Further study on association of 5SrRNA-L5 protein complex and methionyl-tRNA to methionyl-tRNA synthetase in the macromolecular aminoacyl-tRNA synthetase complex.
To obtain direct evidence for the attachment of 5SrRNA-ribosomal L5 protein particles (5SRNP) and methionine-tRNA (tRNA(met)) to methionyl-tRNA synthetase (MetRS) in the macromolecular aminoacyl-tRNA synthetase (ARS) complex of rat liver, a MetRS-5SRNP-tRNA(met) complex was dissociated from the macromolecular ARS complex fraction by n-octyl-beta-D-glucoside (Method I) or by omega-aminooctyl agarose (Method II) chromatography. The dissociated MetRS complex fraction was purified by gel filtration followed by tRNA-Sepharose chromatography using partially purified tRNA(met) in Method I, and by hydrophobic interaction chromatography in Method II. In both methods, final Superdex200 chromatography showed that MetRS activity was present in the region corresponding to the molecular weight of the MetRS-5SRNP-tRNA(met) complex (M(r) 200,000). One main protein band corresponding to the molecular weight of MetRS was observed on SDS-PAGE of the final product, which was concentrated by lyophilizing after dialysis against water. Using serum albumin as an inhibitor of adhesion of L5 to the microconcentrators which was used to concentrate the final product, a distinct L5 band was detected on SDS-PAGE, the intensity of which was comparable to that of the MetRS band. Northern blot analysis of RNA prepared from the tRNA-Sepharose fraction showed the presence of 5SrRNA. Dot blot analysis using an antibody against ribosomal protein L5 showed that L5 was present in the Superdex200 fractions prepared by both methods. The MetRS specific activities in MetRS complex fractions incubated without tRNA increased during the purification procedures, indicating that endogeneous tRNA(met) exists stably in the MetRS complex. 5SRNP and 5SrRNA markedly enhanced the MetRS activity in the MetRS complex, indicating that 5SRNP(A) plays a role as a positive effector of MetRS.
Ogata
K
K
Institute for Gene Expression, Dobashi Kyoritsu Hospital, Ehime.
Ohno
R
R
Morioka
S
S
Terao
K
K
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D000818:Animals; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides / Q000494:pharmacology; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D046911:Macromolecular Substances; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D011487:Protein Conformation; D012341:RNA, Ribosomal, 5S / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000378:metabolism*; D051381:Rats; D012269:Ribosomal Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae
D005960:Glucosides; D046911:Macromolecular Substances; D012341:RNA, Ribosomal, 5S; D012358:RNA, Transfer, Met; D012269:Ribosomal Proteins; C029742:ribosomal protein L5; C018619:octyl-beta-D-glucoside; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase