DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract.

TLDR

RNA polymerase II transcription requires promoter‑containing eukaryotic DNA. The cell‑free system uses a dialyzed, concentrated HeLa cell extract with necessary cofactors and added DNA. The system yields full‑length, capped adenovirus transcripts, accurately initiating from late, early, and intermediate promoters and producing RNAs over 4000 nucleotides.

Abstract

We have developed a cell-free system for studying the synthesis of mRNA in mammalian cells. The system consists of a dialyzed and concentrated whole-cell extract derived from HeLa cells, small molecules and cofactors needed for transcription, and exogenously added DNA. Accurate transcription by RNA polymerase II is entirely dependent upon addition of promoter-containing eukaryotic DNA. At optimal DNA and extract concentrations, transcription initiation from the adenovirus serotype 2 late promoter is readily detectable, and specific transcripts over 4000 nucleotides in length are observed. The RNA synthesized in vitro contains the same 5' capped RNase T1 undecanucleotide as does the in vivo transcript. RNA synthesis also initiates accurately at both an early and an intermediate adenovirus promoter site.