Studies with tRNA adenylyl(cytidylyl)transferase from Escherichia coli B

Best, Audrey N.; Novelli, G. David

doi:10.1016/0003-9861(71)90516-9

Cited by 29 publications

(12 citation statements)

References 30 publications

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“…Cu 2ϩ stimulated the hydrolysis of pyrophosphate and ATP and completely inhibited the hydrolysis of 2Ј-AMP, suggesting some difference in the active sites involved in these reactions. Because the nucleotidyltransferase activity of the E. coli tRNA-NT (which also involves the hydrolysis of ATP and CTP) had an alkaline pH optimum (pH 9.0 -9.5) and required Mg 2ϩ (16,17), it is not related to the Ni 2ϩ -dependent neutral phosphatase and 2Ј-nucleotidase activities observed in our experiments.…”

Section: Resultsmentioning

confidence: 76%

“…All three fractions (monomers, dimers, and oligomers) had approximately the same level of phosphatase activity with pNPP. In contrast to the characterized nucleotidyltransferase activity of this protein (16,17), the phosphatase activity had a neutral pH optimum, pH 7.0, and was greatly stimulated by Ni 2ϩ (Fig. 1).…”

Section: Resultsmentioning

confidence: 97%

“…3; Table I). For the nucleotidyltransferase reaction, 1 mM PP i caused only 20% inhibition (16). This insensitivity to PP i can be explained by the presence of pyrophosphatase activity in the E. coli tRNA-NT.…”

Section: Resultsmentioning

confidence: 98%

“…Class II enzymes have similar sizes (400 -470 amino acids) and share a highly conserved 25-kDa N-terminal region with characteristic active-site signature motifs (DxD and RRD), but differ from one another in their C-terminal regions. The E. coli tRNA-NT belongs to the class II enzymes, and its nucleotidyltransferase activity has been characterized (16,17). This protein has two domains, the N-terminal nucleotidyltransferase domain and * The costs of publication of this article were defrayed in part by the payment of page charges.…”

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confidence: 99%

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The HD Domain of the Escherichia coli tRNA Nucleotidyltransferase Has 2′,3′-Cyclic Phosphodiesterase, 2′-Nucleotidase, and Phosphatase Activities

Yakunin¹,

Proudfoot²,

Kuznetsova³

et al. 2004

Journal of Biological Chemistry

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In all mature tRNAs, the 3-terminal CCA sequence is synthesized or repaired by a template-independent nucleotidyltransferase (ATP(CTP):tRNA nucleotidyltransferase; EC 2.7.7.25). The Escherichia coli enzyme comprises two domains: an N-terminal domain containing the nucleotidyltransferase activity and an uncharacterized C-terminal HD domain. The HD motif defines a superfamily of metal-dependent phosphohydrolases that includes a variety of uncharacterized proteins and domains associated with nucleotidyltransferases and helicases from bacteria, archaea, and eukaryotes. The C-terminal HD domain in E. coli tRNA nucleotidyltransferase demonstrated Ni 2؉ -dependent phosphatase activity toward pyrophosphate, canonical 5-nucleoside tri-and diphosphates, NADP, and 2-AMP. Assays with phosphodiesterase substrates revealed surprising metal-independent phosphodiesterase activity toward 2,3-cAMP, -cGMP, and -cCMP. Without metal or in the presence of Mg 2؉ , the tRNA nucleotidyltransferase hydrolyzed 2,3-cyclic substrates with the formation of 2-nucleotides, whereas in the presence of Ni 2؉ , the protein also produced some 3-nucleotides. Mutations at the conserved His-255 and Asp-256 residues comprising the C-terminal HD domain of this protein inactivated both phosphodiesterase and phosphatase activities, indicating that these activities are associated with the HD domain. Low concentrations of the E. coli tRNA (10 nM) had a strong inhibiting effect on both phosphatase and phosphodiesterase activities. The competitive character of inhibition by tRNA suggests that it might be a natural substrate for these activities. This inhibition was completely abolished by the addition of Mg 2؉ , Mn 2؉ , or Ca 2؉ , but not Ni 2؉ . The data suggest that the phosphohydrolase activities of the HD domain of the E. coli tRNA nucleotidyltransferase are involved in the repair of the 3-CCA end of tRNA.

