Fluorescence studies on association of human translation initiation factor eIF4E with mRNA cap-analogues.
Binding of a long series of mono- and dinucleotide analogues of the 7-methylguanosine containing 5'-mRNA-cap to human protein translation initiation factor eIF4E has been investigated by means of fluorescence. A new methodological approach in gathering and analysis of the fluorescence data provided us with very accurate values of the association equilibrium constant K and normalized, maximal quenching of the protein fluorescence delta Fmax, during titration of eIF4E by various cap-analogues. The results confirm participation of at least two conserved tryptophan residues of eIF4E in interaction with 7-methylguanine, as has been described recently for murine eIF4E, complexed with 7-methyl-GDP in crystal (Marcotrigiano et al., 1997, Cell 89, 951), and for yeast eIF4E, complexed with the same ligand in solution (Matsuo et al., 1997, Nature Struct. Biol. 4, 717). On the other hand binding by eIF4E of unmethylated guanine nucleotides and N2,N2,7-trimethylguanine containing nucleotides differ substantially from the way of binding of the regular mRNA-cap. Influence of the structural features of the cap-analogues, especially the type of the second nucleoside in the dinucleotide caps, on their association with eIF4E and biological activities in in vitro protein translation systems has been discussed in light of the known structures of the eIF4E-7-methyl-GDP complexes in crystal and solution.
Wieczorek
Z
Z
Department of Physics and Biophysics, University of Agriculture and Technology, Olsztyn, Poland.
Niedźwiecka-Kornaś
A
A
Chlebicka
L
L
Jankowska
M
M
Kiraga
K
K
Stepiński
J
J
Dadlez
M
M
Drabent
R
R
Darzynkiewicz
E
E
Stolarski
R
R
D000818:Animals; D039561:Eukaryotic Initiation Factor-4E; D006150:Guanine Nucleotides / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000378:metabolism*; D012315:RNA Caps / Q000737:chemistry* / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D039561:Eukaryotic Initiation Factor-4E; D006150:Guanine Nucleotides; D010448:Peptide Initiation Factors; D012315:RNA Caps; D012333:RNA, Messenger