The process whereby ribosomes attach to an mRNA involves the recognition of specific nucleotide sequences by the components of a system that includes six initiation factors and ATP. The long-range objective of this project is to describe this process in terms of a series of sequential interactions between the factors, the ribosomes, and the mRNA. The more immediate aims are: 1) to resolve the subunits of factor CSF and purify factor eIF4B; 2) relate the need for different components (of the initiation system) to specific structural features of an mRNA; 3) define the regions of an mRNA to which factors CSF and eIF4B bind; 4) determine the specific role of the cap of an mRNA in the ribosome attachment reaction; and 5) determine those factors whose presence at limiting levels can bring about translational discrimination between mRNAs. For the resolution and further purification of the factors, denaturing conditions and antibody affinity columns will be used. The antibodies will also be used directly to test the involvement of specific components in the ribosome mRMA attachment reaction. The structural features of an mRNA that will be related to the need for specific factors are the presence or absence of an m7Gppp... or an m7Ippp... cap and the presence of secondary structure in the 5' leader region of the mRNA. One idea to be tested is that the need for the 5' cap is not intrinsic to the ribosome attachment reaction, but rather to provide a specific affinity for CSF that allows it to overcome competitive interaction by other basic proteins. We will look for such competitive proteins in the total extract using the preferential inhibition of the binding to noncapped mRNAs as an assay. To define the regions of an mRNA with which the factors interact, T1 digests of a ribosome-protected fragment of reovirus RNA will be analyzed before and after appropriate incubations with CSF and eIF4B. Finally, a dipeptide synthesis assay will be used to follow translation of two competitive mRNAs. Using the fractionated factors, the levels of different components of the translation system will be made limiting and the effect of such limitation on the relative translation of the two mRNAs will be monitored.
| Seal, S N; Schmidt, A; Marcus, A (1989) Ribosome binding to inosine-substituted mRNAs in the absence of ATP and mRNA factors. J Biol Chem 264:7363-8 |
