Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.1 (S1 nuclease)
 3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Site-directed mutations were introduced in the connecting loops and one of the two stem regions of the RNA pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA. The kinetic parameters of valylation for each mutated RNA were determined in a cell-free extract from wheat germ. Structure mapping was performed on most mutants with enzymic probes, like RNase T1, nuclease S1 and cobra venom ribonuclease. An insertion of four A residues in the four-membered connecting loop L1 that crosses the deep groove of the pseudoknot reduces aminoacylation efficiency. Deletions up to three nucleotides do not affect aminoacylation or RNA pseudoknot formation. Deletion of the entire loop abolishes aminoacylation. Although elimination of the pseudoknot is presumed, this could not be demonstrated. Unlike the mutations in loop L1, all mutations in the three-membered connecting loop L2 that crosses the shallow groove of the RNA pseudoknot decrease the aminoacylation efficiency considerably. Nonetheless, the RNA pseudoknot is still present in most mutated RNAs. These results indicate that a number of mutations can be introduced in both loops without abolishing aminoacylation. Results obtained with the introduction of mismatches and A.U base-pairs in stem S1 of the pseudoknot, containing three G.C base-pairs in wild-type RNA, indicate that the pseudoknot is only marginally stable. Our estimation of the gain of free energy due to the pseudoknot formation is at most 2.0 kcal/mol. The pseudoknot structure can, however, be stabilized upon binding the valyl-tRNA synthetase.
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PMID:Mutational analysis of the pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA. Aminoacylation efficiency and RNA pseudoknot stability. 173 Oct 70

We report the subcloning and characterization of the molecular elements necessary for the expression of the Escherichia coli valS gene which encodes the enzyme valyl-tRNA synthetase (EC 6.1.1.9). The valS gene was subcloned from the Clarke-Carbon plasmid pLC26-22 by genetic complementation of the valS temperature-sensitive mutant strain, AB4141. The protein-coding region of the valS structural gene was determined by in vitro DNA directed coupled transcription-translation assays. Assays of cellular extracts of cells transformed with a plasmid containing a full length copy of the valS gene enhanced in vivo valyl-tRNA synthetase-specific activity 12-fold. The DNA sequences of the 5'- and 3'-terminal regions of the valS structural gene are presented. The transcription initiation sites of the valS gene were determined, in vivo and in vitro, by S1 nuclease protection studies, primer-extension analysis and both alpha-32P labeled and gamma-32P-end-labeled in vitro transcription assays. In vivo, valS transcription initiates from tandem overlapping promoters separated by seven nucleotides. In vitro, only the upstream promoter is active. The presence of several regions of hyphenated dyad symmetry overlapping the tandem promoter region are noted. The valS translational start codon (AUG) is located 93 base pairs downstream from the major transcription initiation site. The valS transcriptional unit encodes only the valyl-tRNA synthetase gene since the 3' terminus of the amino acid-coding region of this gene is followed closely (26 base pairs) by an efficient rho-independent transcription termination site.
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PMID:Valyl-tRNA synthetase gene of Escherichia coli K12. Molecular genetic characterization. 327 59

The tRNA-like structure of turnip yellow mosaic virus is known to be efficiently recognized and aminoacylated by valyl-tRNA synthetase. The present work reports domains in the isolated tRNA-like fragment (159 terminal nucleotides at the 3'-end of the two viral RNAs) in contact with purified yeast valyl-tRNA synthetase. These domains were determined in protection experiments using chemical and enzymatic structural probes. In addition, new data, re-enforcing the validity of the tertiary folding model for the native RNA, are given. In particular, at the level of the amino acid accepting arm it was found that the two phosphate groups flanking the three guanine residues of loop I are inaccessible to ethylnitrosourea. This is in agreement with a higher-order structure of this loop involving "pseudo knotting", as proposed by Rietveld et al. (1982). Valyl-tRNA synthetase efficiently protects the viral RNA against digestion by single-strand-specific S1 nuclease at the level of the anticodon loop. With cobra venom ribonuclease, specific for double-stranded regions of RNA, protection was detected on both sides of the anticodon arm and at the 5'-ends of loop I, a region that is involved in the building up of the acceptor arm. Loop II, which is topologically homologous to the T-loop of canonical tRNA was likewise protected. Weak protection was observed between arms I and II, and at the 3'-side of arm V. This arm, located at the 5'-side of arm IV (homologous to the D-arm of tRNA), does not participate in the pseudo-knotted model of the valine acceptor arm. Ethylnitrosourea was used to determine the phosphates of the tRNA-like structure in close contact with the synthetase. These are grouped in several stretches scattered over the RNA molecule. In agreement with the nuclease digestion results, protected phosphates are located in arms I, II, and III. Additionally, this chemical probe permits detection of other protected phosphates on the 3'-side of arm IV and on both sides of arm V. When displayed in the three-dimensional model of the tRNA-like structure, protected areas are localized on both limbs of the L-shaped RNA. It appears that valyl-tRNA synthetase embraces the entire tRNA-like structure. This is reminiscent of the interaction model of canonical yeast tRNAVal with its cognate synthetase.
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PMID:Contact areas of the turnip yellow mosaic virus tRNA-like structure interacting with yeast valyl-tRNA synthetase. 354 Mar 11

