SYNTHESIS OF A FLUORESCENT 7-METHYLGUANOSINE ANALOG AND A FLUORESCENCE SPECTROSCOPIC STUDY OF ITS REACTION WITH WHEATGERM CAP BINDING PROTEINS

摘要

In the initiation of protein synthesis, the mRNA 5'-terminal 7-methylguanosine cap structure and several recognition proteins play a pivotal role. For the study of this cap binding reaction, one approach is to use fluorescence spectroscopy. A ribose diol-modified fluorescent cap analog, anthraniloyl-m(7)GTP (Antm(7)GTP), was designed and synthesized for this purpose. This fluorescent cap analog was found to have a high quantum yield, resistance to photobleaching and avoided overlap of excitation and emission wavelengths with those of proteins, The binding of Ant-m(7)GTP with wheatgerm initiation factors eIF-4F and eIF-(iso)4F was determined. The fluorescent cap analog and m(7)GTP had similar interactions with both cap binding proteins. Fluorescence quenching experiments showed that the microenvironment of Ant-m(7)GTP when bound to protein was hydrophobic.