Role of low-molecular-weight substrates in functional binding of the tRNAPhe acceptor end by phenylalanyl-tRNA synthetase.
The functional roles of phenylalanine and ATP in productive binding of the tRNA(Phe) acceptor end have been studied by photoaffinity labeling (cross-linking) of T. thermophilus phenylalanyl-tRNA synthetase (PheRS) with tRNA(Phe) analogs containing the s(4)U residue in different positions of the 3'-terminal single-stranded sequence. Human and E. coli tRNA(Phe)s used as basic structures differ by efficiency of the binding and aminoacylation with the enzyme under study. Destabilization of the complex with human tRNA(Phe) caused by replacement of three recognition elements decreases selectivity of labeling of the alpha- and beta-subunits responsible for the binding of adjacent nucleotides of the CCA-end. Phenylalanine affects the positioning of the base and ribose moieties of the 76th nucleotide, and the recorded effects do not depend on structural differences between bacterial and eukaryotic tRNA(Phe)s. Both in the absence and presence of phenylalanine, ATP more effectively inhibits the PheRS labeling with the s(4)U76-substituted analog of human tRNA(Phe) (tRNA(Phe)-s(4)U76) than with E. coli tRNA(Phe)-s(4)U76: in the first case the labeling of the alpha-subunits is inhibited more effectively; the labeling of the beta-subunits is inhibited in the first case and increased in the second case. The findings analyzed with respect to available structural data on the enzyme complexes with individual substrates suggest that the binding of phenylalanine induces a local rearrangement in the active site and directly controls positioning of the tRNA(Phe) 3'-terminal nucleotide. The effect of ATP on the acceptor end positioning is caused by global structural changes in the complex, which modulate the conformation of the acceptor arm. The rearrangement of the acceptor end induced by small substrates results in reorientation of the 3'-OH-group of the terminal ribose from the catalytic subunit onto the noncatalytic one, and this may explain the unusual stereospecificity of aminoacylation in this system.
Vasil'eva
I A
IA
Institute of Chemical Biology and Fundamental Medicine, Siberian Division, Russian Academy of Sciences, Novosibirsk 630090, Russia.
Bogachev
V S
VS
Favre
A
A
Lavrik
O I
OI
Moor
N A
NA
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D010652:Phenylalanine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012360:RNA, Transfer, Phe / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017385:Sequence Homology; D013379:Substrate Specificity / Q000235:genetics; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics; D013891:Thiouridine / Q000737:chemistry
D001426:Bacterial Proteins; D012360:RNA, Transfer, Phe; D013891:Thiouridine; D010652:Phenylalanine-tRNA Ligase