Abstract:
:Aminoacylation of a transfer RNA (tRNA) by its cognate aminoacyl-tRNA synthetase relies upon the recognition of specific nucleotides as well as conformational features within the tRNA by the synthetase. In Escherichia coli, the aminoacylation of tRNA(His) by histidyl-tRNA synthetase (HisRS) is highly dependent upon the recognition of the unique G-1:C73 base pair and the 5'-monophosphate. This work investigates the RNA-protein interactions between the HisRS active site and these critical recognition elements. A homology model of the tRNA(His)-HisRS complex was generated and used to design site-specific mutants of possible G-1:C73 contacts. Aminoacylation assays were performed with these HisRS mutants and N-1:C73 tRNA(His) and microhelix(His) variants. Complete suppression of the negative effect of 5'-phosphate deletion by R123A HisRS, as well as the increased discrimination of Q118E HisRS against a 5'-triphosphate, suggests a possible interaction between the 5'-phosphate and active-site residues Arg123 and Gln118 in which these residues create a sterically and electrostatically favorable pocket for the binding of the negatively charged phosphate group. Additionally, a network of interactions appears likely between G-1 and Arg116, Arg123, and Gln118 because mutation of these residues significantly reduced the sensitivity of HisRS to changes at G-1. Our studies also support an interaction previously proposed between Gln118 and C73. Defining the RNA-protein interactions critical for efficient aminoacylation by E. coli HisRS helps to further characterize the active site of this enzyme and improves our understanding of how the unique identity elements in the acceptor stem of tRNA(His) confer specificity.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Connolly SA,Rosen AE,Musier-Forsyth K,Francklyn CSdoi
10.1021/bi035708fsubject
Has Abstractpub_date
2004-02-03 00:00:00pages
962-9issue
4eissn
0006-2960issn
1520-4995journal_volume
43pub_type
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