R G Roeder

We have developed a procedure for preparing extracts from nuclei of human tissue culture cells that directs accurate transcription initiation in vitro from class II promoters. Conditions of extraction and assay have been optimized for maximum activity using the major late promoter of adenovirus 2. The extract also directs accurate transcription initiation from other adenovirus promoters and cellular promoters. The extract also directs accurate transcription initiation from class III promoters (tRNA and Ad 2 VA).

We describe a new assay system that allows a rapid, direct, and quantitative detection of promoter-dependent in vitro transcription by RNA polymerase II. The template used is a hybrid plasmid containing the adenovirus major late promoter linked to a synthetic 400-base-pair DNA fragment that lacks cytidine residues on the transcribed strand--i.e., generates a transcript with no guanosine residues. In vitro transcriptions are carried out in the absence of GTP or, if the reactions contain GTP, in the presence of RNase T1 and the chain terminator 3'-0-methyl-GTP. Under these conditions the only RNAs that can accumulate, whether from a circular or linearized DNA template, are the 400-nucleotide RNase T1-resistant transcripts resulting from accurate initiation at the major late promoter. Thus, specific transcription can be directly monitored by conventional RNA quantitation methods. Using this fast assay, we show that three basic transcription factors, TFIIB, TFIID, and TFIIE, are absolutely required, in addition to the RNA polymerase II, for specific transcription initiation from the adenovirus major late promoter. Units of activity can be defined for each of these individual components. The applicability of this kind of assay to other systems is discussed.

Immediate early genes of herpes simplex viruses contain one or more copies of the conserved TAAT-GARAT (where R is purine) DNA motif. A virus-encoded regulatory protein (Vmw65) is believed to stimulate transcription via this element, although the protein does not bind directly to DNA. Overlapping the TAATGARAT element in many cases is an octamer sequence (ATGCAAAT) that is involved both in transcription by RNA polymerases II and III and in adenovirus DNA replication. So far at least two proteins (OTF-1 and OTF-2) have been identified that bind to the octamer. We show that both affinity-purified OTF-1 and OTF-2 bind to the TAATGARAT sequence and that Vmw65 induces the formation of an additional complex that involves OTF-1 and that is further retarded in a band-shift gel assay. Complementation experiments involving addition of purified OTF-1 to nuclear extracts that have been depleted of endogenous OTF-1 show that at least one other cellular factor(s) is required for complex formation. This cellular factor may be involved in recognition of the GARAT sequence.