Amino acid sequence analysis of the beta- and gamma-subunits of eukaryotic initiation factor eIF-2. Identification of regions interacting with GTP.
By affinity labelling using two different GTP photoaffinity analogues we previously demonstrated that both the beta- and gamma-subunits of eukaryotic initiation factor eIF-2 are involved in GTP binding (Bommer, U.-A. and Kurzchalia, T.V. (1989) FEBS Lett. 244, 323-327). We have now applied the same method in combination with CNBr cleavage and microsequence analysis in order investigate which part of the polypeptide chain of eIF-2 beta is in close contact to the bound GTP. From the three main CNBr fragments of eIF-2 beta, the C-terminal one was found to be labelled by the applied GTP photoaffinity analogue, Guo(2',3'-TDBH)ppp. Because the cDNA sequence of the gamma-subunit of eIF-2 has not yet been published and because cDNA sequence analysis of eIF-2 beta revealed only two out of three consensus sequence elements of a GTP-binding domain, we also sequenced the CNBr fragments of eIF-2 gamma. In this way, sequences containing about 50 amino acid residues were obtained. Taken together with the recently published N-terminal sequences of tryptic peptides of eIF-2 gamma from pig liver (Suzuki et al. 1990, J. Biochem. 108, 635-641), about 30% of the total sequence is now known. One of the CNBr fragments from rabbit eIF-2 gamma contains a sequence (AXXAXXGK) which in several respects resembles that of the consensus sequence element absent from the beta-subunit.
Bommer
U A
UA
Institute of Molecular Biology, Berlin-Buch, F.R.G.
Kraft
R
R
Kurzchalia
T V
TV
Price
N T
NT
Proud
C G
CG
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D015151:Immunoblotting; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013552:Swine
D000345:Affinity Labels; D015852:Eukaryotic Initiation Factor-2; D046911:Macromolecular Substances; D010446:Peptide Fragments; D006160:Guanosine Triphosphate; D003488:Cyanogen Bromide