Our goal is to determine whether or not bacterial translational initiation factor 3 protein (IF3) associates with mRNA ribosome binding site sequences and destibilizes their secondary structures. Control of gene expression comprises the central problem of cancer, viral infection, hypertension, contraception, muscular dystrophy, developmental anomalies, arthritis and other autoimmune diseases. Gene expression is controlled at the levels of transcription and translation. IF3 is the simplest system to study in order to obtain a detailed and fundamental understanding of protein-nucleic acid interactions in the regulation of translation. IF3 is crucial for mRNA binding to ribosomes. IF3 is a strongly binding RNA helix destabilizing protein, with sequence specificity for the initiation codan AUG, and a sequence near the 3' end of 16S ribosomal RNA which is virtually conserved in all forms of life, from bacteria to humans, in both mitochondrial and cytoplasmic ribosomes. Hence, elucidating the function of bacterial IF3 will be of value in studying the control of translation in higher organisms. It is thus important to ask whether the functions of IF3 include discrimination among efficient and inefficient mRNAs. Known mRNA ribosome binding site sequences of varying strength and IF3 dependence, and ribosomal RNA sequences, will be subcloned into high efficiency transcription plasmids, transcribed and labelled in vitro. RNA/IF3 mixtures will be analyzed by nitrocellulose blotting to determine RNA affinity for IF3. RNA secondary structures will be determined by neclease mapping and circular dischroism spectra. The site and strength of IF3 binding to such RNA sequences will be assessed by the degree to which IF3 protects a stretch of RNA from degradative enzymes, and the degree to which IF3 reduces the circular dichroism of a region of RNA. IF3 tertiary structure, and sidechain interactions with necleotides of bound RNAs, will be probed by two dimensional nmr COSY and NOESY spectra, and by X-ray crystallography.
