Affinity modification of phenylalanyl-tRNA synthetase from Thermus thermophilus by tRNAPhe transcripts containing 4-thiouridine.
Photoreactive derivatives of tRNAPhe containing residues of 4-thiouridine (s4U) were synthesized by the transcription system of T7 RNA polymerase. Complete substitution of s4U for 16 uridine residues ([16s4U]-tRNAPhe) caused a 14-fold decrease in the catalytic efficiency of aminoacylation of the tRNAPhe transcript by phenylalanyl-tRNA synthetase from T. thermophilus. [1s4U]-tRNAPhe obtained by random incorporation of s4U residues with further isolation of s4U-monosubstituted RNA molecules on an affinity gel has the same kinetic parameters in aminoacylation as the tRNAPhe transcript. The s4U-containing tRNAPhe transcripts were shown to bind covalently to phenylalanyl-tRNA synthetase, and the specificity of modification was demonstrated. The modification stoichiometry determined in this work suggests that the enzyme is a functional dimer. The modification labels both alpha- and beta-subunits of the enzyme, which has an oligomeric structure of alpha2beta2, and forms "cross-linking" products of subunits upon modification with [16s4U]-tRNAPhe. The prevalence of modification of the alpha-subunit suggests that tRNA has contacts with the enzyme, which have not been deciphered previously by X-ray analysis.
Moor
N A
NA
Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia. moor@niboch.nsc.ru.
Stepanov
V G
VG
Ankilova
V N
VN
Favre
A
A
Lavrik
O I
OI
D001483:Base Sequence; D001665:Binding Sites; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010652:Phenylalanine-tRNA Ligase / Q000737:chemistry / Q000378:metabolism*; D019920:Photoaffinity Labels; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics*; D013891:Thiouridine / Q000378:metabolism; D014158:Transcription, Genetic
D019920:Photoaffinity Labels; D012329:RNA, Bacterial; D012360:RNA, Transfer, Phe; D013891:Thiouridine; D010652:Phenylalanine-tRNA Ligase