Effect of elongation factor Tu on the conformation of phenylalanyl‐tRNA<sup>Phe</sup>

Riehl, Nadine; Giegé, Richard; Ebel, Jean‐Pierre; Ehresmann, Bernard

doi:10.1016/0014-5793(83)80871-0

Cited by 27 publications

(12 citation statements)

References 22 publications

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“…Most probably, these degradations are induced by the synthetase interacting with tRNATh' and may be ascribed to a conformational change in the nucleic acid. Similar effects were observed with other tRNAs interacting with aminoacyl-tRNA synthetases [17] or elongation factor Tu [49]. Fig.…”

Section: Complex Formation Between Trnath' and Threonyl-trna Syn Thetasesupporting

confidence: 84%

Tertiary structure of Escherichia coli tRNA^Thr₃ in solution and interaction of this tRNA with the cognate threonyl‐tRNA synthetase

Theobald

Springer

Grunberg‐Manago

et al. 1988

European Journal of Biochemistry

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The solution structure of Escherichiu coli tRNA$' (anticodon GGU) and the residues of this tRNA in contact with the a2 dimeric threonyl-tRNA synthetase were studied by chemical and enzymatic footprinting experiments. Alkylation of phosphodiester bonds by ethylnitrosourea and of N-7 positions in guanosines and N-3 positions in cytidines by dimethyl sulphate as well as carbethoxylation of N-7 positions in adenosines by diethyl pyrocarbonate were conducted on different conformers of tRNA:hr. The enzymatic structural probes were nuclease S1 and the cobra venom ribonuclease. Results will be compared to those of three other tRNAs, tRNAAspp, tRNAPhe and tRNATrp, already mapped with these probes.The reactivity of phosphates towards ethylnitrosourea of the unfolded tRNA was compared to that of the native molecule. The alkylation pattern of tRNATh' shows some similarities to that of yeast tRNAPhe and mammalian tRNATrp, especially in the D-arm (positions 19 and 24) and with tRNATrp, at position 50, the junction between the variable region and the T-stem. In the T-loop, tRNAThr, similarly to the three other tRNAs, shows protections against alkylation at phosphates 59 and 60. However, tRNA:h' is unique as far as very strong protections are also found for phosphates 55 to 58 in the T-loop. Compared with yeast tRNAAspp, the main differences in reactivity concern phosphates 19, 24 and 50.Mapping of bases with dimethyl sulphate and diethyl pyrocarbonate reveal conformational similarities with yeast tRNAPhe. A striking conformational feature of tRNATh' is found in the 3'-side of its anticodon stem, where G40, surrounded by two G residues, is alkylated under native conditions, in contrast to other G residues in stem regions of tRNAs which are unreactive when sandwiched between two purines. This data is indicative of a perturbed helical conformation in the anticodon stem at the level of the 30-40 base pairs.Footprinting experiments, with chemical and enzymatic probes, on the tRNA complexed with its cognate threonyl-tRNA synthetase indicate significant protections in the anticodon stem and loop region, in the extraloop, and in the amino acid accepting region. The involvement of the anticodon of tRNA:h' in the recognition process with threonyl-tRNA synthetase was demonstrated by nuclease S1 mapping and by the protection of G34 and G35 against alkylation by dimethyl sulphate. These data are discussed in the light of the tRNA/synthetase recognition problem and of the structural and functional properties of the tRNA-like structure present in the operator region of the thrS mRNA.The understanding of the specific recognition and aminoacylation of tRNAs by their cognate aminoacyl-tRNA synthetases requires a precise structural knowledge of a number of different tRNA/synthetase systems. With such information it is expected one will find the conformational differences and similarities existing between such different systems and reveal structural rules responsible for the recognition process. From the tRNA point of view, many experimental (see...

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Section: Complex Formation Between Trnath' and Threonyl-trna Syn Thetasesupporting

confidence: 84%

Tertiary structure of Escherichia coli tRNA^Thr₃ in solution and interaction of this tRNA with the cognate threonyl‐tRNA synthetase

Theobald

Springer

Grunberg‐Manago

et al. 1988

European Journal of Biochemistry

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“…Although both enzymes belong to the nucleotidyltransferase superfamily (Yue et al ., 1996), the eubacterial and archaeal CCA‐adding enzymes belong to different subfamilies which apparently share little or no sequence homology outside the active site signature sequence (Martin and Keller, 1996; Yue et al ., 1996). ENU has been used previously to examine tRNA tertiary structure (Vlassov et al ., 1981), as well as the interaction of tRNAs with cognate aminoacyl‐tRNA synthetases (Garret et al ., 1983; Vlassov et al ., 1983; Romby et al ., 1985), with elongation factor Tu (Riehl et al ., 1983) and with retroviral reverse transcriptase (Garret et al ., 1984). Although data obtained by ENU alkylation of phosphates could, in principle, be compromised by concurrent modification of protein amino or thiol groups (Margison and O'Connor, 1978), this has not proved to be a problem.…”

Section: Discussionmentioning

confidence: 99%

“…Ethylnitrosourea (ENU) preferentially alkylates phosphate groups in RNA and DNA, and has been used widely to study RNA structure and protein–nucleic acid interactions (Vlassov et al ., 1981, 1983; Garret et al ., 1983, 1984; Riehl et al ., 1983; Romby et al ., 1985; Cavarelli et al ., 1993). We have now used ENU to identify phosphates in tRNA Asp of Bacillus subtilis that are essential for addition of CTP to tRNA‐C, and ATP to tRNA‐CC.…”

Section: Introductionmentioning

confidence: 99%

CCA addition by tRNA nucleotidyltransferase: polymerization without translocation?

