tRNA discrimination at the binding step by a class II aminoacyl-tRNA synthetase.
Aminoacyl-tRNA synthetases preserve the fidelity of decoding genetic information by accurately joining amino acids to their cognate transfer RNAs. Here, tRNA discrimination at the level of binding by Escherichia coli histidyl-tRNA synthetase is addressed by filter binding, analytical ultracentrifugation, and iodine footprinting experiments. Competitive filter binding assays show that the presence of an adenylate analogue 5'-O-[N-(L-histidyl)sulfamoyl]adenosine, HSA, decreased the apparent dissociation constant (K(D)) for cognate tRNA(His) by more than 3-fold (from 3.87 to 1.17 microM), and doubled the apparent K(D) for noncognate tRNA(Phe) (from 7.3 to 14.5 microM). By contrast, no binding discrimination against mutant U73 tRNA(His) was observed, even in the presence of HSA. Additional filter binding studies showed tighter binding of both cognate and noncognate tRNAs by G405D mutant HisRS [Yan, W., Augustine, J., and Francklyn, C. (1996) Biochemistry 35, 6559], which possesses a single amino acid change in the C-terminal anticodon binding domain. Discrimination against noncognate tRNA was also observed in sedimentation velocity experiments, which showed that a stable complex was formed with the cognate tRNA(His) but not with noncognate tRNA(Phe). Footprinting experiments on wild-type versus G405D HisRS revealed characteristic alterations in the pattern of protection and enhancement of iodine cleavage at phosphates 5' to tRNA nucleotides in the anticodon and hinge regions. Together, these results suggest that the anticodon and core regions play major roles in the initial binding discrimination between cognate and noncognate tRNAs, whereas acceptor stem nucleotides, particularly at position 73, influence the reaction at steps after binding of tRNA.
Bovee
M L
ML
Department of Biochemistry, University of Vermont College of Medicine, Burlington 05405, USA.
Yan
W
W
Sproat
B S
BS
Francklyn
C S
CS
D000241:Adenosine / Q000737:chemistry; D000926:Anticodon / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018983:DNA Footprinting; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006643:Histidine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry; D012354:RNA, Transfer, His / Q000737:chemistry / Q000235:genetics; D013873:Thionucleotides / Q000737:chemistry; D014461:Ultracentrifugation
D000926:Anticodon; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; D012354:RNA, Transfer, His; D013873:Thionucleotides; D006643:Histidine-tRNA Ligase; D000241:Adenosine