Discrete functional stages of vaccinia virus early transcription during a single round of RNA synthesis in vitro.
We have developed a system for analysis of discrete steps in vaccinia virus early mRNA synthesis during a single round of transcription in vitro. A synthetic early promoter is used to direct transcription by vaccinia RNA polymerase of a G-less cassette in linear duplex DNA. Omission of GTP from transcription reactions leads to the formation of ternary elongation complexes paused stably at the end of the G-less cassette. These complexes can be induced to elongate by provision of GTP. While initiation of transcription is sensitive to low concentrations of salt and Sarkosyl, elongation is relatively resistant to these agents. Termination can be studied in a single synthetic cycle by forming transcription complexes paused just proximal to the termination signal TTTTTNT that can subsequently elongate and terminate. By selectively incorporating the termination-inhibiting analog BrUMP into proximal and distal portions of the nascent transcript, we localize the termination signal within or near the sequence UUUUUNU in the nascent RNA. We show that access of the vaccinia termination factor (VTF/capping enzyme) to the transcriptional apparatus can occur subsequent to initiation and synthesis of a 390-nucleotide nascent RNA. Termination is more sensitive to inhibition by salt and Sarkosyl than in elongation. This sensitivity is not reversed by preincubation of VTF with the transcription complex. Finally, we confirm the identity of VTF and vaccinia mRNA capping enzyme by demonstration of VTF activity associated with capping enzyme expressed in Escherichia coli.
Luo
Y
Y
Program in Molecular Biology, Sloan-Kettering Institute, New York, New York 10021.
Hagler
J
J
Shuman
S
S
D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009994:Osmolar Concentration; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012521:Sarcosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000187:drug effects; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics*
D003902:Detergents; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; C025231:sarkosyl; D012321:DNA-Directed RNA Polymerases; D012521:Sarcosine