A fluorescence spectroscopic study on the binding of mRNA 5'-cap-analogs to human translation initiation factor eIF4E: a critical evaluation of the sources of error.
Equilibrium constants for the association of human protein translation initiation factor eIF4E with two mRNA 5'-cap analogs, namely 7-methylguanosine 5'-triphosphate and P1-(7-methylguanosine-5') P3-(guanosine-5') triphosphate, and with guanosine 5'-monophosphate have been redetermined by the fluorescence quenching method taking the inner filter effect of the cap-analog into account. It has been shown that neglecting the latter correction may lead to either underestimation or overestimation of the association constant obtained by applying the Eadie-Hofstee plot: the reasonably firm binding of 7-methylated cap-analogs becomes underestimated, while the weak binding of non-methylated nucleotides becomes overestimated.
Wieczorek
Z
Z
Department of Physics and Biophysics, University of Agriculture and Technology, Olsztyn, Poland. zbyszek@art.olsztyn.pl
Darzynkiewicz
E
E
Lönnberg
H
H
D001665:Binding Sites; D004912:Erythrocytes / Q000378:metabolism; D004926:Escherichia coli; D039561:Eukaryotic Initiation Factor-4E; D006801:Humans; D007700:Kinetics; D008745:Methylation; D010448:Peptide Initiation Factors / Q000378:metabolism*; D012315:RNA Caps / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods; D013329:Structure-Activity Relationship
D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D012315:RNA Caps; D012333:RNA, Messenger; D011994:Recombinant Proteins