9604188 Gershon Part 1-Technical RNA methyltransferase, viz. vaccinia virus protein VP39, methylates the ribose 2'-O- of the penultimate nucleotide of the mRNA cap structure leading to formation of the type I mRNA cap. The three-dimensional structure of this enzyme, recently determined by the P.I., affords a structural basis on which to investigate the catalytic mechanism of RNA methylation. Since other structurally-defined methyltransferases methylate base rather than sugar moieties, this system provides also an unusual opportunity to examine nucleic acid ribose methylation. VP39 is a bifunctional protein whose second function is as a poly(A) polymerase processivity factor, interacting with both mRNA ends. This study investigates whether VP39 possesses more than a single RNA binding site to satisfy its dual function, using competition assays between polyadenylation and methyltransferase substrates for wild-type VP39 and mutants with substituted surface residues at possible RNA binding sites. Functional and RNA binding assays will also be done. Due to the greater affinity of monomeric VP39 for capped than uncapped RNA, the effect on capped RNA binding on VP39 interaction with its dimerization partner, VP55, will also be investigated. Further experiments will aim at the study of the bimolecular methylation reaction sequence for the interaction of VP39 with capped RNA, such as the involvement of substrate ionization, and of specific residues in catalysis. Whether cap 2'-0-methylation correlates with inversion at the methyl carbon center will also be determined. Part 2 Non -Technical Vaccinia virus is a benign poxvirus whose notoriety stems from its extensive use as the vaccine that led to the first global eradication of a human disease, namely smallpox. Vaccinia is virtually unique in its ability to express its genes autonomously, without needing most of the machinery present in the cells it infects. It does this by carrying its own analogs of host cell enzymes that it would other requi re. Many of these analogs are much easier to study than the host cell equivalent, because they can more easily be produced in large quantities and purified, yet maintain an equally informative, molecular information. One of these enzymes, named "cap specific 2'-O-methyltransferase", plays a role in the expression of all genes, and is considered a prototype for this class of enzymes. This study is aimed at understanding the precise mechanism by which the enzyme operates.The three-dimensional structure of RNA methyltransferase, viz. vaccinia virus protein VP39, affords a physical basis on which to investigate the catalytic mechanism of RNA methylation. The elucidation of the interaction of VP39 with capped RNA, structurally and biochemically, is one example of advances in Molecular Biochemistry towards the understanding of reaction mechanisms.
