Document Detail

Effects of nucleotide substitutions within the T-loop of precursor tRNAs on interaction with ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and yeast.
MedLine Citation:
PMID:  8660309     Owner:  NLM     Status:  MEDLINE    
Recognition of tRNA and tRNA-like substrates by the enzyme ATP/CTP:tRNA nucleotidyltransferase requires chemically intact nucleotides within the T-loop, especially at positions 57 and 58, which are invariant purines among naturally occurring tRNAs. To test the effects of base substitutions at these positions, which are distant from the site of catalysis, we synthesized mutant tRNA(Glu) molecules. These in vitro-synthesized RNAs also contained an extra 33 bases at the 5' end and lacked post-transcriptionally modified bases. The precursor tRNAs were used as substrates for nucleotidyltransferases from Escherichia coli and yeast. Substitution of cytidines at either position 57 or 58 had dramatic inhibitory effects on recognition by both enzymes, including raising the apparent Km and lowering the apparent Vmax.; substitution of an adenosine at position 57 or a uridine at position 58 inhibited the reaction only slightly by comparison. Our results demonstrate that the identities of nucleotides at positions 57 and 58 are relevant to recognition by nucleotidyltransferase, and that a purine is required at position 57. The extra bases at the 5' end and the lack of post-transcriptionally modified bases did not substantially inhibit interaction with the enzyme, as judged by the wild-type precursor tRNA(Glu) acting as an effective substrate for both enzymes in the presence of equal concentrations of appropriate tRNA substrates isolated from E. coli.
Z Li; K A Gillis; L A Hegg; J Zhang; D L Thurlow
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Biochemical journal     Volume:  314 ( Pt 1)     ISSN:  0264-6021     ISO Abbreviation:  Biochem. J.     Publication Date:  1996 Feb 
Date Detail:
Created Date:  1996-07-26     Completed Date:  1996-07-26     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  49-53     Citation Subset:  IM    
Department of Chemistry, Clark University, Worcester, MA 01610, USA.
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MeSH Terms
Adenosine Triphosphate / metabolism
Cytidine Triphosphate / metabolism
Electrophoresis, Polyacrylamide Gel
Escherichia coli / enzymology*,  genetics
Molecular Sequence Data
Nucleic Acid Conformation
RNA Nucleotidyltransferases / metabolism*
RNA Precursors / chemistry*,  genetics,  metabolism
RNA, Transfer, Glu / chemistry*,  genetics,  metabolism
Saccharomyces cerevisiae / enzymology*,  genetics
Substrate Specificity
Grant Support
Reg. No./Substance:
0/RNA Precursors; 0/RNA, Transfer, Glu; 56-65-5/Adenosine Triphosphate; 65-47-4/Cytidine Triphosphate; EC 2.7.7.-/RNA Nucleotidyltransferases; EC 2.7.7.-/tRNA nucleotidyltransferase

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