A fluorescence study of the interactions of protein synthesis initiation factors with messenger RNA and applications of fluorescence energy transfer techniques in biopolymers

Most eukaryotic mRNAs contain a 5{dollar}spprime{dollar} cap (m{dollar}sp7{dollar}GpppX) and a 3{dollar}spprime{dollar} poly(A) tail to increase synergistically the translational efficiency. In this report the poly(A) binding protein (PABP) and cap-binding protein, eIF-4F, were found to interact. These data suggest that PABP may exert its effect on translational efficiency either by increasing the formation of initiation factor-mRNA complex or by enhancing ribosome recycling. In order to investigate the functional consequences of these interactions, the fluorescent cap analog, ant-m{dollar}sp7{dollar}GTP, which is an environmentally sensitive fluorescent probe was used to investigate the cap-binding affinity. Our data show that the binding of eIF-(iso)4F or eIF-4F to cap analog enhanced their binding affinity towards PABP approximately 40-fold. Similarly, the eIF-4F/PABP or eIF-(iso)4F/PABP complexes show a 40-fold enhancement of cap analog binding compared to eIF-4F or eIF-(iso)4F alone. At least part of the enhancement of the translational initiation by PABP can be accounted for by direct changes in cap binding affinity. The interactions of these components also suggest a mechanism whereby the poly(A) tail is brought into close proximity with m{dollar}sp7{dollar}G cap. This effect was examined by fluorescence energy transfer and it was determined that the PABP/eIF-4F complex could bind both poly(A) and 5{dollar}spprime{dollar} cap simultaneously. Fluorescence resonance energy transfer techniques are also very powerful in studies of biopolymers. In this report, two models are constructed and good for studies of pseudoknotted RNA and RNA helicase activity.