Alternative design of a tRNA core for aminoacylation

• Published in: Journal of molecular biology Vol. 303; no. 4; pp. 503 - 514
• Main Authors: Christian, T, Lipman, R S, Evilia, C, Hou, Y M
• Format: Journal Article
• Language: English
• Published: England 03-11-2000
• Subjects: Acylation; Amino Acyl-tRNA Synthetases - metabolism; Anticodon - genetics; Base Pairing - genetics; Base Sequence; Escherichia coli - enzymology; Escherichia coli - genetics; Kinetics; Models, Molecular; Molecular Sequence Data; Mutation - genetics; Nucleic Acid Conformation; RNA, Bacterial - chemistry; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; RNA, Transfer, Cys - chemistry; RNA, Transfer, Cys - genetics; RNA, Transfer, Cys - metabolism; RNA, Transfer, Gln - chemistry; RNA, Transfer, Gln - genetics; Substrate Specificity; Sulfuric Acid Esters - metabolism; Thermodynamics
• ISSN: 0022-2836
• DOI: 10.1006/jmbi.2000.4169

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.