In vitro analysis of translational rate and accuracy with an unmodified tRNA

Harrington, Kristi M.; Nazarenko, Irina; Dix, Daniel B.; Thompson, Robert C.; Uhlenbeck, Olke C.

doi:10.1021/bi00081a003

Cited by 78 publications

(94 citation statements)

References 41 publications

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“…The precipitate was dissolved in 5 mM NaOAc (pH 5.3) and stored at Ϫ80°C. In the case of tRNA Phe , the product of the aminoacylation reaction was used directly in EF-Tu binding experiments without phenol extraction and ethanol precipitation, because this procedure denatures unmodified E. coli tRNA Phe (15).…”

Section: Methodsmentioning

confidence: 99%

The tRNA Specificity of Thermus thermophilus EF-Tu

Asahara

Uhlenbeck

2002

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

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By introducing a GAC anticodon, 21 different Escherichia coli tRNAs were misacylated with either phenylalanine or valine and assayed for their affinity to Thermus thermophilus elongation factor Tu (EF-Tu)⅐GTP by using a ribonuclease protection assay. The presence of a common esterified amino acid permits the thermodynamic contribution of each tRNA body to the overall affinity to be evaluated. The E. coli elongator tRNAs exhibit a wide range of binding affinities that varied from ؊11.7 kcal͞mol for Val-tRNA Glu to ؊8.1 kcal͞mol for Val-tRNA Tyr , clearly establishing EF-Tu⅐GTP as a sequence-specific RNA-binding protein. Because the ionic strength dependence of koff varied among tRNAs, some of the affinity differences are the results of a different number of phosphate contacts formed between tRNA and protein. Because EF-Tu is known to contact only the phosphodiester backbone of tRNA, the observed specificity must be a consequence of an indirect readout mechanism. E longation factor Tu (EF-Tu) binds GTP and aminoacyl tRNA (aa-tRNA) to form a ternary complex that subsequently binds ribosome and participates in codon-directed binding of the aa-tRNA to the ribosomal A site. Although EFTu⅐GTP binds poorly to tRNAs lacking the esterified amino acid (1), the protein is generally considered to lack specificity because it binds all elongator aa-tRNAs with a similar affinity (2-4). However, recent experiments have shown that EF-Tu⅐GTP exhibits substantial specificity for both the esterified amino acid and the tRNA body of aa-tRNAs (5). This specificity was previously unappreciated because the contributions of the amino acid and the tRNA body to the overall affinity are arranged in a compensatory manner such that the cognate aa-tRNAs all bind with similar affinities. However, misacylated tRNAs were found to bind EF-Tu⅐GTP with a wide range of affinities that were either tighter or weaker than the cognate aa-tRNAs (5). The four different tRNA bodies that were tested displayed about a 100-fold range of K D values with Thermus thermophilus EFTu⅐GTP when each was esterified with the same amino acid. The same range of K D values was observed when the four tRNAs were esterified with a different common amino acid, clearly establishing that EF-Tu shows specificity toward the tRNA body. However, the four tRNAs used in these experiments (Escherichia coli tRNA Ala , tRNA Gln , tRNA Val , and yeast tRNA Phe ) have very similar overall architectures, raising the possibility that EFTu⅐GTP could exhibit a much larger range of affinities with tRNAs of different architectures.Experiments presented here take advantage of the important role of the anticodon as a identity element for valyl-tRNA synthetase (ValRS) and phenylalanyl-tRNA synthetase (PheRS) (6-10) to prepare 21 different E. coli tRNAs that were misacylated with either valine or phenylalanine. By comparing the affinities of the different tRNAs esterified with a common amino acid, the specificity of EF-Tu⅐GTP for the different tRNA bodies was established. Materials and MethodsE. coli...

