Substrate recognition by the tRNAHis-guanylyltransferase: a kinetic investigation with model RNA substrates
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Date
2015-05
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The Ohio State University
Abstract
All tRNAs undergo post-transcriptional modifications before being aminoacylated with a specific amino acid. In the case of tRNAHis, this includes the addition of a required guanosine at the -1 position (G-1), required for histidyl-tRNA synthetase (HisRS) to recognize the tRNA and charge it with histidine. The tRNAHis guanylyltransferase (Thg1) enzyme, with family members found in all domains of life, completes this reaction via its novel ability to add nucleotides in the 3'-5' direction in a chemically similar reaction to that of canonical 5'-3' polymerases. Eukaryotic Thg1 enzymes selectively recognize tRNAHis for modification with G-1, however the molecular basis for this observed substrate selectivity is not completely understood. Therefore, the long-term goal of this project is to elucidate the molecular features that participate in tRNA recognition by Thg1. Insight into tRNA recognition by HisRS has been achieved by using small RNA stem-loop substrates and a similar approach is applied here to probe tRNA recognition by Thg1. The substrates to be tested are a substrate with a seven base pair stem that mimics the seven base pair acceptor stem of tRNAHis and a 12 base pair substrate that mimics the acceptor stem and T-stem, which coaxially stack in full-length tRNA. The three Thg1 family enzymes tested for activity with these model RNAs are from Methanobrevibacter smithii, an archaeal species, Bacillus thuringiensis, a bacterial species, and Saccharomyces cerevisiae, a eukaryote. Here we report the results of kinetic assays with each of these purified enzymes and 5'-triphosphorylated substrates, all of which are analyzed using single-turnover assays that evaluate the rate of 3'-5' addition. Data from these assays will be used to evaluate the dependence of diverse Thg1 family enzymes on the length of the model RNA stem and its potential for forming Watson-Crick vs. non-Watson-Crick base pairs during the 3'-5' addition reaction.
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tRNA modifying enzyme, tRNAHis-guanylyltransferase (Thg1), 3'-5' nucleotide addition reaction, Single turnover kinetic data