Alternative design of a tRNA core for aminoacylation.
The core of Escherichia coli tRNA(Cys) is important for aminoacylation of the tRNA by cysteine-tRNA synthetase. This core differs from the common tRNA core by having a G15:G48, rather than a G15:C48 base-pair. Substitution of G15:G48 with G15:C48 decreases the catalytic efficiency of aminoacylation by two orders of magnitude. This indicates that the design of the core is not compatible with G15:C48. However, the core of E. coli tRNA(Gln), which contains G15:C48, is functional for cysteine-tRNA synthetase. Here, guided by the core of E. coli tRNA(Gln), we sought to test and identify alternative functional design of the tRNA(Cys) core that contains G15:C48. Although analysis of the crystal structure of tRNA(Cys) and tRNA(Gln) implicated long-range tertiary base-pairs above and below G15:G48 as important for a functional core, we showed that this was not the case. The replacement of tertiary interactions involving 9, 21, and 59 in tRNA(Cys) with those in tRNA(Gln) did not construct a functional core that contained G15:C48. In contrast, substitution of nucleotides in the variable loop adjacent to 48 of the 15:48 base-pair created functional cores. Modeling studies of a functional core suggests that the re-constructed core arose from enhanced stacking interactions that compensated for the disruption caused by the G15:C48 base-pair. The repacked tRNA core displayed features that were distinct from those of the wild-type and provided evidence that stacking interactions are alternative means than long-range tertiary base-pairs to a functional core for aminoacylation.
Christian
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Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, BLSB 222, 19107, USA.
Lipman
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Evilia
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C
Hou
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D000215:Acylation; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D000926:Anticodon / Q000235:genetics; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012350:RNA, Transfer, Cys / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012351:RNA, Transfer, Gln / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity; D013463:Sulfuric Acid Esters / Q000378:metabolism; D013816:Thermodynamics
D000926:Anticodon; D012329:RNA, Bacterial; D012350:RNA, Transfer, Cys; D012351:RNA, Transfer, Gln; D013463:Sulfuric Acid Esters; D000604:Amino Acyl-tRNA Synthetases; C019975:cysteinyl-tRNA synthetase; C007482:dimethyl sulfate