Recognition of Capped RNA Substrates by VP39, the Vaccinia Virus-Encoded mRNA Cap-Specific 2'-O-Methyltransferase

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• 作者：Steve W. Lockless ; Hui-Teng Cheng ; Alec E. Hodel ; Florante A. Quiocho ; and Paul D. Gershon
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：June 9, 1998
• 年：1998
• 卷：37
• 期：23
• 页码：8564 - 8574
• 全文大小：166K
• 年卷期：v.37,no.23(June 9, 1998)
• ISSN：1520-4995

文摘

We have investigated the interaction of VP39, the vaccinia-encodedmRNA cap-specific 2'-O-methyltransferase, with its capped RNA substrate. Twosites on the protein surface, responsible forbinding the terminal cap nucleotide (m⁷G) and cap-proximalRNA, were characterized, and a third(downstream RNA binding) site was identified. Regarding thecrystallographically defined m⁷G bindingpocket, VP39 showed significant activity with adenine-capped RNA.Although VP39 mutants lackingspecific m⁷G-contact side chains within the pocket showedreduced catalytic activity, none was transformedinto a cap-independent RNA methyltransferase. Moreover, eachretained a preference for m⁷G and Aover unmethylated G as the terminal cap nucleotide, indicating aredundancy of m⁷G-contact residuesable to confer cap-type specificity. Despite containing the2'-O-methylation site, m⁷GpppG (capdinucleotide) could not be methylated by VP39, butm⁷GpppGU^biotinp could. This indicatedthe minimum-length 2'-O-methyltransferase substrate to be eitherm⁷GpppGp, m⁷GpppGpN, orm⁷GpppGpNp. RNA-protein contacts immediately downstream of the m⁷GpppGmoiety were found to be pH-sensitive. Thiswas detectable only in the context of a weakened interaction ofnear-minimum-length substrates withVP39's m⁷G binding pocket (through the use of eitheradenine-capped substrate or a VP39 pocket mutant),as a dramatic elevation of K_M at pH values above7.5. K_M values for substrates with RNAchain lengthsof 2-6 nt were between 160 and 230 nM, but dropped to 9-15 nM uponincreasing chain lengths to20-50 nt. This suggested the binding of regions of the RNAsubstrate >6 nt from the 5' terminus to apreviously unknown site on the VP39 surface.