Role of the mammalian transcription factors IIF, IIS, and IIX during elongation by RNA polymerase II.

Abstract

We have used a recently developed system that allows the isolation of complexes competent for RNA polymerase II elongation (E. Bengal, A. Goldring, and Y. Aloni, J. Biol. Chem. 264:18926-18932, 1989). Pulse-labeled transcription complexes were formed at the adenovirus major late promoter with use of HeLa cell extracts. Elongation-competent complexes were purified from most of the proteins present in the extract, as well as from loosely bound elongation factors, by high-salt gel filtration chromatography. We found that under these conditions the nascent RNA was displaced from the DNA during elongation. These column-purified complexes were used to analyze the activities of different transcription factors during elongation by RNA polymerase II. We found that transcription factor IIS (TFIIS), TFIIF, and TFIIX affected the efficiency of elongation through the adenovirus major late promoter attenuation site and a synthetic attenuation site composed of eight T residues. These factors have distinct activities that depend on whether they are added before RNA polymerase has reached the attenuation site or at the time when the polymerase is pausing at the attenuation site. TFIIS was found to have antiattenuation activity, while TFIIF and TFIIX stimulated the rate of elongation. In comparison with TFIIF, TFIIS is loosely bound to the elongation complex. We also found that the activities of the factors are dependent on the nature of the attenuator. These results indicate that at least three factors play a major role during elongation by RNA polymerase II.RNA polymerase II sequentially utilizes several accessory factors during the multistage process of transcription (27). These factors can be functionally classified into three groups: (i) regulatory factors, which either directly bind to specific DNA sequences (18,26) or regulate the activity of specific DNA binding proteins (18); (ii) general transcription factors, which together with RNA polymerase II constitute the basic transcription machinery required for basal levels of transcription (27, 44); and (iii) factors that affect elongation and termination by RNA polymerase 11 (21,30,34,37,50).Recent studies have shown that the expression of several genes can be regulated by premature termination (1,32,41). Interestingly, it has been shown that premature termination during transcription of the mouse 13-major globin gene (33), the c-myc gene (5), and the Drosophila heat shock gene hsp-70 (43) can vary with the physiological state of the cells. Premature termination termed attenuation, has been shown to regulate the quantity of mRNA of several animal viruses, including simian virus 40 (SV40) (1,14,15,48,49), polyomavirus (47), the parvovirus minute virus of mice (2, 40), adenovirus type 2 (Ad2) (23,24,45), and human immunodeficiency virus types 1 and 2 (21, 54). These observations indicate that regulation of gene expression can be exerted at the level of elongation by RNA polymerase II. The findings that 3'-end formation of Ul and U2 small nuclear RNAs requires promoter elements...

DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs

“…Antitermination by the N protein involves the host proteins, NusA, NusB, NusG, ribosomal protein S10 and RNA elements at the 5Ј end of nascent viral transcripts. Whereas processive mechanisms found in phage have been extensively characterized and references therein), the mechanisms in eukaryotes are much less understood, except for the Drosophila hsp70 gene (Rougvie and Lis 1988;O'Brien and Lis 1991), the c-myc gene (Krumm et al 1992;Strobl and Eick 1992), and factor TFIIF (Price et al 1989;Bengal et al 1991), TFIIS (Sekimizu et al 1976), SIII (Elongin) (Bradsher et al 1993a,b;Aso et al 1995), P-TEFb (Marshall andPrice 1995;Marshall et al 1996), and HIV-1 Tat (Marciniak and Sharp 1991;Kato et al 1992;Zhou and Sharp 1995;Mancebo et al 1997;Zhu et al 1997). Tat activation of HIV-1 transcription is interesting because Tat enhances the processivity of transcription complexes in a manner reminiscent of N , and Tat activation is sensitive to DRB (Marciniak and Sharp 1991;Zhou and Sharp 1995;Mancebo et al 1997;Zhu et al 1997).…”

The block to transcription elongation at the minute virus of mice attenuator is regulated by cellular elongation factors.

“…The results shown in Fig. 6 clearly demonstrate that the concentrations of TFIIF and TFIIX shown to elongate the Ad2 attenuator RNA (6) can also prevent the elongation block at the MVM attenuator. It is interesting, however, that the lowest concentrations of TFIIS which are able to affect the extent of the elongation block at the MVM attenuator are about 10 times higher than those affecting the Ad2 attenuator.…”

“…Interestingly, an effect similar to that of TFIIS and TFIIF was observed with TFIIX, an activity initially identified as stimulating transcription from the Ad2 major late promoter when sequences downstream of +33 (relative to the major late promoter cap site) were present (27). The observed effect on elongation was specific for TFIIS, TFIIF, and TFIIX; the addition of TFIIA, TFIIB, TFIID, or TFIIE did not stimulate read-through of the Ad2 attenuation site (6).…”

“…We found that the addition of highly purified preparations of TFIIS (26) or TFIIF (14) to isolated transcriptional complexes affected the efficiency of elongation. The 185-nt Ad2 attenuator RNA disappeared, while the runoff transcripts increased-proportionally as a function of factor concentration (6). Interestingly, an effect similar to that of TFIIS and TFIIF was observed with TFIIX, an activity initially identified as stimulating transcription from the Ad2 major late promoter when sequences downstream of +33 (relative to the major late promoter cap site) were present (27).…”

“…Elongation complexes are then partially purified by using gel filtration chromatography at high KCl concentrations. The column-purified transcription complexes, which are depleted of elongation factors, can serve as a model system for determining the activities of factors during elongation (6).…”