This project focuses on the relationship of eukaryotic mRNA primary and secondary structure to intrinsic mRNA translation efficiency. Our studies are aimed toward defining structural features of the 5' -cap, 5' -noncoding region and 5' -proximal coding sequence of mRNA required for efficient recognition, binding and translation by initiation factors and ribosomes. We are employing a biochemical approach to this problem by use of mRNA molecules synthesized from plasmid DNA templates in vitro with bacteriophage SP6 and T7 RNA polymerases. Use of a defined mRNA substrate will allow a systematic study of protein- mRNA interactions since we will be able to introduce base substitutions, insertions and deletions. These alterations will be directed toward modifying either secondary structure or specific sequences. The effects of such modifications on mRNA activity will be examined. Thus far we have used defined coding regions, in our model mRNAs, taken from rabbit beta globin, bacterial chloramphenicol acetyltransferase (CAT) and herpes simplex virus thymidine kinase (TK). We are also using artifical and naturally occurring bicistronic coding sequences to study elements of mRNA structure and host-factors responsible for initiation of internal start sites. The bicistronic mRNA used have two or more in-frame start codons. We will also isolate and characterize host factors which are responsible for invoking start site preference in dual, in-frame start sites mRNAs. Lastly we are analyzing structural elements of the 5' -cap structure of mRNA necessary for ligand binding interactions with cytoplasmic cap binding proteins which participate in the initiation process of translation. This is being approached by competitive inhibition studies using synthetic cap analogs.