We have studied the interactions between Escherichia coli tRNAVal and valyl-tRNA synthetase (ValRS) by enzymatic footprinting with nuclease S1 and ribonuclease V1, and by analysis of the aminoacylation kinetics of mutant tRNAVal transcripts. Valyl-tRNA synthetase specifically protects the anticodon loop, the 3' side of the stacked T-stem/acceptor-stem helix, and the 5' side of the anticodon stem of tRNAVal against cleavage by double- and single-strand-specific nucleases. Increased nuclease susceptibility at the ends of the anticodon- and T-stems in the tRNAVal.ValRS complex is indicative of enzyme-induced conformational changes in the tRNA. The most important synthetase recognition determinants are the middle and 3' anticodon nucleotides (A35 and C36, respectively); G20, in the variable pocket, and G45, in the tRNA central core, are minor recognition elements. The discriminator base, position 73, and the anticodon stem also are recognized by ValRS. Replacing wild-type A73 with G73 reduces the aminoacylation efficiency more than 40-fold. However, the C73 and U73 mutants remain good substrates for ValRS, suggesting that guanosine at position 73 acts as a negative determinant. The amino acid acceptor arm of tRNAVal contains no other synthetase recognition nucleotides, but regular A-type RNA helix geometry in the acceptor stem is essential [Liu, M., et al. (1997) Nucleic Acids Res. 25, 4883-4890]. In the anticodon stem, converting the U29:A41 base pair to C29:G41 reduces the aminoacylation efficiency 50-fold. This is apparently due to the rigidity of the anticodon stem caused by the presence of five consecutive C:G base pairs, since the A29:U41 mutant is readily aminoacylated. Identity switch experiments provide additional evidence for a role of the anticodon stem in synthetase recognition. The valine recognition determinants, A35, C36, A73, G20, G45, and a regular A-RNA acceptor helix are insufficient to transform E. coli tRNAPhe into an effective valine acceptor. Replacing the anticodon stem of tRNAPhe with that of tRNAVal, however, converts the tRNA into a good substrate for ValRS. These experiments confirm G45 as a minor ValRS recognition element.
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PMID:Synthetase recognition determinants of E. coli valine transfer RNA. 1038 13

The secondary structure of the isolated tRNA-like sequence (n=159) present at the 3' OH terminus of turnip yellow mosaic virus RNA has been established from partial nuclease digestion with S1 nuclease and T1, CL(3), and Naja oxiana RNases. The fragment folds into a 6-armed structure with two main domains. The first domain, of loose structure and nearest the 5' OH terminus, is composed of one large arm which extends into the coat protein cistron. The second, more compact domain, is composed of the five other arms and most probably contains the structure recognized by valyl-tRNA synthetase. In this domain three successive arms strikingly resemble the T[unk], anticodon, and D arms found in tRNA. Near the amino-acid accepting terminus, however, there is a new stem and loop region not found in standard tRNA. This secondary structure is compatible with a L-shaped three-dimensional organization in which the corner of the L and the anticodon-containing limb are similar to, and the amino-acid accepting region different from, that in tRNA. Ethylnitrosourea accessibility studies have shown similar tertiary structure features in the T[unk] loop of tRNA and in the homologous region of the viral RNA.
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PMID:The tRNA-like structure of turnip yellow mosaic virus RNA: structural organization of the last 159 nucleotides from the 3' OH terminus. 1645 15