MONOCLONAL-ANTIBODIES TO YEAST POLY(A) POLYMERASE (PAP) PROVIDE EVIDENCE FOR ASSOCIATION OF PAP WITH CLEAVAGE FACTOR-I

Purified yeast poly(A) polymerase (PAP) was used to produce monoclonal antibodies which recognize the enzyme in immunoblots. Epitope mapping using truncated forms of PAP and cyanogen bromide cleavage products r evealed two classes of antibodies. One class (N-term) recognizes an ep tiope in the first 100 amino acids, and a second class (C-term) is spe cific for a determinant located in the last 20 amino acids of PAP. The se C-terminal 20 amino acids can be removed without affecting the nons pecific poly(A) addition activity of the purified enzyme. Neither anti body inhibits the nonspecific poly(A) polymerase activity or the seque nce-specific activity observed in processing extracts. The antibodies show species specificity and cannot recognize mammalian, Xenopus, or v accinia PAP. The C-term antibodies can deplete PAP from yeast whole ce ll extracts, resulting in loss of poly(A) addition activity. This immu nodepletion also causes a reduction in the cleavage activity which can be restored by addition of yeast cleavage factor I [CF I; Chen, J., a nd Moore, C. (1992) Mol. Cell Biol. 12, 3470-3481], a factor needed fo r both the cleavage and poly(A) addition reactions. This demonstrates that a complex of PAP and CF I exists in extracts in the absence of AT P or exogenous RNA substrate. The monoclonal antibodies against yeast PAP will be a useful tool for further study of factors required for ye ast mRNA 3' end processing.