Recognition of Capped RNA Substrates by VP39, the Vaccinia Virus-Encoded mRNA Cap-Specific 2‘-O-Methyltransferase
We have investigated the interaction of VP39, the vaccinia-encoded mRNA cap-specific 2‘-O-methyltransferase, with its capped RNA substrate. Two sites on the protein surface, responsible for binding the terminal cap nucleotide (m7G) and cap-proximal RNA, were characterized, and a third (downstream RN...
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Published in | Biochemistry (Easton) Vol. 37; no. 23; pp. 8564 - 8574 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Chemical Society
09.06.1998
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Subjects | |
Online Access | Get full text |
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Summary: | We have investigated the interaction of VP39, the vaccinia-encoded mRNA cap-specific 2‘-O-methyltransferase, with its capped RNA substrate. Two sites on the protein surface, responsible for binding the terminal cap nucleotide (m7G) and cap-proximal RNA, were characterized, and a third (downstream RNA binding) site was identified. Regarding the crystallographically defined m7G binding pocket, VP39 showed significant activity with adenine-capped RNA. Although VP39 mutants lacking specific m7G-contact side chains within the pocket showed reduced catalytic activity, none was transformed into a cap-independent RNA methyltransferase. Moreover, each retained a preference for m7G and A over unmethylated G as the terminal cap nucleotide, indicating a redundancy of m7G-contact residues able to confer cap-type specificity. Despite containing the 2‘-O-methylation site, m7GpppG (cap dinucleotide) could not be methylated by VP39, but m7GpppGUbiotinp could. This indicated the minimum-length 2‘-O-methyltransferase substrate to be either m7GpppGp, m7GpppGpN, or m7GpppGpNp. RNA−protein contacts immediately downstream of the m7GpppG moiety were found to be pH-sensitive. This was detectable only in the context of a weakened interaction of near-minimum-length substrates with VP39's m7G binding pocket (through the use of either adenine-capped substrate or a VP39 pocket mutant), 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 from the 5‘ terminus to a previously unknown site on the VP39 surface. |
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Bibliography: | Supported by National Science Foundation Grant MCB-9604188 to P.D.G. ark:/67375/TPS-HLC2DGSB-F istex:2AAC21471E2A759BBE03175E02B5C62A504FECAF ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 |
ISSN: | 0006-2960 1520-4995 |
DOI: | 10.1021/bi980178m |