Determinant nucleotides of yeast tRNA(Asp) interact directly with aspartyl-tRNA synthetase.

Rudinger, Joëlle; Puglisi, Joseph D.; Pütz, Joern; Schatz, Dagmar; Eckstein, Fritz; Florentz, Catherine; Giegé, Richard

doi:10.1073/pnas.89.13.5882

Cited by 59 publications

(28 citation statements)

References 24 publications

(19 reference statements)

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“…Transcribed RNA containing phosphorothioates has about the same biological activity as its unmodified counterpart; this could be demonstrated with tRNAs and mRNAs [5][6][7]12]. Here 5S rRNA was used containing six or less phosphorothioate residues per molecule.…”

Section: Resultsmentioning

confidence: 99%

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5S rRNA sugar‐phosphate backbone protection in complexes with specific ribosomal proteins

et al. 1996

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5S ribosomal RNA forms stable specific complexes with ribosomal proteins LI8, L25 and L5. In this work, interaction of phosphate residues of E. coli 5S rRNA within 5S rRNA-protein complexes has been studied. For this purpose 5S rRNA with statistically distributed phosphorothioate residues has been used for complex formation and the accessibility of phosphorothioates to iodine cleavage in the complex and in the free state has been studied. In free 5S rRNA, the phosphate residue at A73 was partially protected, probably due to being involved in the organization of the spatial structure of 5S rRNA. This protection is stronger in the complex with three proteins when the 5S rRNA structure is stabilized. In the 5S rRNA-L18 complex only two phosphate groups, G7 and A34, were protected. L25 in a complex with 5S rRNA protects large numbers of phosphorothioate groups coneentrating in two clusters, indicating the possibility of two binding sites for this protein on 5S rRNA. The protection pattern differs from that for individual proteins because of the possible rearrangement of the structure.

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Section: Resultsmentioning

confidence: 99%

“…This approach was successfully applied in studies concerning interactions of sugarphosphate backbones of tRNAs with aminoacyl-tRNA synthetases [5,6] and of tRNA as well as of mRNA with ribosomes [7,8].…”

Section: Introductionmentioning

confidence: 99%

5S rRNA sugar‐phosphate backbone protection in complexes with specific ribosomal proteins

et al. 1996

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“…These results support the notion that the loop of the hairpin structure is involved in binding the translation factor but does not prove it, since due to their size nucleases may be prevented from cleavage by a binding protein via steric hindrance from a more remote site. Therefore, to define the SELB binding site in more detail, we employed a footprinting technique based on cleavage of phosphorothioate-containing transcripts by iodine (26,31). Phosphate ester bonds protected from iodine cleavage are believed to be in close contact with a binding protein because of the small size of this cleaving reagent.…”

Section: Resultsmentioning

confidence: 99%

Interaction of translation factor SELB with the formate dehydrogenase H selenopolypeptide mRNA.

