RECOGNITION OF CAPPED RNA SUBSTRATES BY VP39, THE VACCINIA VIRUS-ENCODED MESSENGER-RNA CAP-SPECIFIC 2'-O-METHYLTRANSFERASE

We have investigated the interaction of VP39, the vaccinia-encoded mRN A cap-specific 2'-O-methyltransferase, with its capped RNA substrate. Two sites on the protein surface, responsible for binding the terminal cap nucleotide (m(7)G) and cap-proximal RNA, were characterized, and a third (downstream RNA binding) site was identified, Regarding the cr ystallographically defined m(7)G binding pocket, VP39 showed significa nt activity with adenine-capped RNA. Although VP39 mutants lacking spe cific m(7)G-contact side chains within the pocket showed reduced catal ytic activity, none was transformed into a cap-independent RNA methylt ransferase. Moreover, each retained a preference for m(7)G and A over unmethylated G as the terminal cap nucleotide, indicating a redundancy of m(7)G-contact residues able to confer cap-type specificity. Despit e containing the 2'-O-methylation site, m(7)GpppG (cap dinucleotide) c ould not be methylated by VP39, but m(7)GpppGU(biotin)p could, This in dicated the minimum-length 2'-O-methyltransferase substrate to be eith er m(7)GpppGp, m(7)GpppGpN, or m(7)GpppGpNp. RNA-protein contacts imme diately downstream of the m(7)GpppG moiety were found to be pH-sensiti ve. This was detectable only in the context of a weakened interaction of near-minimum-length substrates with VP39's m(7)G binding pocket (th rough the use of either adenine-capped substrate or a VP39 pocket muta nt), as a dramatic elevation of K-M at pH values above 7.5. K-M values for substrates with RNA chain lengths of 2-6 nt were between 160 and 230 nM, but dropped to 9-15 nM upon increasing chain lengths to 20-50 nt. This suggested the binding of regions of the RNA substrate >6 nt f rom the 5' terminus to a previously unknown site on the VP39 surface.