STRUCTURE-FUNCTION-RELATIONSHIPS IN THE SACCHAROMYCES-CEREVISIAE POLY(A) POLYMERASE - IDENTIFICATION OF A NOVEL RNA-BINDING SITE AND A DOMAIN THAT INTERACTS WITH SPECIFICITY FACTOR(S)

We have constructed deletions in the nonconserved regions at the amino and carboxyl ends of the poly(A) polymerase (PAP) of Saccharomyces ce revisiae and examined the effects of these truncations on function of the enzyme, PAP synthesizes a poly(A) tail onto the 3'-end of RNA with out any primer specificity but, in the presence of cellular factors, i s directed specifically to the cleaved ends of mRNA precursors, The la st 31 amino acids of PAP are dispensable for both nonspecific and spec ific activities, Removal of the next 36 amino acids affects an RNA bin ding domain, which is essential for the activity of the enzyme and for cell viability. This novel RNA binding site was further localized usi ng additional deletions, cyanogen bromide cleavage of PAP cross-linked with RNA or 8-azido-ATP, and a monoclonal antibody against a COOH-ter minal PAP epitope, A deletion that partially disrupts this domain has reduced nonspecific activity but functions in specific polyadenylation . In contrast, deletion of the first 18 amino acids of PAP has no effe ct on nonspecific polyadenylation but completely eliminates specific a ctivity. This region is essential for enzyme function in vivo and is p robably involved in the interaction of PAP with other protein(s) of th e polyadenylation machinery.