Enzymatic aminoacylation of tRNA acceptor stem helices with cysteine is dependent on a single nucleotide.
The discriminator base U73 at the acceptor terminus of Escherichia coli tRNA(Cys) is a determinant for the specific aminoacylation of this tRNA by the cognate cysteine tRNA synthetase. Substitution of U73 has a major deleterious effect on the catalytic efficiency of aminoacylation. Here, we show that an RNA hairpin minihelix and an RNA hairpin microhelix that recreate, respectively, the 12-base pair acceptor-T psi C stem and the 7-base pair acceptor helix of E. coli tRNA(Cys) were aminoacylated with cysteine. As in tRNA(Cys), alteration of U73 to A73, C73, or G73 in the cysteine mini- and microhelices eliminated aminoacylation. This established that the strong influence of U73 on aminoacylation is fully retained from the full-length tRNA(Cys) to the mini- and microhelixCys. Transfer of U73 to the noncognate minihelixAla conferred cysteine acceptance to the latter, despite the presence of the major determinant for alanine tRNA synthetase. Even minihelixGly, which shares U73 with minihelixCys, was an efficient substrate for aminoacylation with cysteine. Conversely, as long as U73 was present in minihelixCys, introduction of the glycine or alanine determinant could not block charging by cysteine tRNA synthetase. Although the catalytic efficiency of aminoacylation of these small RNA helices with cysteine was reduced by orders of magnitude from that of tRNA(Cys), the single nucleotide U73 determines the ability of these RNA helices to be aminoacylated with cysteine. These results demonstrated a dominant role of U73 for aminoacylation of small RNA helices by cysteine tRNA synthetase.
Hamann
C S
CS
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Hou
Y M
YM
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001483:Base Sequence; D003545:Cysteine / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012350:RNA, Transfer, Cys / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D046249:Transfer RNA Aminoacylation
D017931:DNA Primers; D012350:RNA, Transfer, Cys; D000604:Amino Acyl-tRNA Synthetases; D003545:Cysteine