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Section: Resultsmentioning

confidence: 76%

Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 98%

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confidence: 99%

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The HD Domain of the Escherichia coli tRNA Nucleotidyltransferase Has 2′,3′-Cyclic Phosphodiesterase, 2′-Nucleotidase, and Phosphatase Activities

Yakunin¹,

Proudfoot²,

Kuznetsova³

et al. 2004

Journal of Biological Chemistry

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“…"?N-C was prepared and converted to tRNAPheN-C-A. Analysis of the base sequence of the terminal T, RNAase fragment established that tRNA?h!N-C-A is indeed formed but is unable to accept phenylalanine (Fig.3, 4, 5) or 15 other amino acids. Thus, the inability of tRNA.. .…”

Section: Discussionmentioning

confidence: 99%

Biological Activity of Escherichia coli tRNA^Phe Modified in Its C‐C‐A Terminus

Tal

Littauer

Deutscher

1972

European Journal of Biochemistry

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An improved method for the stepwise degradation of tRNA by means of periodate oxidation, amine‐catalyzed elimination and alkaline phosphatase treatment was developed. Alkaline phosphatase was found to hydrolyze the exposed 3′‐phosphate group of tRNA…N‐C‐Cp at a much faster rate than that of tRNA…N‐Cp. This difference in rate of dephosphorylation was manifested at several temperatures. Terminally modified Escherichia coli tRNA, from which all or part of the C‐C‐A was removed was characterized by its ability to accept AMP and CMP using rat liver tRNA nucleotidyltransferase. It was found that tRNA…N‐C‐C accepts AMP but not CMP; tRNA…N‐C accepts CMP and AMP in a molar ratio of 1:1 and tRNA…N accepts CMP and AMP in a molar ratio of 2:1. In addition, it was observed that tRNA…N‐C accepts AMP in the absence of CTP in the reaction mixture. The product of AMP incorporation into tRNAPhe…N‐C was shown to be tRNAPhe…‐N‐C‐A, by electrophoretic analysis of the 3′‐terminal fragment obtained by T1 ribonuclease digestion. It was also shown that tRNA…N‐C‐A can not be acylated with a mixture of 15 amino acids, suggesting that an intact C‐C‐A sequence is an absolute requirement for aminoacylation. Under conditions of high enzyme concentrations, in vitro, tRNA nucleotidyltransferase also misincorporates AMP residues into tRNA…N. The ability of various terminally modified tRNAs to inhibit phenylalanine charging of tRNA was determined by kinetic measurements. tRNAox, obtained by periodate oxidation of tRNA, served as a competitive inhibitor for phenylalanine acylation of intact tRNA. The measured Ki value of 40 nM equaled the apparent Km for tRNAPhe. tRNA…N‐C‐Cp, tRNA…N‐C‐C, tRNA…N‐C, tRNA…N and tRNA…N‐C‐A were found to be less competent competitive inhibitors, their Ki values being one order of magnitude higher. These results suggest that the terminal adenosine participates in the binding of tRNAPhe to phenylalanyl‐tRNA synthetase and that the relative position of this adenosine with respect to the rest of the tRNA molecule is probably critical for an effective binding to the enzyme. The reduction of tRNAox with sodium borohydride converts the terminal dialdehyde group to a dialcohol lacking a covalent bond between the C′2 and C′3 of the terminal adenosine. Whereas unfractionated tRNAox could not serve as an acceptor for any amino acids, reduction to tRNAox‐red restored the abilities to accept phenylalanine, methionine and tyrosine, On the other hand, acceptor activities for arginine, isoleucine, aspartic acid, histidine, serine, tryptophan and valine were not regained. It is concluded that the covalent bond between the C′2 and C′3 of the terminal adenosine is a decisive determinant for the aminoacylation of some Escherichia coli tRNAs but plays a minor role in other species. Terminally modified tRNAs inhibited poly(U)‐directed [14C]phenylalanyl‐tRNA binding to 30‐S or a mixture of 30‐S and 50‐S ribosomes as effectively as intact, unacylated tRNA. Thus, the C‐C‐A terminus does not contribute significantly to the binding of tRNAPhe t...

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