Effects of nucleotide substitutions within the T-loop of precursor tRNAs on interaction with ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and yeast

Abstract

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.

CCA addition by tRNA nucleotidyltransferase: polymerization without translocation?

“…Alkylation of several phosphates in the T‐loop interferes with CCA‐adding activity (data summarized in Figure 5). Modification of nucleotides responsible for tertiary interactions between the D‐ and T‐loops (Spacciapoli et al ., 1989) and cytidine substitutions at nucleotides 57 or 58 of the TΨCG loop (Li et al ., 1996) have also been shown to inhibit the CCA‐adding enzyme. However, it is not yet clear whether inhibition of activity resulting from alkylation of T‐loop phosphates blocks interactions between the T‐loop and the enzyme directly, or inhibits enzyme activity indirectly by locally distorting the RNA backbone.…”

Key Elements in Maintenance of the tRNA L-shape

“…For example, it was shown, that disruption of base-pairs between the D and T-loops had a negative effect on the 3 0 and 5 0 processing of tRNA His from Drosophila, 11 while positions 57 and 58 of the T-loop were involved in the recognition by the CCA-adding enzyme. 13 However, there has been no evidence that these enzymes are sensitive to the juxtaposition of the two helical domains in the tRNA rather than to the identity of particular nucleotides or to the integrity of particular base pairs.…”

This Is the End: Processing, Editing and Repair at the tRNA 3-Terminus

“…In accordance with these results, footprinting analysis revealed that most of the contacts between tRNA and the enzyme were found in the top-half domain of the tRNA (Shi et al, 1998b). Thereby, nucleotides at position 56, 57 and 58 play a prominent role in recognition and interaction with the nucleotidyltransferase (Hegg and Thurlow, 1990;Li et al, 1996;Li et al, 1997). Thereby, nucleotides at position 56, 57 and 58 play a prominent role in recognition and interaction with the nucleotidyltransferase (Hegg and Thurlow, 1990;Li et al, 1996;Li et al, 1997).…”