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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...

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RNA DNA hybridization experiments utilizing separated strands of HeLa mitochondrial DNA and mit-RNA from HeLa cells exposed to short pulses of have shown that the labeled RNA hybridizes with both the light (L) and the heavy (H) strand, though to a different relative extent depending upon the labeling time. Thus, hybridization of pulse-labeled RNA is about equal with the two strands when the pulse is very short (1-5 min), and becomes more and more predominant with the H strand with increasing pulse length. Pulse-labeled fast-sedimenting mit-RNA forms RNase-resistant doublestranded structures up to more than 5 jAm long when selfannealed or annealed with an excess of unlabeled mit-RNA. These observations and the previous evidence of complete transcription of the H strand strongly suggest that mit-DNA is transcribed in HeLa cells symmetrically over a considerable portion of its length, with the transcript of the L strand being rapidly degraded or otherwise removed from the mitochondrial fraction.RNA -DNA hybridization experiments utilizing separated strands of HeLa mit-DNA and mit-RNA from HeLa cells uniformly labeled with [5-3H]uridine have shown that this RNA contains sequences complementary to the whole or almost whole length of the heavy (H) strand and to a short portion of the light (L) strand (1, 2). These results have been interpreted to indicate that mit-DNA in HeLa cells is completely or almost completely transcribed, and, furthermore, that this transcription occurs almost exclusively from the H strand, as previously reported for rat liver mit-DNA (3). However, these results did not exclude the possibility that the L strand is also transcribed to a considerable extent, if not completely, with the majority of this transcript being subsequently degraded or otherwise removed from the mitochondrial fraction. In the course of investigations on the synthesis and processing of mit-RNA, an analysis of the hybridization properties of RNA from HeLa cells labeled for a short term with [5-3H]uridine has unexpectedly revealed that the above mentioned possibility is indeed true. This paper describes these experiments. MATERIALS AND METHODS 1757and extraction of mitochondrion-associated RNA have been previously described (4) (the isopycnic centrifugation was performed here for 40 min at 25,000 rpm in the Spinco SW25.1 rotor).The RNA samples, fractionated on a sucrose gradient, to be used in RNA DNA or RNA RNA hybridizations were subjected to DNase digestion and phenol-sodium dodecylsulfate (SDS) extraction and either sedimented again through a sucrose gradient or fractionated on Sephadex G-100 (1).Hybridization of mit-RNA with H or L mit-DNA strands (which had been separated in an alkaline CsCl density gradi- for 4 hr; after rapid cooling, the mixtures were treated with 5 ,ug/ml of previously heated pancreatic RNase for 20 min at room temperature, then filtered through nitrocellulose membranes, which were washed with 100 ml of 2X SSC (SSC = 0.15 M NaCl-0.015 M Na citrate). Unless otherwise specified, the RNA pre...