A model for the aminoacyl-tRNA binding site of eukaryotic elongation factor 1 alpha.
Eukaryotic elongation factor 1 alpha (EF-1 alpha) binds all the aminoacyl-tRNAs except the initiator tRNA in a GTP-dependent manner. While the GTP binding site is delineated by the three GTP binding consensus elements, less is known about the aminoacyl-tRNA binding sites. In order to better understand this site, we have initiated cross-linking and protease mapping studies of the EF-1 alpha-GTP-aminoacyl-tRNA complex. Two different chemical cross-linking reagents, trans-diaminedichloroplatinum(II) and diepoxybutane, were used to cross-link four different aminoacyl-tRNA species to EF-1 alpha. A series of peptides were obtained, located predominantly in domains II and III. The ability of aminoacyl-tRNA to protect protease digestion sites was also monitored, and domain II was found to be protected from digestion by aminoacyl-tRNA. Last, an aminoacyl-tRNA analog with a reactive group on the aminoacyl side chain, N epsilon-bromoacetyl-Lys-tRNA, was cross-linked to EF-1 alpha. This reagent cross-liked to histidine 296 in a GTP-dependent manner and thus localizes the aminoacyl group adjacent to domain II. A model is developed for aminoacyl-tRNA binding to EF-1 alpha based on its similarity to the prokaryotic factor EF-Tu, for which an x-ray crystal structure is available.
Kinzy
T G
TG
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106.
Freeman
J P
JP
Johnson
A E
AE
Merrick
W C
WC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D012343:RNA, Transfer / Q000302:isolation & purification / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012350:RNA, Transfer, Cys / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012361:RNA, Transfer, Pro / Q000378:metabolism; D012366:RNA, Transfer, Val / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism
D003432:Cross-Linking Reagents; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D012346:RNA, Transfer, Amino Acyl; D012350:RNA, Transfer, Cys; D012360:RNA, Transfer, Phe; D012361:RNA, Transfer, Pro; D012366:RNA, Transfer, Val; D012343:RNA, Transfer