We have shown that an extract made from HeLa cells harvested 6 hr after infection with vaccinia virus can transcribe a duplex DNA template containing a late viral gene. S1 nuclease analyses using genomic and synthetic probes indicated that the 5' ends of RNA synthesized in vitro are similar to those of RNA made in vivo and contain 5' poly(A) sequences contiguous with the translation initiation codon. Kinetic analysis of RNA synthesized in vitro demonstrated that a correctly initiated and 5' polyadenylylated product appeared within 5 min after transcription reactions were started. A cis-splicing mechanism of poly(A) addition can be ruled out because the DNA template used in vitro had no poly(dT) sequence and could contain as few as 37 base pairs upstream of the start of the RNA. In addition, we found that a point mutation in the first of two consecutively encoded adenylate residues preceding the ATG initiation codon abolished transcription in vitro. These data are consistent with at least three models: (i) RNA polymerase initiates RNA synthesis with a run of adenylate residues; (ii) a poly(A) primer is used for initiation; or (iii) the poly(A) leader is rapidly and efficiently attached to the RNA by ligation.
Cell-Free System Endonucleases