Molecular recognition of tRNA(Pro) by Escherichia coli proline-tRNA synthetase.
We have investigated the molecular recognition of tRNA(Pro) by Escherichia coli proline tRNA synthetase (ProRS) in vitro using semi-synthetic tRNAs and site-directed mutagenesis of full-length tRNA transcripts. These studies have led to an improved understanding of how this class II synthetase interacts with its tRNA substrate. The ability to efficiently aminoacylate a tRNA(Pro) molecule assembled by annealing together a shorter, chemically synthesized oligonucleotide and a 3/4 tRNA prepared enzymatically, has facilitated the identification of RNA structural features that are critical for aminoacylation by ProRS. This approach has been successful using either a 3'-3/4 tRNA annealed to a 5'-oligonucleotide or a 5'-3/4 tRNA annealed to a 3'-oligonucleotide. These studies show that ProRS appears to be particularly sensitive to mutations that result in structural changes in the core region of tRNA(Pro). Moreover, the so-called "variable pocket" nucleotides appear to be dispensable for aminoacylation. We have also identified a specific 2'-hydroxyl-base interaction between the ribose of U8 and the 2-amino group of G46 that makes a thermodynamically significant contribution to tRNA(Pro) aminoacylation by E. coli ProRS.
Liu
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Department of Chemistry, University of Minnesota, Minneapolis 55455, USA.
Yap
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LP
Stehlin
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Musier-Forsyth
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D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006900:Hydroxylation; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D012361:RNA, Transfer, Pro / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics
D012361:RNA, Transfer, Pro; D000604:Amino Acyl-tRNA Synthetases; C100134:prolyl T RNA synthetase