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 revealed two classes of antibodies. One class (N-term) recognizes an epitope in the first 100 amino acids, and a second class (C-term) is specific for a determinant located in the last 20 amino acids of PAP. These C-terminal 20 amino acids can be removed without affecting the nonspecific poly(A) addition activity of the purified enzyme. Neither antibody inhibits the nonspecific poly(A) polymerase activity or the sequence-specific activity observed in processing extracts. The antibodies show species specificity and cannot recognize mammalian, Xenopus, or vaccinia PAP. The C-term antibodies can deplete PAP from yeast whole cell extracts, resulting in loss of poly(A) addition activity. This immunodepletion also causes a reduction in the cleavage activity which can be restored by addition of yeast cleavage factor I [CF I; Chen, J., & Moore, C. (1992) Mol. Cell Biol. 12, 3470-3481], a factor needed for 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 ATP or exogenous RNA substrate. The monoclonal antibodies against yeast PAP will be a useful tool for further study of factors required for yeast mRNA 3' end processing.
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111-1800.
M M
MM
Kessler
A M
AM
Zhelkovsky
A
A
Skvorak
C L
CL
Moore
D000911:Antibodies, Monoclonal / Q000302:isolation & purification* / Q000494:pharmacology; D001666:Binding Sites, Antibody; D004789:Enzyme Activation / Q000187:drug effects; D005656:Fungal Proteins / Q000737:chemistry*; D015151:Immunoblotting; D011062:Polynucleotide Adenylyltransferase / Q000737:chemistry* / Q000276:immunology; D016601:RNA-Binding Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000276:immunology; D013045:Species Specificity; D039221:mRNA Cleavage and Polyadenylation Factors
D000911:Antibodies, Monoclonal; D005656:Fungal Proteins; D016601:RNA-Binding Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; D011062:Polynucleotide Adenylyltransferase