Role of the extra G-C pair at the end of the acceptor stem of tRNA^Hbin aminoacylation

Abstract

All sequenced histidine tRNAs have one additional nucleotide at the 5' end compared with other tRNA species. To investigate the role of this unique structure in aminoacylation, we constructed in vitro transcripts corresponding to the E. coli histidine tRNA sequence and its variants at the G-1-C73 base pair, by using T7 RNA polymerase transcription system. A transcript having a wild-type sequence with no modified bases was a good substrate for histidyl-tRNA synthetase (HisRS), and aminoacylation activity was affected by introduction of a triphosphate at the 5' terminus. Base replacements at position 73 caused a marked decrease of Vmax, and deletion and substitution of the G-1 had a remarkable effect on the aminoacylation. A mutant having an A-1-U73 pair was also not a good substrate for HisRS. Comparison among G-1-deficient mutants showed that A was preferable rather than C as the base at position 73. These data demonstrate that the set of the G-1-C73 pair at the end of the acceptor stem of histidine tRNA is crucial for the catalytic process of aminoacylation.

tRNAHis maturation: An essential yeast protein catalyzes addition of a guanine nucleotide to the 5′ end of tRNAHis

“…In most cases p-tRNA was generated by inclusion of 20 mM GMP during in vitro transcription, which results in transcripts mostly beginning with p-tRNA (Himeno et al 1989;Sampson et al 1989). For the experiment shown in Figure 5B, substrate beginning only with p-tRNA was prepared by transcription of pGu14 to produce tRNA with 18 additional nucleotides (GAACCCTGTGCAAG CAAC) at its 5Ј end, followed by gel purification of the product, and treatment with RNase H in the presence of a chimeric 2Ј-O-methylated/deoxy oligonucleotide (HHMI/Keck Center, Yale Univ.)…”

Efficient aminoacylation of a yeast tRNAAsp transcript with a 5' extension

“…Despite this structural peculiarity, viral tRNA-like molecules are efficiently aminoacylated. In contrast however, E. coli tRNAHi" which is known to have an extra G-l residue at the 5 ' end of its acceptor stem has its aminoacylation activity affected by introduction of a triphosphate at the 5 ' terminus [29], but this behaviour may be explained by the fact that G-l base-pairs with the discriminator residue C73 and thus brings the triphosphate group in closer vicinity to the catalytic site of the synthetase. It is interesting to note that the arginylation ability of extended tRNAASp is reduced as compared to that of the normal-length tRNA*"* transcript, while its aspartylation ability is similar.…”

Identity elements of Thermus thermophilus tRNAThr

“…Synthetic DNA oligomers carrying the T7 promoter and tRNA genes were ligated into pUC19 and transformed into E. coli strain JM109 [3,23,24]. The template DNA sequences were confirmed by dideoxy sequencing [25].…”

“…Each template DNA for the discriminator base-substituted mutant was prepared from a plasmid carrying the normal tRNA sequence and two synthetic primers by mutation using the polymerase chain reaction [26]. Transcripts of the tRNA genes were prepared in a reaction mixture containing 40 mM Tris-HCl (pH 8.1), 5.0 mM dithiothreitol, 2.0 mM spermidine, 10 mM magnesium chloride, bovine serum albumin (50 I.tg/ml), 2.0 mM of each NTP, 20 mM 5' GMP, BstNI-digested template DNA (0.2 mg/ml), 2 units of inorganic pyrophosphatase (Sigma) and pure T7 RNA polymerase (50 I.tg/ml) [3,23,27]. Transcripts initiated with A were prepared in a reaction mixture containing 20 mM 5' AMP instead of 5' GMP [23].…”

“…Transcripts of the tRNA genes were prepared in a reaction mixture containing 40 mM Tris-HCl (pH 8.1), 5.0 mM dithiothreitol, 2.0 mM spermidine, 10 mM magnesium chloride, bovine serum albumin (50 I.tg/ml), 2.0 mM of each NTP, 20 mM 5' GMP, BstNI-digested template DNA (0.2 mg/ml), 2 units of inorganic pyrophosphatase (Sigma) and pure T7 RNA polymerase (50 I.tg/ml) [3,23,27]. Transcripts initiated with A were prepared in a reaction mixture containing 20 mM 5' AMP instead of 5' GMP [23]. The transcripts were purified by 20% polyacrylamide gel electrophoresis.…”