Interstice mutations that block site-to-site translocation of a misactivated amino acid bound to a class I tRNA synthetase.
Class I aminoacyl-tRNA synthetases catalyze editing reactions that prevent ambiguity from entering the genetic code. Misactivated amino acids are translocated in cis from the active site for aminoacylation to the center for editing, located approximately 30 A away. Mutational analysis has functionally separated the two sites by creating mutations that disrupt the catalytic center for editing but not for aminoacylation and vice versa. What is not known is whether translocation per se can be disrupted without an effect on either catalytic center. Here we describe mutations in a presumptive "hinge region" of isoleucyl-tRNA synthetase that is situated between the two sites. Interstice mutations had little or no effect on either catalytic center. In contrast, the same specific mutations disrupted translocation. Thus, with these mutations all three functions, translocation, catalysis of aminoacylation, and editing, have been mutationally separated. The results are consistent with translocation involving a hinge-region conformational shift that does not perturb the two catalytic centers.
Bishop
Anthony C
AC
The Skaggs Institute for Chemical Biology, The Scripps Research Institute, Beckman Center, BCC379, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Beebe
Kirk
K
Schimmel
Paul R
PR
D001692:Biological Transport; D002384:Catalysis; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D007533:Isoleucine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D017393:RNA Editing; D012355:RNA, Transfer, Ile / Q000378:metabolism; D014633:Valine / Q000378:metabolism*; D014637:Valine-tRNA Ligase / Q000378:metabolism
D003838:Deoxyadenine Nucleotides; D012355:RNA, Transfer, Ile; D007533:Isoleucine-tRNA Ligase; D014637:Valine-tRNA Ligase; D014633:Valine; C026600:2'-deoxyadenosine triphosphate