1998

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The CCA-adding enzyme repairs the 3Ј-terminal CCA sequence of all tRNAs. To determine how the enzyme recognizes tRNA, we probed critical contacts between tRNA substrates and the archaeal Sulfolobus shibatae class I and the eubacterial Escherichia coli class II CCAadding enzymes. Both CTP addition to tRNA-C and ATP addition to tRNA-CC were dramatically inhibited by alkylation of the same tRNA phosphates in the acceptor stem and TΨC stem-loop. Both enzymes also protected the same tRNA phosphates in tRNA-C and tRNA-CC. Thus the tRNA substrate must remain fixed on the enzyme surface during CA addition. Indeed, tRNA-C cross-linked to the S.shibatae enzyme remains fully active for addition of CTP and ATP. We propose that the growing 3Ј-terminus of the tRNA progressively refolds to allow the solitary active site to reuse a single CTP binding site. The ATP binding site would then be created collaboratively by the refolded CC terminus and the enzyme, and nucleotide addition would cease when the nucleotide binding pocket is full. The template for CCA addition would be a dynamic ribonucleoprotein structure.

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“…The phosphate backbone of the tRNA was ethylated with ENU according to (19,22,23) and the modification interference assay using ENU was performed according to (24,25) with slight modifications. The overall scheme of this assay is shown in Figure 1A.…”

Section: Methodsmentioning

confidence: 99%

A unique tRNA recognition mechanism of Caenorhabditis elegans mitochondrial EF-Tu2

Suematsu¹,

Sato²,

Sakurai³

et al. 2005

Nucleic Acids Research

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Nematode mitochondria expresses two types of extremely truncated tRNAs that are specifically recognized by two distinct elongation factor Tu (EF-Tu) species named EF-Tu1 and EF-Tu2. This is unlike the canonical EF-Tu molecule that participates in the standard protein biosynthesis systems, which basically recognizes all elongator tRNAs. EF-Tu2 specifically recognizes Ser-tRNASer that lacks a D arm but has a short T arm. Our previous study led us to speculate the lack of the D arm may be essential for the tRNA recognition of EF-Tu2. However, here, we showed that the EF-Tu2 can bind to D arm-bearing Ser-tRNAs, in which the D–T arm interaction was weakened by the mutations. The ethylnitrosourea-modification interference assay showed that EF-Tu2 is unique, in that it interacts with the phosphate groups on the T stem on the side that is opposite to where canonical EF-Tu binds. The hydrolysis protection assay using several EF-Tu2 mutants then strongly suggests that seven C-terminal amino acid residues of EF-Tu2 are essential for its aminoacyl-tRNA-binding activity. Our results indicate that the formation of the nematode mitochondrial (mt) EF-Tu2/GTP/aminoacyl-tRNA ternary complex is probably supported by a unique interaction between the C-terminal extension of EF-Tu2 and the tRNA.

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Effect of elongation factor Tu on the conformation of phenylalanyl‐tRNA^Phe

Cited by 27 publications

References 22 publications

Tertiary structure of Escherichia coli tRNA^Thr₃ in solution and interaction of this tRNA with the cognate threonyl‐tRNA synthetase

Tertiary structure of Escherichia coli tRNA^Thr₃ in solution and interaction of this tRNA with the cognate threonyl‐tRNA synthetase

CCA addition by tRNA nucleotidyltransferase: polymerization without translocation?

A unique tRNA recognition mechanism of Caenorhabditis elegans mitochondrial EF-Tu2

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Effect of elongation factor Tu on the conformation of phenylalanyl‐tRNAPhe

Cited by 27 publications

References 22 publications

Tertiary structure of Escherichia coli tRNAThr3 in solution and interaction of this tRNA with the cognate threonyl‐tRNA synthetase

Tertiary structure of Escherichia coli tRNAThr3 in solution and interaction of this tRNA with the cognate threonyl‐tRNA synthetase

CCA addition by tRNA nucleotidyltransferase: polymerization without translocation?

A unique tRNA recognition mechanism of Caenorhabditis elegans mitochondrial EF-Tu2

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Product

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Effect of elongation factor Tu on the conformation of phenylalanyl‐tRNA^Phe

Tertiary structure of Escherichia coli tRNA^Thr₃ in solution and interaction of this tRNA with the cognate threonyl‐tRNA synthetase

Tertiary structure of Escherichia coli tRNA^Thr₃ in solution and interaction of this tRNA with the cognate threonyl‐tRNA synthetase