center (24). Taken together with our observations, the specific interaction between H69 and the D-stem of tRNA might be an important factor modulating translational fidelity independently of the codon-anticodon interaction in the decoding center (24,25). O'Connor and Dahlberg (26) carried out a genetic selection of plasmid-encoded rRNA that promotes frameshifting of trpE91 and identified three mutations in the H69 loop, C1914U, a deletion of A1916 (⌬1916), and an insertion of two adenosines between A1916 and U1917 (ϩAA1916).…”

Section: Discussionsupporting

confidence: 69%

“…In the crystal structure of the 70 S ribosome complexed to both A-and P-site tRNAs, H69 is positioned between the two tRNAs (1). The minor groove of H69 (positions 1908 -1909, 1922-1923) interacts with the minor groove of the D-stem of the P-site tRNA (positions [12][13][25][26] (Fig. 1B), whereas the conserved loop (positions 1913-1915) of H69 makes a contact with the D-stem of A-site tRNA (positions 11-12 and 25-26) (Fig.…”

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confidence: 99%

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Conserved Loop Sequence of Helix 69 in Escherichia coli 23 S rRNA Is Involved in A-site tRNA Binding and Translational Fidelity

Hirabayashi

Sato²,

Suzuki

2006

Journal of Biological Chemistry

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Ribosomes translate the genetic information contained in mRNAs into proteins. The large (50 S) subunit of the ribosome catalyzes the formation of a peptide bond between the aminoacyl-tRNA (aa-tRNA) 3 bound to the A-site and the peptidyl-tRNA at the P-site. This peptide bond formation takes place at the peptidyltransferase center of the 50 S subunit. Codon-anticodon pairing occurs at the decoding center of the small (30 S) subunit. The aa-tRNA is delivered to the ribosome as a ternary complex of aa-tRNA, EF-Tu, and GTP. Cognate codon recognition is strictly monitored by 16 S rRNA and triggers GTP hydrolysis and dissociation of EF-Tu. This allows aa-tRNA to be accommodated by the A site of the 50 S subunit. Thus, accuracy of protein synthesis is based on the synergistic interplay of the large and small subunits of the ribosome. However, mechanistic insights into the ribosome dynamics during decoding are still rudimentary.The intersubunit bridges of the ribosome are functional sites that are not only necessary for subunit connection but also play roles in translation. Helix 69 (H69) (position 1906 -1924) is a highly conserved stemloop in domain IV of 23 S rRNA of the bacterial 50 S subunit (Fig. 1A). In fact, each base in the loop of H69 shows more than 98% conservation in 436 bacterial rRNAs (www.rna.icmb.utexas.edu). Crystallographic studies revealed that H69 is located on the surface involved in intersubunit association with the 30 S subunit by connecting with helix 44 (h44) of 16 S rRNA forming the bridge B2a; A1912, A1913, A1914, A1918 and A1919 in H69 make contact with positions 1407-1410 and 1494 -1495 in h44 and G1517 in h45 (1, 2) (see Fig. 7A). In the free 50 S subunit, H69 makes a compact structure and interacts with H71 of 23 S rRNA (3), whereas in the 70 S ribosome H69 stretches toward the small subunit and interacts with h44 of 16 S rRNA (1). The tip of H69 moves about 13.5 Å during this structural change. In addition, H69 directly interacts with A/T-, A-, and P-site tRNAs during each translation step (1). During the decoding step, aa-tRNA is brought into the A/T-site as a complex with EF-Tu/GTP (ternary complex). Cryoelectron microscopy analyses showed a kinked conformation for aa-tRNA at the A/T state (molecular spring) (4, 5). The tip of H69 makes a contact with the hinge of the kink between D-and anticodon-stems in tRNA. This interaction is supposed to facilitate the structural distortion of tRNA that enables the anticodon-stem to fit into the decoding center of 16 S rRNA. In the crystal structure of the 70 S ribosome complexed to both A-and P-site tRNAs, H69 is positioned between the two tRNAs (1). The minor groove of H69 (positions 1908 -1909, 1922-1923) interacts with the minor groove of the D-stem of the P-site tRNA (positions 12-13, 25-26) (Fig. 1B), whereas the conserved loop (positions 1913-1915) of H69 makes a contact with the D-stem of A-site tRNA (positions 11-12 and 25-26) (Fig. 1B). Moreover, it has been reported that H69 interacts with various translational factors. In the post-t...

