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.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				V. Vethantham, N. Rao, and J. L. Manley Sumoylation regulates multiple aspects of mammalian poly(A) polymerase function Genes & Dev., February 15, 2008; 22(4): 499 - 511. [Abstract] [Full Text] [PDF]

				V. Vethantham, N. Rao, and J. L. Manley Sumoylation Modulates the Assembly and Activity of the Pre-mRNA 3' Processing Complex Mol. Cell. Biol., December 15, 2007; 27(24): 8848 - 8858. [Abstract] [Full Text] [PDF]

				G. Martin and W. Keller RNA-specific ribonucleotidyl transferases RNA, November 1, 2007; 13(11): 1834 - 1849. [Abstract] [Full Text] [PDF]

				R. Trippe, E. Guschina, M. Hossbach, H. Urlaub, R. Luhrmann, and B.-J. Benecke Identification, cloning, and functional analysis of the human U6 snRNA-specific terminal uridylyl transferase RNA, August 1, 2006; 12(8): 1494 - 1504. [Abstract] [Full Text] [PDF]

				L. S. Chen and T. L. Sheppard Chain Termination and Inhibition of Saccharomyces cerevisiae Poly(A) Polymerase by C-8-modified ATP Analogs J. Biol. Chem., September 24, 2004; 279(39): 40405 - 40411. [Abstract] [Full Text] [PDF]

				A. ZHELKOVSKY, S. HELMLING, A. BOHM, and C. MOORE Mutations in the middle domain of yeast poly(A) polymerase affect interactions with RNA but not ATP RNA, April 1, 2004; 10(4): 558 - 564. [Abstract] [Full Text] [PDF]

				Y. Tacahashi, S. Helmling, and C. L. Moore Functional dissection of the zinc finger and flanking domains of the Yth1 cleavage/polyadenylation factor Nucleic Acids Res., March 15, 2003; 31(6): 1744 - 1752. [Abstract] [Full Text] [PDF]

				S. L. Topalian, S. Kaneko, M. I. Gonzales, G. L. Bond, Y. Ward, and J. L. Manley Identification and Functional Characterization of Neo-Poly(A) Polymerase, an RNA Processing Enzyme Overexpressed in Human Tumors Mol. Cell. Biol., August 15, 2001; 21(16): 5614 - 5623. [Abstract] [Full Text] [PDF]

				S. Helmling, A. Zhelkovsky, and C. L. Moore Fip1 Regulates the Activity of Poly(A) Polymerase through Multiple Interactions Mol. Cell. Biol., March 15, 2001; 21(6): 2026 - 2037. [Abstract] [Full Text]

				J. Bard, A. M. Zhelkovsky, S. Helmling, T. N. Earnest, C. L. Moore, and A. Bohm Structure of Yeast Poly(A) Polymerase Alone and in Complex with 3'-dATP Science, August 25, 2000; 289(5483): 1346 - 1349. [Abstract] [Full Text]

				J. Zhao, L. Hyman, and C. Moore Formation of mRNA 3' Ends in Eukaryotes: Mechanism, Regulation, and Interrelationships with Other Steps in mRNA Synthesis Microbiol. Mol. Biol. Rev., June 1, 1999; 63(2): 405 - 445. [Abstract] [Full Text] [PDF]

				A. Zhelkovsky, S. Helmling, and C. Moore Processivity of the Saccharomyces cerevisiae Poly(A) Polymerase Requires Interactions at the Carboxyl-Terminal RNA Binding Domain Mol. Cell. Biol., October 1, 1998; 18(10): 5942 - 5951. [Abstract] [Full Text]

				D. F. Colgan and J. L. Manley Mechanism and regulation of mRNA polyadenylation Genes & Dev., November 1, 1997; 11(21): 2755 - 2766. [Full Text] [PDF]

				M. M. Kessler, M. F. Henry, E. Shen, J. Zhao, S. Gross, P. A. Silver, and C. L. Moore Hrp1, a sequence-specific RNA-binding protein that shuttles between the nucleus and the cytoplasm, is required for mRNA 3'-end formation in yeast Genes & Dev., October 1, 1997; 11(19): 2545 - 2556. [Abstract] [Full Text] [PDF]

				J. Zhao, M. M. Kessler, and C. L. Moore Cleavage Factor II of Saccharomyces cerevisiae Contains Homologues to Subunits of the Mammalian Cleavage/ Polyadenylation Specificity Factor and Exhibits Sequence-specific, ATP-dependent Interaction with Precursor RNA J. Biol. Chem., April 18, 1997; 272(16): 10831 - 10838. [Abstract] [Full Text] [PDF]

				G. Stumpf and H. Domdey Dependence of Yeast Pre-mRNA 3'-End Processing on CFT1: A Sequence Homolog of the Mammalian AAUAAA Binding Factor Science, November 29, 1996; 274(5292): 1517 - 1520. [Abstract] [Full Text]

				M. M. Kessler, J. Zhao, and C. L. Moore Purification of the Saccharomyces cerevisiae Cleavage/Polyadenylation Factor I. SEPARATION INTO TWO COMPONENTS THAT ARE REQUIRED FOR BOTH CLEAVAGE AND POLYADENYLATION OF mRNA 3prime ENDS J. Biol. Chem., October 25, 1996; 271(43): 27167 - 27175. [Abstract] [Full Text] [PDF]

				K. Perumal, K. Sinha, D. Henning, and R. Reddy Purification, Characterization, and Cloning of the cDNA of Human Signal Recognition Particle RNA 3'-Adenylating Enzyme J. Biol. Chem., June 8, 2001; 276(24): 21791 - 21796. [Abstract] [Full Text] [PDF]