CCA addition by tRNA nucleotidyltransferase: polymerization without translocation?
The CCA-adding enzyme repairs the 3'-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 CCA-adding 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 TPsiC 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 3'-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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Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520-8024, USA.
Maizels
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Weiner
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D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003432:Cross-Linking Reagents; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D000431:Ethanol / Q000494:pharmacology; D005038:Ethylnitrosourea / Q000494:pharmacology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010710:Phosphates / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D010710:Phosphates; D000431:Ethanol; D012343:RNA, Transfer; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase; D005038:Ethylnitrosourea