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 cerevisiae and examined the effects of these truncations on function of the enzyme. PAP synthesizes a poly(A) tail onto the 3'-end of RNA without any primer specificity but, in the presence of cellular factors, is directed specifically to the cleaved ends of mRNA precursors. The last 31 amino acids of PAP are dispensable for both nonspecific and specific activities. Removal of the next 36 amino acids affects an RNA binding domain, which is essential for the activity of the enzyme and for cell viability. This novel RNA binding site was further localized using additional deletions, cyanogen bromide cleavage of PAP cross-linked with RNA or 8-azido-ATP, and a monoclonal antibody against a COOH-terminal 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 effect on nonspecific polyadenylation but completely eliminates specific activity. This region is essential for enzyme function in vivo and is probably involved in the interaction of PAP with other protein(s) of the polyadenylation machinery.
Zhelkovsky
A M
AM
Department of Molecular Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111-1800, USA.
Kessler
M M
MM
Moore
C L
CL
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites; D002417:Cattle; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D000939:Epitopes / Q000032:analysis; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011062:Polynucleotide Adenylyltransferase / Q000737:chemistry* / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014466:Ultraviolet Rays; D014981:Xenopus
D000911:Antibodies, Monoclonal; D001386:Azides; D003432:Cross-Linking Reagents; D000939:Epitopes; D010446:Peptide Fragments; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C017040:8-azidoadenosine 5'-triphosphate; D012313:RNA; D000255:Adenosine Triphosphate; D011062:Polynucleotide Adenylyltransferase; D003488:Cyanogen Bromide