Translation ability of mitochondrial tRNAs<sup>Ser</sup> with unusual secondary structures in an <i>in vitro</i> translation system of bovine mitochondria

Hanada, Takao; Suzuki, Tsutomu; Yokogawa, Takashi; Takemoto-Hori, Chie; Sprinzl, Mathias; Watanabe, Kimitsuna

doi:10.1046/j.1365-2443.2001.00491.x

Cited by 53 publications

(45 citation statements)

References 45 publications

(80 reference statements)

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“…The lysate was subjected to ultracentrifugation to obtain the crude ribosome. 70 S tight-coupled (TC) ribosomes were then purified from the crude ribosome preparation by 14 -16 h of ultracentrifugation in a 6 -38% (w/v) SDG as described (18). Recombinant elongation factors were prepared as described (19).…”

Section: Methodsmentioning

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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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: Methodsmentioning

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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“…In Vitro Mitochondrial Translation. The in vitro translation assay was carried out according to methods described in the literature (28). Briefly, the aminoacyl-tRNA Leu(UUR) was prepared at 37°C for 10 min in a reaction mixture (50 l Leu(UUR) , and 20 g of human mt leucyl-tRNA synthetase.…”

Section: Molecular Surgery To Construct the Artificial Mt Trna Leu(uumentioning

confidence: 99%

“…We examined whether the mt tRNA Leu(UUR) lacking the wobble modification could function in the translation process. The translational activity was measured by using an in vitro mitochondrial translation system (28). We used an mRNA transcribed in vitro that has 30 triplet repeats for the leucine codons UUA and UUG, as well as UUC as a negative control.…”

Section: Uug Codon-specific Translational Defect Of Mt Trnas Leu(uur)mentioning

confidence: 99%

Codon-specific translational defect caused by a wobble modification deficiency in mutant tRNA from a human mitochondrial disease

Kirino

Yasukawa

Ohta

et al. 2004

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

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Point mutations in the mitochondrial (mt) tRNA Leu(UUR) gene are responsible for mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), a subgroup of mitochondrial encephalomyopathic diseases. We previously showed that mt tRNA Leu(UUR) with an A3243G or T3271C mutation derived from patients with MELAS are deficient in a normal taurinecontaining modification ( m 5 U; 5-taurinomethyluridine) at the anticodon wobble position. To examine decoding disorder of the mutant tRNA due to the wobble modification deficiency independent of the pathogenic point mutation itself, we used a molecular surgery technique to construct an mt tRNA Leu(UUR) molecule lacking the taurine modification but without the pathogenic mutation. This ''operated'' mt tRNA Leu(UUR) without the taurine modification showed severely reduced UUG translation but no decrease in UUA translation. We thus concluded that the UUG codon-specific translational defect of the mutant mt tRNAs Leu(UUR) is the primary cause of MELAS at the molecular level. This result could explain the complex I deficiency observed clinically in MELAS.

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“…The lysate was subjected to ultracentrifugation to obtain the crude ribosome. 70 S tight-coupled ribosomes were then purified from the crude ribosome preparation by 14 -16 h of ultracentrifugation in a 6 -38% (w/v) sucrose density gradient (SDG) as described (16). Recombinant E. coli elongation factors EF-G and EF-Tu were prepared as described (17).…”

Section: Construction Of Plasmids Generating 23 S Rrna With Shortenedmentioning

confidence: 99%

“…Poly(U)-directed Polyphenylalanine Synthesis-In vitro translation was performed as described previously (16,18) with slight modifications. The reaction mixture contained 75 fmol/l ribosome, 0.3 pmol/l [ 14 C]Phe-tRNA, and 0.5 g/l poly(U).…”

Section: Construction Of Plasmids Generating 23 S Rrna With Shortenedmentioning

confidence: 99%

The A-site Finger in 23 S rRNA Acts as a Functional Attenuator for Translocation

Komoda

Sato

Phelps

et al. 2006

Journal of Biological Chemistry

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Helix 38 (H38) in 23 S rRNA, which is known as the "A-site finger (ASF)," is located in the intersubunit space of the ribosomal 50 S subunit and, together with protein S13 in the 30 S subunit, it forms bridge B1a. It is known that throughout the decoding process, ASF interacts directly with the A-site tRNA. Bridge B1a becomes disrupted by the ratchet-like rotation of the 30 S subunit relative to the 50 S subunit. This occurs in association with elongation factor G (EF-G)-catalyzed translocation. To further characterize the functional role(s) of ASF, variants of Escherichia coli ribosomes with a shortened ASF were constructed. The E. coli strain bearing such ASF-shortened ribosomes had a normal growth rate but enhanced ؉1 frameshift activity. ASF-shortened ribosomes showed normal subunit association but higher activity in poly(U)-dependent polyphenylalanine synthesis than the wild type (WT) ribosome at limited EF-G concentrations. In contrast, other ribosome variants with shortened bridge-forming helices 34 and 68 showed weak subunit association and less efficient translational activity than the WT ribosome. Thus, the higher translational activity of ASF-shortened ribosomes is caused by the disruption of bridge B1a and is not due to weakened subunit association. Single round translocation analyses clearly demonstrated that the ASF-shortened ribosomes have higher translocation activity than the WT ribosome. These observations indicate that the intrinsic translocation activity of ribosomes is greater than that usually observed in the WT ribosome and that ASF is a functional attenuator for translocation that serves to maintain the reading frame.Ribosomes are universally conserved ribonucleoproteins that translate the genetic information contained in mRNAs into proteins. In bacteria, the large (50 S) and the small (30 S) subunits associate to form functional 70 S ribosomes. Both subunits are connected by 12 intersubunit bridges formed by RNA-

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Translation ability of mitochondrial tRNAs^Ser with unusual secondary structures in an in vitro translation system of bovine mitochondria

Cited by 53 publications

References 45 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

Codon-specific translational defect caused by a wobble modification deficiency in mutant tRNA from a human mitochondrial disease

The A-site Finger in 23 S rRNA Acts as a Functional Attenuator for Translocation

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Translation ability of mitochondrial tRNAsSer with unusual secondary structures in an in vitro translation system of bovine mitochondria

Cited by 53 publications

References 45 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

Codon-specific translational defect caused by a wobble modification deficiency in mutant tRNA from a human mitochondrial disease

The A-site Finger in 23 S rRNA Acts as a Functional Attenuator for Translocation

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Translation ability of mitochondrial tRNAs^Ser with unusual secondary structures in an in vitro translation system of bovine mitochondria