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Section: Methodsmentioning

confidence: 99%

The tRNA Specificity of Thermus thermophilus EF-Tu

Asahara

Uhlenbeck

2002

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

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112

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“…Pseudouridylation of tRNA does not in uence the overall three-dimensional structure of tRNAs, is not essential for cell viability, and is not generally required for aminoacylation (13,16,46). However, W does affect the local structure of the domains in which it resides (see 6).…”

Section: Transfer Rnamentioning

confidence: 99%

“…Maintenance of the proper conformation of the three anticodon residues undoubtedly helps to foster correct codonanticodon interactions. This may increase translational accuracy by decreasing the rate of peptide bond formation, thereby allowing more time for rejection of incorrect codon-anticodon pairs (46).…”

Section: Transfer Rnamentioning

confidence: 99%

“…In these contexts, W 34, W 35, and W 36 are postulated to strengthen codon-anticodon base-pairing, particularly noncanonical interactions involving G-A or G-G wobble base pairs (11). Thus, pseudouridylation plays an important biological role in ne-tuning the structure of those tRNAs in which it occurs, thereby in uencing their decoding activity, improving the delity of protein biosynthesis, and helping to maintain the proper reading frame (46).…”

Section: Transfer Rnamentioning

confidence: 99%

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Pseudouridine in RNA: What, Where, How, and Why

2000

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SummaryPseudouridine (5-ribosyluracil ) is a ubiquitous yet enigmatic constituent of structural RNAs (transfer, ribosomal, small nuclear, and small nucleolar). Although pseudouridine (W ) was the rst modi ed nucleoside to be discovered in RNA, and is the most abundant, its biosynthesis and biological roles have remained poorly understood since its identi cation as a " fth nucleoside" in RNA. Recently, a combination of biochemical, biophysical, and genetic approaches has helped to illuminate the structural consequences of W in polyribonucleotides, the biochemical mechanism of U! W

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“…Rppto-tRNAs were generated by in vitro transcription in the presence of one of the four ␣-S-triphosphate nucleotides (15) at 1% of the corresponding unsubstituted ribonucleotide. In vitro-transcribed tRNAs are substrates for aminoacylation and are active in translation (16,29). The pto-tRNAs were labeled at the 5Ј end with 32 P and bound under P-site binding conditions to 70S ribosomes or 30S ribosomal subunits in the presence or absence of mRNA.…”

Section: Resultsmentioning

confidence: 99%

Identification of specific Rp-phosphate oxygens in the tRNA anticodon loop required for ribosomal P-site binding

Schnitzer

Ahsen

1997

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

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ABSTRACTtRNA binding to the ribosomal P site is dependent not only on correct codon-anticodon interaction but also involves identification of structural elements of tRNA by the ribosome. By using a phosphorothioate substitutioninterference approach, we identified specific nonbridging Rp-phosphate oxygens in the anticodon loop of tRNA Phe from Escherichia coli which are required for P-site binding. Stereospecific involvement of phosphate oxygens at these positions was confirmed by using synthetic anticodon arm analogues at which single Rp-or Sp-phosphorothioates were incorporated. Identical interference results with yeast tRNA Phe and E. coli tRNA f Met indicate a common backbone conformation or common recognition elements in the anticodon loop of tRNAs. N-ethyl-N-nitrosourea modification-interference experiments with natural tRNAs point to the importance of the same phosphates in the loop. Guided by the crystal structure of tRNA Phe , we propose that specific Rp-phosphate oxygens are required for anticodon loop (''U-turn'') stabilization or are involved in interactions with the ribosome on correct tRNAmRNA complex formation.

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In vitro analysis of translational rate and accuracy with an unmodified tRNA

Cited by 78 publications

References 41 publications

The tRNA Specificity of Thermus thermophilus EF-Tu

The tRNA Specificity of Thermus thermophilus EF-Tu

Pseudouridine in RNA: What, Where, How, and Why

Identification of specific Rp-phosphate oxygens in the tRNA anticodon loop required for ribosomal P-site binding

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