Interaction of wheat germ protein synthesis initiation factor eIF-(iso)4F and its subunits p28 and p86 with m7GTP and mRNA analogues.
The binding of p28, p86, and native wheat germ eIF-(iso)4F with m7GTP and oligonucleotides was measured and compared. The purified subunits (p28, 28 kDa and p86, 86 kDa) of wheat germ protein synthesis initiation factor eIF-(iso)4F have been obtained from Escherichia coli expression of the cloned DNA (van Heerden, A., and Browning, K. S. (1994) J. Biol. Chem. 269, 17454-17457). The binding of the 5'-terminal cap analogue m7GTP to the small subunit (p28) of eIF-(iso)4F as a function of pH, temperature, and ionic strength is described. The mode of binding of p28 to cap analogues is very similar to the intact protein. Assuming that all tryptophan residues contribute to p28 and eIF-(iso)4F fluorescence, iodide quenching shows that all 9 tryptophan residues in p28 are solvent-accessible, while only 6 out of 16 tryptophan residues are solvent-accessible on the intact eIF-(iso)4F. The fluorescence stopped-flow studies of eIF-(iso)4F and p28 with cap show a concentration-independent conformational change. The rate of this conformational change was approximately 10-fold faster for the isolated p28 compared with the native eIF-(iso)4F. From these studies it appears that cap recognition resides in the p28 subunit. However, p86 enhances the interaction with capped oligonucleotides and probably is involved in protein-protein interactions as well. Both subunits are required for helicase activity.
Department of Chemistry, Hunter College, City University of New York, New York 10021-5024, USA.
M
M
Sha
Y
Y
Wang
T
T
Xiang
A
A
van Heerden
K S
KS
Browning
D J
DJ
Goss
D001483:Base Sequence; D039562:Eukaryotic Initiation Factor-4F; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism*; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000378:metabolism*; D012314:RNA Cap Analogs / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D012639:Seeds / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014908:Triticum / Q000378:metabolism*
D039562:Eukaryotic Initiation Factor-4F; D046911:Macromolecular Substances; D009843:Oligoribonucleotides; D010448:Peptide Initiation Factors; D012314:RNA Cap Analogs; D012333:RNA, Messenger; C017516:7-methylguanosine triphosphate