TY - JOUR
T1 - Contributions of discrete tRNA domains to aminoacylation by E.coli seryl-tRNA synthetase
T2 - A kinetic analysis using model RNA substrates
AU - Sampson, Jeffrey R.
AU - Saks, Margaret E.
N1 - We are very grateful to John N.Abelson, in whose laboratory this work was performed, for his encouragement and support of this project. We wish to thank Giuseppe Tocchini Valentini for constructing the T7-CUA and T7-AXL genes and for providing purified SerRS for our initial experiments. We also thank Barry Cooperman for supplying purified E.coli inorganic pyrophosphatase. This work was supported by NIH grant GM48560 (J.N.A.) and by NIH (GM13776; M.E.S.) and American Cancer Society (PF-3404; J.R.S.) postdoctoral fellowships.
PY - 1993/9/25
Y1 - 1993/9/25
N2 - 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.
AB - 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.
UR - https://www.scopus.com/pages/publications/0027442959
UR - https://www.scopus.com/pages/publications/0027442959#tab=citedBy
U2 - 10.1093/nar/21.19.4467
DO - 10.1093/nar/21.19.4467
M3 - Article
C2 - 8233780
AN - SCOPUS:0027442959
SN - 0305-1048
VL - 21
SP - 4467
EP - 4475
JO - Nucleic acids research
JF - Nucleic acids research
IS - 19
ER -