Baron¹,

Heider²,

Böck³

1993

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

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The SELB protein from Escherichia coli is a specialized elongation factor required for the UGA-directed insertion of the amino acid selenocysteine into selenopolypeptides. Discrimination of the UGA codon requires the presence of a recognition element within the mRNA, which is located at the 3' side of the UGA codon; a hairpin structure can be formed within this mRNA region. By gel shift assays, a specific interaction between SELB and the mRNA recognition element could be demonstrated. Footprinting experiments, using nucleases or iodine as cleaving agents, showed that SELB binds to the loop region of the hairpin structure. In the presence of selenocysteinyl-tRNA, SELB formed a complex with the charged tRNA and the mRNA. The results indicate that targeted insertion of selenocysteine is accomplished by the binding of the SELB protein to this mRNA recognition element, resulting in the formation of a selenocysteinyl-tRNA-SELB complex at the mRNA in the immediate neighborhood of the UGA codon.A number of unusual coding events, which contradict the hitherto paradigmatic scheme ofprotein synthesis, have been discovered during the last few years (for review, see refs. 1 and 2). Besides ribosome hopping and frameshifting, a particularly intriguing finding was that the genetic capacity of a cell can be expanded in such a way that a nonstandard amino acid, selenocysteine, is incorporated into polypeptides. This amino acid is present in several proteins from organisms belonging to all three lines of descent, archaea, bacteria, and eukarya (for review, see refs. 3 and 4). The first indication that selenocysteine is inserted cotranslationally was provided by the finding that the genes for two selenoproteins-namely, fdhF coding for a formate dehydrogenase from Escherichia coli (5) and gpx coding for a glutathione peroxidase from mouse (6)-contain an in-frame TGA (UGA) codon. Since then, a number of genes coding for other selenoproteins also have been found to possess an in-frame TGA (UGA) codon (7-15). With the exception of the gene for plasma selenoprotein P, which contains 10 TGA (UGA) codons, all these genes contain only 1 such codon.A biologically basic question bearing considerable relevance for the understanding of the translation and the evolution of the genetic code is how the translational machinery can cope with the situation that one and the same triplet can signal either chain termination or selenocysteine insertion. The best-examined systems up to now are those of the E. coli formate dehydrogenase H (fdhF) and N (fdnG) genes (5, 10).Their analysis has revealed that a sequence of 40 bases in the mRNA at the 3' side of the UGA codon, which can be folded into a putative hairpin structure, is required for selenocysteine insertion (16, 17). Mutagenesis of this hairpin structure showed that the sequence of the loop region is particularly important and that it may serve as a recognition element for some putative factor directing the selenocysteine-inserting tRNA species (tRNASec) to decode that particular UGA (18)...

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“…Protein contacts on an RNA molecule can be identified by protection of phosphorothioate linkages from chemical cleavage by iodine (Schatz et al 1991;Rudinger et al 1992). In vitro-transcribed RNAs, with randomly incorporated phosphorothioate nucleotide analogs, were subjected to iodine-mediated cleavage in the presence or absence of the purified Ku heterodimer (Fig.…”

Section: Ku Interacts With the Terminal Stem-loop Of The Template Boumentioning

confidence: 99%

RNA recognition by the DNA end-binding Ku heterodimer

Dalby

Goodrich

Pfingsten

et al. 2013

RNA

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Most nucleic acid-binding proteins selectively bind either DNA or RNA, but not both nucleic acids. The Saccharomyces cerevisiae Ku heterodimer is unusual in that it has two very different biologically relevant binding modes: (1) Ku is a sequence-nonspecific double-stranded DNA end-binding protein with prominent roles in nonhomologous end-joining and telomeric capping, and (2) Ku associates with a specific stem-loop of TLC1, the RNA subunit of budding yeast telomerase, and is necessary for proper nuclear localization of this ribonucleoprotein enzyme. TLC1 RNA-binding and dsDNA-binding are mutually exclusive, so they may be mediated by the same site on Ku. Although dsDNA binding by Ku is well studied, much less is known about what features of an RNA hairpin enable specific recognition by Ku. To address this question, we localized the Ku-binding site of the TLC1 hairpin with single-nucleotide resolution using phosphorothioate footprinting, used chemical modification to identify an unpredicted motif within the hairpin secondary structure, and carried out mutagenesis of the stem-loop to ascertain the critical elements within the RNA that permit Ku binding. Finally, we provide evidence that the Ku-binding site is present in additional budding yeast telomerase RNAs and discuss the possibility that RNA binding is a conserved function of the Ku heterodimer.

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Determinant nucleotides of yeast tRNA(Asp) interact directly with aspartyl-tRNA synthetase.

Cited by 59 publications

References 24 publications

5S rRNA sugar‐phosphate backbone protection in complexes with specific ribosomal proteins

5S rRNA sugar‐phosphate backbone protection in complexes with specific ribosomal proteins

Interaction of translation factor SELB with the formate dehydrogenase H selenopolypeptide mRNA.

RNA recognition by the DNA end-binding Ku heterodimer

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