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Section: Discussionsupporting

confidence: 69%

mentioning

confidence: 99%

Conserved Loop Sequence of Helix 69 in Escherichia coli 23 S rRNA Is Involved in A-site tRNA Binding and Translational Fidelity

Hirabayashi

Sato²,

Suzuki

2006

Journal of Biological Chemistry

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“…Such changes could be transduced down the body of the aa-tRNA to the decoding center, destabilizing the codon-anticodon interactions and thus helping to facilitate frameshifting. A second possibility arises from the observation that stronger interactions between the Hirsch suppressor tRNA and the ribosome serve to increase the rate of EF-Tu activation and GTPase hydrolysis and aa-tRNA accommodation, resulting in increased rates of nonsense suppression (8,9). The 9-Å model of Ϫ1 PRF predicts that increased rates of accommodation would promote increased Ϫ1 PRF frequencies (42), and increased affinities for aa-tRNAs have been demonstrated for ribosomes containing the mak8-1, W255C, P257T, and I282T forms of L3 (41).…”

Section: Discussionmentioning

confidence: 99%

Identification of Functionally Important Amino Acids of Ribosomal Protein L3 by Saturation Mutagenesis

Meškauskas

Petrov

Dinman

2005

Molecular and Cellular Biology

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There is accumulating evidence that many ribosomal proteins are involved in shaping rRNA into their functionally correct conformations through RNA-protein interactions. Moreover, although rRNA seems to play the central role in all aspects of ribosome function, ribosomal proteins may be involved in facilitating communication between different functional regions in ribosome, as well as between the ribosome and cellular factors. In an effort to more fully understand how ribosomal proteins may influence ribosome function, we undertook large-scale mutational analysis of ribosomal protein L3, a core protein of the large subunit that has been implicated in numerous ribosome-associated functions in the past. A total of 98 different rpl3 alleles were genetically characterized with regard to their effects on killer virus maintenance, programmed ؊1 ribosomal frameshifting, resistance/hypersensitivity to the translational inhibitor anisomycin and, in specific cases, the ability to enhance translation of a reporter mRNA lacking the 5 7 mGppp cap structure and 3 poly(A) tail. Biochemical studies reveal a correlation between an increased affinity for aminoacyl-tRNA and the extent of anisomycin resistance and a decreased peptidyltransferase activity and increased frameshifting efficiency. Immunoblot analyses reveal that the superkiller phenotype is not due to a defect in the ability of ribosomes to recruit the Ski-complex, suggesting that the defect lies in a reduced ability of mutant ribosomes to distinguish between cap Early in the history of modern yeast genetics, two mutants were independently identified based on separate phenotypes: resistance to the peptidyltransferase inhibitors trichodermin and anisomycin (tcm1-1) (25, 47) and inability to maintain the M 1 killer virus (mak8-1 [maintenance of killer]) (63). Subsequently, they were shown to be allelic to RPL3, the gene that encodes ribosomal protein L3, a 44-kDa essential ribosomal protein in the large subunit of the ribosome (16, 64). Independently, ribosome reconstitution experiments demonstrated that L3 is one of a few proteins essential for peptidyltransferase center (PTC) activity (49), and L3 and L24 are the only proteins capable of initiating in vitro assembly of the Escherichia coli 50S ribosomal subunit (35). These early findings implicated L3 as a central component of peptidyltransfer activity and ribosome assembly and as an important factor in the interaction between host cells and viruses.Previous studies demonstrating that peptidyltransferase inhibitors had antiviral activities by promoting altered efficiencies of programmed Ϫ1 ribosomal frameshifting (Ϫ1 PRF) (11) provoked more in-depth characterization of the mak8-1 mutants. An initial study (i) demonstrated that increased Ϫ1 PRF efficiency constituted the basis for the loss of killer phenotype in mak8-1 cells, (ii) showed these cells were immune to further changes in Ϫ1 PRF by anisomycin or sparsomycin, two drugs that were previously shown to alter Ϫ1 PRF efficiencies, and (iii) identified that the origin...

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“…GTP-hydrolyzing regulatory factors help direct the initiation, elongation, and completion of translation [16,17]. Proteins are also required to charge tRNA molecules with the appropriate amino acid [18] and adjust their binding affinity to the ribosome [19]. Here we examine the early evolution of these proteins by a survey of conserved structural architectures.…”

Section: Introductionmentioning

confidence: 99%

The evolution and functional repertoire of translation proteins following the origin of life

2010

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BackgroundThe RNA world hypothesis posits that the earliest genetic system consisted of informational RNA molecules that directed the synthesis of modestly functional RNA molecules. Further evidence suggests that it was within this RNA-based genetic system that life developed the ability to synthesize proteins by translating genetic code. Here we investigate the early development of the translation system through an evolutionary survey of protein architectures associated with modern translation.ResultsOur analysis reveals a structural expansion of translation proteins immediately following the RNA world and well before the establishment of the DNA genome. Subsequent functional annotation shows that representatives of the ten most ancestral protein architectures are responsible for all of the core protein functions found in modern translation.ConclusionsWe propose that this early robust translation system evolved by virtue of a positive feedback cycle in which the system was able to create increasingly complex proteins to further enhance its own function.ReviewersThis article was reviewed by Janet Siefert, George Fox, and Antonio Lazcano (nominated by Laura Landweber)

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A Renewed Focus on Transfer RNA

Cited by 15 publications

References 18 publications

Conserved Loop Sequence of Helix 69 in Escherichia coli 23 S rRNA Is Involved in A-site tRNA Binding and Translational Fidelity

Conserved Loop Sequence of Helix 69 in Escherichia coli 23 S rRNA Is Involved in A-site tRNA Binding and Translational Fidelity

Identification of Functionally Important Amino Acids of Ribosomal Protein L3 by Saturation Mutagenesis

The evolution and functional repertoire of translation proteins following the origin of life

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