Antitermination of vaccinia virus early transcription: possible role of RNA secondary structure.
Transcription of vaccinia early genes by the viral RNA polymerase terminates downstream of a signal sequence TTTTTNT in the nontemplate DNA strand. Signal recognition occurs at the level of the sequence UUUUUNU in nascent RNA and depends on a virus-encoded termination factor (VTF). The presence of TTTTTNT elements within protein encoding regions of some early genes requires that these 5' proximal signals be ignored in order to achieve early expression of the full-sized proteins. In the case of the A18R gene, which contains a proximal terminator that is not utilized in vivo (Pacha et al., J. Virol. 64, 3853-3863 (1990)), the TTTTTNT sequence can be folded into a potential hairpin structure such that UUUUUNU would be part of a duplex stem in the nascent RNA. We find that the A18R putative hairpin is unable to promote factor-dependent termination in a purified in vitro transcription system. Sequence manipulations that abrogate the potential to form an RNA hairpin restore the activity of the TTTTTNT motif. The in vitro studies suggest that antitermination at the proximal site of the A18R gene may be mediated by secondary structure in the nascent RNA, and that early termination involves recognition by VTF and/or RNA polymerase of the UUUUUNU sequence in single-stranded form.
Luo
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Y
Program in Molecular Biology, Sloan-Kettering Institute, New York, New York 10021.
Shuman
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S
D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000235:genetics*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics*; D015183:Restriction Mapping; D013728:Terminator Regions, Genetic; D013937:Thymidine Kinase / Q000235:genetics; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000235:genetics*
D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral; D013937:Thymidine Kinase; D012321:DNA-Directed RNA Polymerases