Determination of tRNA(Phe) nucleotides contacting the subunits of Thermus thermophilus phenylalanyl-tRNA synthetase by photoaffinity crosslinking.
The nucleotides of tRNA(Phe) interacting with the subunits of Thermus thermophilus phenylalanyl-tRNA synthetase (the alpha(2)beta(2) heterotetramer) have been determined by photoaffinity crosslinking of randomly s(4)U-monosubstituted tRNA(Phe) transcripts which retain aminoacylation parameters closely similar to those of the native tRNA(Phe). The thiolated transcripts have been fractionated by affinity electrophoresis and separately crosslinked to the enzyme. Sites of crosslinking to the beta subunit have been identified at positions 33 and 39 and crosslinking sites to the alpha subunit have been localized at positions 20, 45 and 47, using alkaline hydrolysis analysis of the crosslinked proteinase K-treated tRNAs. An additional crosslink to the beta subunit, not identified in the full-length crosslinked tRNAs, has been deduced to occur at position 12, based on the analysis of an unusual (fast migrating) crosslinked product. Nucleotide s(4)U8 of native tRNA(Phe) has been shown to form a minor crosslink to the alpha subunit. Four of the seven crosslinking sites, namely nucleotides 8, 12, 20 and 39, are among those shown to be protected against cleavage by iodine in footprinting experiments; in contrast, only nucleotide 12 is among the contact sites defined in the crystal structure. The data of independent biochemical approaches strongly suggest conformational flexibility of the complex under functional conditions, thus reflecting the importance of macromolecular dynamics for the interaction.
Moor
N A
NA
Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences, Novosibirsk 630090, Russia. moor@niboch.nsc.ru
Ankilova
V N
VN
Lavrik
O I
OI
Favre
A
A
D001665:Binding Sites; D008958:Models, Molecular; D010652:Phenylalanine-tRNA Ligase / Q000737:chemistry / Q000378:metabolism*; D019920:Photoaffinity Labels; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000378:metabolism*; D016963:Thermus thermophilus / Q000201:enzymology / Q000378:metabolism*; D013891:Thiouridine / Q000737:chemistry
D019920:Photoaffinity Labels; D012360:RNA, Transfer, Phe; D013891:Thiouridine; D010652:Phenylalanine-tRNA Ligase