TRANSLATION-COMPETENT EXTRACTS FROM SACCHAROMYCES-CEREVISIAE - EFFECTS OF L-A-RNA, 5'-CAP, AND 3'-POLY(A) TAIL ON TRANSLATIONAL EFFICIENCY OF MESSENGER-RNAS

Yeast genetics has proven fruitful in the identification of key player s that are involved in translational initiation. However, the exact ro les of many translation initiation factors in translation initiation r emain unknown. This has been due to lack of a suitable in vitro transl ation system in which the mode of action of certain translation factor s can be studied. This report describes the preparation of cell-free S accharomyces cerevisiae lysates that can mediate the translation of ex ogenously added mRNAs. Optimal translation required the absence of vir al L-A RNA in the lysate and the presence of both a 5' cap and a 3' po ly(A) tail on the mRNAs. A cooperative effect of cap and poly(A) tail on translation initiation was observed, a property that has been found to operate in intact yeast cells as well. In addition, the yeast lysa tes mediated translational initiation through several viral internal r ibosome entry sites, demonstrating that the yeast translation apparatu s can perform internal initiation. Thus, these lysates may be useful i n the biochemical analysis of cap-dependent and cap-independent transl ation events. (C) 1997 Academic Press.