Alternative design of a tRNA core for aminoacylation

The core of Escherichia coli tRNA(Cys) is important for aminoacylation of t he 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 n ot compatible with G15:C48. However, the core of E. coli tRNA(Gln), which c ontains G15:C48, is functional for cysteine-tRNA synthetase. Here, guided b y 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 an alysis 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 functi onal core, we showed that this was not the case. The replacement of tertiar y interactions involving 9, 21, and 59 in tRNA(Cys) With those in tRNA(Gln) did not construct a functional core that contained G15:C48. Ln contrast, s ubstitution 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 interact ions that compensated for the disruption caused by the G15:C48 base-pair. T he repacked tRNA core displayed features that were distinct from those of t he wild-type and provided evidence that stacking interactions are alternati ve means than long-range tertiary base-pairs to a functional core for amino acylation. (C) 2000 Academic Press.