Specific transcription from the adenovirus E2E promoter by RNA polymerase III requires a subpopulation of TFIID

Abstract

The early E2 (E2E) promoter of adenovirus type 2 possesses a TATA-like element and binding sites for the factors E2F and ATF. This promoter is transcribed by RNA polymerase II in high salt nuclear extracts, but by RNA polymerase III in standard nuclear extracts, as judged by sensitivity to low and high, respectively, concentrations of alpha-amanitin. Transcription by the two RNA polymerases initiated at the same site and depended, in both cases, on the TATA-like sequence and upstream elements. However, RNA polymerase III transcripts, unlike those synthesized by RNA polymerase II, terminated at two runs of Ts downstream of the initiation site. Although they are not essential, sequences downstream of the initiation site increased the efficiency of E2E transcription by RNA polymerase III. Such RNA polymerase III dependent transcription required a subpopulation of the general transcription factor, TFIID: TFIID that binds weakly to phosphocellulose (0.3 M eluate) complemented a TFIID-depleted extract to restore RNAp III transcription, whereas TFIID tightly associated with phosphocellulose (1 M eluate) was unable to do so.

RNA Polymerase III Accurately Initiates Transcription from RNA Polymerase II Promoters in Vitro

“…3A, quantified in B). RNA polymerase III initiation from TATA box containing Pol II promoters has been observed previously with fractionated, TFIID-depleted nuclear extract (27,28) or low salt crude nuclear extract with impaired Pol II transcriptional activity (29). It was thus surprising to observe such strong transcriptional activity by Pol III in unfractionated, commonly used nuclear extracts with high Pol II activity on a wide range of Pol II promoters (Figs.…”

“…In the absence of TFIID, human TBP-containing Brf1-TFIIIB stably binds the TATA box and recruits Pol III (28). Transcription of TATA-containing Pol II promoters by Pol III was also described in low salt crude nuclear extract with low Pol II activity (29).…”

A Role for TAF3B2 in the Repression of Human RNA Polymerase III Transcription in Nonproliferating Cells

“…Mouse monoclonal anti-human TBP antibody (MBP-6) was a gift from S. J. Flint (34). The secondary antibodies (goat anti-mouse antibody from Sigma and goat anti-rabbit antibody from Pierce) were both horseradish peroxidase conjugates.…”

“…For TBP depletions, 400 l of beads were incubated with 1 ml of tissue culture supernatant of MBP-6 cells, a hybridoma cell line expressing a mouse monoclonal anti-human TBP antibody (34). 300 l of antibody-coated beads were equilibrated with Pol III transcription buffer (20% (v/v) glycerol, 20 mM HEPES, 100 mM KCl, 5 mM MgCl 2 , 0.2 mM EDTA, 20 M ZnSO 4 , and 2 mM dithiothreitol; adjusted to pH 7.9) and incubated with the same volume of a PC-C fraction for immunodepletion of TBP-containing protein complexes from PC-C. For p53, 10 g of DO-1 antibody were linked to 100 l of protein G-Sepharose beads, and antibody-coated beads were equilibrated with 1ϫ phosphate-buffered saline.…”

Unusual Properties of Adenovirus E2E Transcription by RNA Polymerase III

“…Sequences located between positions ϩ1 and ϩ30 are also necessary for maximally efficient E2E transcription by RNA polymerase II in vitro (16). The results of mutational analysis of the organization of the E2E RNA polymerase III promoter indicate that it too comprises both internal and upstream sequences (16,43). The internal sequences conform to those present in the majority of promoters recognized by RNA polymerase (23): the E2E promoter contains an A box with typical sequence and functional properties and a B box that binds the * Corresponding author.…”