Interaction of T. thermophilus phenylalanyl-tRNA synthetase with the 3'-terminal nucleotide of tRNAPhe.
The interaction of Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) with the 3;-terminal nucleotide of tRNAPhe has been studied by affinity labeling to solve the problem arising from X-ray crystallographic study: the binding sites of phenylalanine and the 3;-terminal nucleotide base were revealed to be identical in the crystal structures of PheRS complexed with the substrates. tRNAPhe derivatives containing a photoreactive 4-thiouridine (tRNAPhe-s4U-76) or 6-thioguanosine residue (tRNAPhe-s6G-76) in the 3;-end have been prepared using terminal tRNA nucleotidyl transferase. Kinetic measurements of aminoacylation provide evidence for a functional role of base-specific interactions of the 3;-terminal adenosine in productive interaction of tRNAPhe with the enzyme: tRNAPhe-s4U-76 cannot be aminoacylated; the replacement of A-76 with s6G results in a 370-fold reduction of catalytic efficiency of aminoacylation mainly due to decreased Vmax value. Relative cross-linking of the s6G-substituted tRNA to the alpha-subunit (69% of the total yield of the cross-linked alpha- and beta-subunits) is two times higher as compared to the cross-linking of tRNAPhe-s4U-76. The dialdehyde derivative, tRNAPhe-Aox-76, with periodate-oxidized 3;-terminal ribose is cross-linked with the same selectivity to the alpha-subunit as tRNAPhe-s6G-76. The results suggest specific binding of the 3;-terminal nucleotide of tRNAPhe by the catalytic subunit of PheRS in the absence of other substrates. Comparative analysis of the cross-linked products in the absence and in the presence of small substrates revealed ATP and aminoacyl-adenylate to effect the interaction of the tRNAPhe acceptor end with PheRS. The correct positioning of the 3;-terminal nucleotide of tRNAPhe corresponding to the structure of the productive complex with PheRS is therefore promoted only in the presence of all three substrates.
Vasil'eva
I A
IA
Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, Novosibirsk, 630090, Russia.
Ankilova
V N
VN
Lavrik
O I
OI
Moor
N A
NA
D000345:Affinity Labels; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010652:Phenylalanine-tRNA Ligase / Q000378:metabolism*; D010777:Photochemistry; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000378:metabolism*; D016963:Thermus thermophilus / Q000201:enzymology*; D013891:Thiouridine / Q000737:chemistry
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D012360:RNA, Transfer, Phe; D013891:Thiouridine; D010652:Phenylalanine-tRNA Ligase