Abstract:
:Known crystal structures of class II aminoacyl-tRNA synthetases complexed to their cognate tRNAs reveal that critical acceptor stem contacts are made by the variable loop connecting the beta-strands of motif 2 located within the catalytic core of class II synthetases. To identify potential acceptor stem contacts made by Escherichia coli prolyl-tRNA synthetase (ProRS), an enzyme of unknown structure, we performed cysteine-scanning mutagenesis in the motif 2 loop. We identified an arginine residue (R144) that was essential for tRNA aminoacylation but played no role in amino acid activation. Cross-linking experiments confirmed that the end of the tRNA(Pro) acceptor stem is proximal to this motif 2 loop residue. Previous work had shown that the tRNA(Pro) acceptor stem elements A73 and G72 (both strictly conserved among bacteria) are important recognition elements for E. coli ProRS. We carried out atomic group "mutagenesis" studies at these two positions of E. coli tRNA(Pro) and determined that major groove functional groups at A73 and G72 are critical for recognition by ProRS. Human tRNA(Pro), which lacks these elements, is not aminoacylated by the bacterial enzyme. An analysis of chimeric tRNA(Pro) constructs showed that, in addition to A73 and G72, transplantation of the E. coli tRNA(Pro) D-domain was necessary and sufficient to convert the human tRNA into a substrate for the bacterial synthetase. In contrast to the bacterial system, base-specific acceptor stem recognition does not appear to be used by human ProRS. Alanine-scanning mutagenesis revealed that motif 2 loop residues are not critical for tRNA aminoacylation activity of the human enzyme. Taken together, our results illustrate how synthetases and tRNAs have coadapted to changes in protein-acceptor stem recognition through evolution.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Burke B,Yang F,Chen F,Stehlin C,Chan B,Musier-Forsyth Kdoi
10.1021/bi001835psubject
Has Abstractpub_date
2000-12-19 00:00:00pages
15540-7issue
50eissn
0006-2960issn
1520-4995pii
bi001835pjournal_volume
39pub_type
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