The poly(A) tail is a characteristic feature of eukaryotic and viral mRNAs,with roles in mRNA translation and stability. However, the mechanisms involved in its addition to mRNA are not fully understood. The goal of the work described herein is to develop a model in which the mechanism of synthesis of a poly(A) tail are defined in detail. Vaccinia virus provides a powerful tool with which to conduct such a study, since its strategy for poly(A) tail elongation is analogous to that employed by cellular polyadenylation systems, yet the viral system is relatively simple due to the absence of coupled processes such as RNA cleavage and signal recognition, and the absence of regulatory decisions involving alternate processing site choice. Furthermore, a detailed mechanistic analysis of poly(A) polymerase, of one of the simplest known RNA-synthetic enzymes, should illuminate a number of more general questions regarding RNA-protein transactions in RNA polymerases. To date, the P. I. has identified the genes encoding the heterodimeric vaccinia virus poly(A) polymerase, and characterized the individual subunits, VP55 and VP39, in highly purified form. VP55 and VP39 were found to be catalytic and stimulatory subunits, respectively, in which VP55 processively adds a 30 - 35 nt poly(A) tail to an mRNA 3' end followed by an abrupt transition to non-processive extension, and VP39 enables VP55 to retain processivity during elongation of the tail beyond 30 - 35 nt. The P. I. has also shown that VP55 interacts with RNA specifically via uridylate residues, and translocates with respect to the RNA during adenylate addition. The abrupt transition in processivity by VP55 thus appears to result from its translocation along the nascent poly(A) tail, which lacks uridylates for stable interaction. vP39 was shown to have an additional function, in 2'- O-methylation of the mRNA 5' cap. In this proposal, a more compete model of poly(A) tail formation will be developed. Initially, studies will be performed to address aspects of poly(A) tail formation in vivo: It will be determined whether the VP55 gene is essential; whether viruses can be made that arc conditionally lethal for poly(A) tail elongation; and whether in vivo tail lengths are regulated by factors other than in vivo levels of PAP. Second, characteristics and specificities of macromolecular interactions will be characterized to determine RNA binding site characteristics, which subunit guides the PAP to the 3'-OH, the contribution of the 5' cap structure to VP39-RNA interaction, and whether one subunit interacts with RNA solely via the other. Third, regions of the proteins required for RNA and nucleotide binding and for dimerization will be identified, and attempts will be made to crystallize the two subunits. Finally, functional experiments will be conducted to determine whether VP39 is a sliding clamp-type processivity factor, whether VP39 interacts with ATP in a functional manner, and to investigate possible links between the 5' and 3' end modification functions of VP39. The above studies are expected to provide a detailed understanding of the mechanism of poly(A) tail synthesis.
Showing the most recent 10 out of 19 publications
