Transcription and accurate polyadenylation in vitro of RNA from the major late adenovirus 2 transcription unit

Abstract

Nucleoprotein complexes with in vitro transcription activity were isolated from HeLa cells late in lytic infection with Adenovirus type 2 (Ad2). Both polymerase II and polymerase III were active in these extracts, and greater than 85% of the labeled RNA was Ad2-specific. Electrophoretic analyses and Southern blot analyses demonstrated that RNA complementary to the entire 30 kb late transcription unit including RNA near the presumed termination site was synthesized. The addition of DRB (5,6,dichloro-l-β-D-ribofuranosyl-benzimidazole) in vivo prior to the isolation of the complexes resulted in accumulation of polymerase II at the promoter proximal sites, but nascent chains started in vivo in DRB were successfully elongated in vitro. Approximately 10% of the RNA labeled in vitro contained poly(A), and the length of poly(A) was very similar to that of nuclear RNA isolated from Ad2-infected cells. The in vitro sites of poly(A) addition were specific—labeled poly(A)-terminated RNA molecules ended at a point on the genome coincident with previously mapped poly(A) sites of mRNAs produced in vivo. In addition, the polyadenylation enzyme (or enzymes) cosediment with the nucleoprotein complexes during sucrose gradient centrifugation since gradient purified complexes synthesize poly(A) containing RNA in vitro in the absence of any added nuclear extract.