Function of the Extra 5‘-Phosphate Carried by Histidine tRNA

Abstract

Among elongator tRNAs, tRNA specific for histidine has the peculiarity to possess one extra nucleotide at position -1. This nucleotide is believed to be responsible for recognition by histidyl-tRNA synthetase. Here, we show that, in fact, it is the phosphate 5' to the extra nucleotide which mainly supports the efficiency of the tRNA aminoacylation reaction catalyzed by Escherichia coli histidyl-tRNA synthetase. In the case of the reaction of E. coli peptidyl-tRNA hydrolase, this atypical phosphate is dispensable. Instead, peptidyl-tRNA hydrolase recognizes the phosphate of the phosphodiester bond between residues -1 and +1 of tRNA(His). Recognition of the +1 phosphate of tRNA(His) by peptidyl-tRNA hydrolase resembles, therefore, that of the 5'-terminal phosphate of other elongator tRNAs.

RNA-binding Site of Escherichia coli Peptidyl-tRNA Hydrolase

“…In the tRNA His structure, this extra acceptor base pair covers the 5Ј-phosphate at the ϩ1 position, which we assume is necessary for recognition by Pth. However, as shown before (15), recognition of the ϩ1 phosphate in tRNA His by E. coli Pth resembles that of the 5Ј-terminal phosphate in the other elongator tRNA molecules, and therefore, peptidyl-tR-NA His behaves as a normal substrate of Pth. After functional comparison of the various prepared small RNAs, a duplex RNA of 13 base pairs mimicking the acceptor and TC stems of tRNA His was chosen to map by NMR the tRNA-binding site on the Pth molecule.…”

“…coli tRNA His was obtained from an overproducing E. coli strain, as already described (15 (15), and 5-50 M of the RNA substrate. After acetylation with acetic anhydride, the N-blocked aminoacylated RNA samples were purified through Chelex 100 (BioRad) and GF05 (IBF) chromatographies (15). Diacetyl-[ 14 C]Lys-tRNA Lys (50 Ci/mol) was prepared as described previously (8).…”

“…15 N-Labeled His-tagged H20A and H20A R133A Pth variants were produced in K37⌬recADE3 cells and purified by affinity chromatography as described previously in the case of the 15 N-labeled His-tagged H20A variant (24). Homogeneity of the samples was estimated to be higher than 95% by SDS-PAGE analysis.…”

“…-C 73 , which is an essential feature to direct aminoacylation by histidyl-tRNA synthetase (13,15). In the tRNA His structure, this extra acceptor base pair covers the 5Ј-phosphate at the ϩ1 position, which we assume is necessary for recognition by Pth.…”

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

“…Thus, absence of G −1 may lead to a lethal level of undercharging of tRNA His in the cell. We note that the corresponding G −1 :C 73 base pair of bacterial tRNA His is also an important determinant of synthetase recognition, although the essential contributions to tRNA identity may be from C 73 and the phosphate at position −1 (Francklyn and Schimmel 1990;Yan and Francklyn 1994;Fromant et al 2000).…”