Directly photocrosslinked nucleotides joining transfer RNA to aminoacyl-tRNA synthetase in methionine and tyrosine systems.
We have used ultraviolet photocrosslinking and 32P post-labeling to help define the contact surface between transfer RNAs and aminoacyl-tRNA synthetases for the methionine and tyrosine systems. Photocrosslinking between tRNAs and synthetases is shown to occur only in cognate complexes. The increased sensitivity of our procedures reduces the amounts of interacting macromolecules and permits lower ultraviolet light doses, thereby minimizing radiation damage. These procedures have detected crosslinks only within the 3'-terminal RNase T1 fragments in yeast tRNAMeti and Escherichia coli tRNATyr2; and although the photoadducts were unstable, we have identified the crosslinked nucleotides. These crosslinks occur at positions C74 and A76 in yeast tRNAMeti and position U64 in E. coli tRNATyr1&2 (conventional tRNA numbering system of Gauss & Sprinzl, 1981). This work demonstrates that even labile photocrosslinks can be exploited for mapping crosslinked nucleotides.
Ackerman
E J
EJ
Joachimiak
A
A
Klinghofer
V
V
Sigler
P B
PB
D000604:Amino Acyl-tRNA Synthetases / Q000528:radiation effects*; D001345:Autoradiography; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis; D008718:Methionine-tRNA Ligase / Q000528:radiation effects*; D012329:RNA, Bacterial / Q000528:radiation effects*; D012331:RNA, Fungal / Q000528:radiation effects*; D012346:RNA, Transfer, Amino Acyl / Q000528:radiation effects*; D012441:Saccharomyces cerevisiae / Q000032:analysis; D014448:Tyrosine-tRNA Ligase / Q000528:radiation effects*; D014466:Ultraviolet Rays
D012329:RNA, Bacterial; D012331:RNA, Fungal; D012346:RNA, Transfer, Amino Acyl; C026464:tRNA(m)(Met), methionine-; D000604:Amino Acyl-tRNA Synthetases; D014448:Tyrosine-tRNA Ligase; D008718:Methionine-tRNA Ligase