Nucleic Acids Research, 1993, Vol. 21, No. 19 4467-4475
© 1993
ORIGINAL ARTICLES |
Contributions of discrete tRNASer domains to aminoacylation by E.coli seryl-tRNA synthetase: a kinetic analysis using model RNA substrates
Division of Biology 147-75, California Institute of Technology Pasadena, CA 91125, USA
Received June 28, 1993. Revised August 13, 1993. Accepted August 13, 1993.
The aminoacylation kinetics of T7 transcripts representing defined regions of Escherichia coli serine tRNAs were determined using purified E.coli seryl-tRNA synthetase (SerRS) and the kinetic values were used to estimate the relative contribution of various tRNA^ domains to recognition by SerRS. The analysis revealed the extra stem/loop structure, characteristic of type II tRNAs such as tRNAser, is the domain which makes the largest contribution to kcat/Km of aminoacylation. Moreover, Km of aminoacylation was increased by a factor of about 1000 when the extra stem/loop was changed to the consensus sequence of type I tRNA extra loops indicating that the stem structure contributes significantly to the binding of tRNASer to SerRS. A model RNA, which represents only the tRNASer coaxial acceptor-T^C stem/loop domain, was also specifically aminoacylated by SerRS having a kcat/Km about 1000-fold greater than background levels. A significant portion of the contribution of this domain to aminoacylation is attributable to the acceptor stem sequence making the acceptor stem the second most important domain for recognition by SerRS. Finally, kcat/Km was essentially unchanged when the entire anticodon stem/loop of tRNASer was deleted indicating that neither the anticodon nucleotides nor the surrounding stem/loop structure are important for recognition by SerRS.
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