Interaction of two cis sites with the RNA replicase of the yeast L-A virus.
L-A is a 4.6-kilobase double-stranded RNA virus of Saccharomyces cerevisiae. The in vitro L-A replication reaction ((-)-strand synthesis) requires an internal site 400 bases from the 3' end in addition to the 3'-terminal 30 nucleotides of the L-A (+)-single-stranded RNA. Elimination of the internal site reduces the template activity 5-10-fold. Here we investigate how the internal site can stimulate the replication reaction which starts at the 3' end of the template. When these two sites are split into two distinct RNA molecules, the internal site can no longer stimulate replication (no trans-activation). However, establishment of an intermolecular hydrogen bonding between these RNAs restored the replication-enhancing activity of the internal site. This result is consistent with a model wherein L-A's RNA polymerase interacts first with the internal site and then with the 3' end site by either looping or by a local dissociation-reassociation mechanism. These results, however, clearly eliminate anchored tracking and sliding models which require continuity of the RNA molecule between these two cis sites.
Fujimura
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Section on Genetics of Simple Eukaryotes, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Wickner
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RB
D001345:Autoradiography; D001483:Base Sequence; D004587:Electrophoresis, Agar Gel; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes; D010957:Plasmids; D012328:RNA Viruses / Q000201:enzymology* / Q000502:physiology; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014779:Virus Replication
D009692:Nucleic Acid Heteroduplexes; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase