A tRNA Identity Switch Mediated by the Binding Interaction between a tRNA Anticodon and the Accessory Domain of a Class II Aminoacyl-tRNA Synthetase

Abstract

Identity elements in tRNAs and the intracellular balance of tRNAs allow accurate selection of tRNAs by aminoacyl-tRNA synthetases. The histidyl-tRNA from Escherichia coli is distinguished by a unique G-1.C73 base pair that upon exchange with other nucleotides leads to a marked decrease in the rate of aminoacylation in vitro. G-1.C73 is also a major identity element for histidine acceptance, such that the substitution of C73 brings about mischarging by glycyl-, glutaminyl-, and leucyl-tRNA synthetases. These identity conversions mediated by the G-1.C73 base pair were exploited to isolate secondary site revertants in the histidyl-tRNA synthetase from E. coli which restore histidine identity to a histidyl-tRNA suppressor carrying U73. The revertant substitutions confer a 3-4 fold reduction in the Michaelis constant for tRNAs carrying the amber-suppressing anticodon and map to the C-terminal domain of HisRS and its interface with the catalytic core. These findings demonstrate that the histidine tRNA anticodon plays a significant role in tRNA selection in vivo and that the C-terminal domain of HisRS is in large part responsible for recognizing this trinucleotide. The kinetic parameters determined also show a small degree of anticooperativity (delta delta G = -1.24 kcal/mol) between recognition of the discriminator base and the anticodon, suggesting that the two helical domains of the tRNA are not recognized independently. We propose that these effects substantially account for the ability of small changes in tRNA binding far removed from the site of a major determinant to bring about a complete conversion of tRNA identity.

Histidyl-tRNA Synthetase

“…The latter two ranges for the Michaelis constants are remarkably large for unknown reasons; a possible explanation would be that the values were obtained over a wide pH range. The K m values of the ATP/PP i exchange reaction are between (Yan et al, 1996;Augustine and Francklyn, 1997;Rühlmann et al, 1997;Francklyn et al, 1998). The enzyme from rabbit reticulocytes shows a turnover of 84 min -1 (= 1.4 s -1 ) and has an isoelectric point of 5.1 (Kane et al, 1978).…”

“…Chromatography on Affi-Gel blue and DNA-agarose (Chen and Somberg, 1980), Blue Sepharose and Poly-U Sepharose (Biswas et al, 1987a) or high pressure liquid chromatography (Biswas et al, 1987a) and preparative gel chromatography (Kalousek and Konigsberg, 1974) have also been used. These and similar methods have also been applied in the purification of several mutant HisRS proteins (Rühlmann et al, 1997;Yan et al, 1996). A stabilization protocol for isolated human HisRS by hemoglobin or hematin has been described by Biswas et al (1988).…”

“…At a high cellular concentration HisRS binds the tRNA-like structure of the mRNA, thereby arresting its own synthesis by preventing the mRNA from further binding to the ribosome. Several vectors for overexpression of the E. coli HisRS gene have been constructed (Eisenbeis and Parker, 1981;Glade and Englisch, 1988;Markmeyer et al, 1990;Englisch et al, 1990;Francklyn et al, 1994;Yan et al, 1996;Rühlmann et al, 1997), some of which have been used to prepare large amounts of purified HisRS Yan et al, 1996;Rühlmann et al, 1997). Additional constructs can be found in the references listed in Table 2.…”

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

“…The tRNA gene typically terminates with a Fok I or Bst N1 restriction site, such that run-off transcription of the linearized plasmid produces a tRNA transcript with the correct CCA end. Reaction conditions for different tRNA sequences can be optimized by fine-tuning of dNTP concentrations, temperature, T7 RNAP concentration, and other variables [21,23,24]. Transcript purification is performed by fractionation on 8M urea/12% polyacrylamide gels, or by reverse phase chromatography on a preparative C4 column.…”

“…Typical purification protocols involve initial purification over a Ni-NTA column (Qiagen), followed by anion exchange chromatography to remove contaminating proteins. Detailed protocols for purification of aaRS from over expression strains have been published previously [23,[34][35][36]. Removal of the His 6 tag by incorporating a specific protease site (e.g.…”