Biomaterial Database

RNA-assisted catalysis in a protein enzyme: The 2'-hydroxyl of tRNA(Thr) A76 promotes aminoacylation by threonyl-tRNA synthetase. 2008

Anand Minajigi, and Christopher S Francklyn

Cell and Molecular Biology Program, College of Medicine, Health Sciences Complex, 89 Beaumont Avenue, University of Vermont, Burlington, VT 05405, USA.

Aminoacyl-tRNA synthetases (aaRSs) join amino acids to 1 of 2 terminal hydroxyl groups of their cognate tRNAs, thereby contributing to the overall fidelity of protein synthesis. In class II histidyl-tRNA synthetase (HisRS) the nonbridging S(p)-oxygen of the adenylate is a potential general base for aminoacyl transfer. To test for conservation of this mechanism in other aaRSs and the role of terminal hydroxyls of tRNA in aminoacyl transfer, we investigated the class II Escherichia coli threonyl-tRNA synthetase (ThrRS). As with other class II aaRSs, the rate-determining step for ThrRS is amino acid activation. In ThrRS, however, the 2'-OH of A76 of tRNA(Thr) and a conserved active-site histidine (His-309) collaborate to catalyze aminoacyl transfer by a mechanism distinct from HisRS. Conserved residues in the ThrRS active site were replaced with alanine, and then the resulting mutant proteins were analyzed by steady-state and rapid kinetics. Nearly all mutants preferentially affected the amino acid activation step, with only a modest effect on aminoacyl transfer. By contrast, H309A ThrRS decreased transfer 242-fold and imposed a kinetic block to CCA accommodation. His-309 hydrogen bonds to the 2'-OH of A76, and substitution of the latter by hydrogen or fluorine decreased aminoacyl transfer by 763- and 94-fold, respectively. The proton relay mechanism suggested by these data to promote aminoacylation is reminiscent of the NAD(+)-dependent mechanisms of alcohol dehydrogenases and sirtuins and the RNA-mediated catalysis of the ribosomal peptidyl transferase center.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008958	Models, Molecular	Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures.	Molecular Models,Model, Molecular,Molecular Model
D002384	Catalysis	The facilitation of a chemical reaction by material (catalyst) that is not consumed by the reaction.	Catalyses
D004252	DNA Mutational Analysis	Biochemical identification of mutational changes in a nucleotide sequence.	Mutational Analysis, DNA,Analysis, DNA Mutational,Analyses, DNA Mutational,DNA Mutational Analyses,Mutational Analyses, DNA
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006639	Histidine	An essential amino acid that is required for the production of HISTAMINE.	Histidine, L-isomer,L-Histidine,Histidine, L isomer,L-isomer Histidine
D012363	RNA, Transfer, Thr	A transfer RNA which is specific for carrying threonine to sites on the ribosomes in preparation for protein synthesis.	Threonine-Specific tRNA,Transfer RNA, Thr,tRNAThr,tRNA(Thr),RNA, Thr Transfer,Thr Transfer RNA,Threonine Specific tRNA,tRNA, Threonine-Specific
D013914	Threonine-tRNA Ligase	An enzyme that activates threonine with its specific transfer RNA. EC 6.1.1.3.	Threonyl T RNA Synthetase,Thr-tRNA Ligase,Threonyl-tRNA Synthetase,Ligase, Thr-tRNA,Ligase, Threonine-tRNA,Synthetase, Threonyl-tRNA,Thr tRNA Ligase,Threonine tRNA Ligase,Threonyl tRNA Synthetase
D017665	Hydroxyl Radical	The univalent radical OH. Hydroxyl radical is a potent oxidizing agent.
D049148	Aminoacylation	A reaction that introduces an aminoacyl group to a molecule. TRANSFER RNA AMINOACYLATION is the first step in GENETIC TRANSLATION.	Activation of Amino Acids,Amino Acylation,Acids Activation, Amino,Acids Activations, Amino,Acylation, Amino,Acylations, Amino,Amino Acids Activation,Amino Acids Activations,Amino Acylations,Aminoacylations