Regulation of mRNA translation constitutes an important aspect in the control of gene expression mammalian cells. Most examples of translational control involve modulation of the initiation phase of protein synthesis. Two kinds of initiation events can be distinguished either of which may be regulated: 1) initiation on polysomes, thereby maintaining a full complement of ribosomes on the mRNAs; and 2) initiation on messenger ribonucleoprotein (mRNP) particles, thereby mobilizing non- active mRNAs into active polysomes. The latter event is poorly understand, yet its regulation is encountered frequently. The PIs propose to characterize proteins that bind to mRNP particles and that mask their activiies. One such protein of 50 kDa has been purified in Pushchino, and its mechanism of action as a translational inhibitor will be probed. Its site of binding to mRNAs will be determined, and the inhibited step of protein synthesis will be identified. They also plan to clone its cDNA and determine the effect of overexpression in transfected mammalian cells. Preliminary evidence from Puschino suggests that the 50 kDa protein may itself be regulated by an "activator"; the "activator" will be purified and characterized. These studies rely on classical biochemical techniques of protein purification and chemistry presently used in both Davis and Puschino, employ in vitro cell-free translatiion systems, some highly fractionated and reconstituted with purified initiatiion factors from Davis, and employ state-of-the-art transfection methods. %%% The project is designed to shed light on the basic molecular mechanisms of how mRNP particles are activated and inhibited for translation, and thus should help elucidate this major poorly understood area of gene regulation.
