Attenuation in SV40 as a mechanism of transcription-termination by RNA polymerase B

We have studied transcription elongation and termination in the human c-myc gene. Transcription of c-myc gene sequences with purified mammalian RNA polymerase II revealed several sites of transcription termination and pausing in the vicinity of the exon 1-intron 1 junction. This region previously has been shown to block transcription elongation in vivo by nuclear run-on analysis (D. Bentley and M. Groudine, Nature [London] 321:702-706, 1986). These sites were recognized by purified RNA polymerase II, and we therefore designated them intrinsic sites of termination and pausing. Two of these sites cause termination of RNA polymerase Ill transcription as well. RNA polymerase II terminated transcription in a cluster of seven consecutive T residues in the nontranscribed strand and paused during transcription at three additional sites in this region. The intrinsic sites of transcription termination and pausing described here correspond closely to the 3' ends of transcripts synthesized in Xenopus oocytes injected with plasmids containing the c-myc termination region (D. Bentley and M. Groudine, Cell 53:245-256, 1988). This correspondence suggests that the intrinsic recognition of these termination and pause sites by purified RNA polymerase II may play a role in the transcription elongation block observed in vivo.

mentioning

confidence: 63%

Intrinsic sites of transcription termination and pausing in the c-myc gene.

Kerppola¹,

Kane²

1988

“…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.…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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

Bengal¹,

Flores²,

Krauskopf³

et al. 1991

149

133

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...

“…Therefore, the strong possibility exists that the study of 3'-end formation of an mRNA in yeast cells will shed light on the actual transcription termination event. Termination is known to be important in the regulation of a variety of bacterial operons (reviewed in references 17 and 24) and appears to be involved in the regulation of at least one eucaryotic gene (9,10,28). We have been investigating the sequence determinants of transcription termination on a DNA segment in S. cerev,isiae.…”

mentioning

confidence: 99%

Sequences responsible for transcription termination on a gene segment in Saccharomyces cerevisiae.

Henikoff¹,

Cohen²

1984

156

We have mapped a signal sequence for mRNA 3'-end formation in Saccharomyces cerevisiae by using a Drosophila melanogaster DNA segment that complements a yeast adenine-8 mutation. That the 3' end of the transcript in S. cerevisiae nearly coincides with that in D. melanogaster is consistent with the possibility that mRNA termini are similarly determined in both organisms. Deletion analysis reveals that the complete signal is no more than 21 base pairs long. Part of the signal is the sequence TTTTTATA, which is seen in the termination region of several yeast genes. TTTTTATA appears to be able to act autonomously as a partial termination signal. The efficiency of the complete signal is affected by substitution of sequences downstream from it. This modulation of the effect of a signal is consistent with termination in S. cerevisiae, resembling rhodependent termination in bacteria.Until recently, transcription termination in eucaryotes has been poorly understood, in part because of the rapid processing that occurs at the 3' ends of transcripts for proteincoding genes. Processing usually involves cleavage of a precursor followed by polyadenylation in higher eucaryotes (6, 21). Histone mRNA 3' ends which lack polyadenylic acid were once thought to result from transcription termination (3) but now appear to be generated by processing (2, 7). The biochemical elucidation of mRNA 3' processing should be possible with the recent development of a cell-free system (23). In contrast to processing, actual transcription termination, which can occur at a considerable distance downstream from the mRNA 3' end (6, 27), has been relatively refractory to analysis.In Saccharomyces cereisisiae, polyadenylic acid addition and transcription termination appear to be coupled, as essentially all transcripts from protein-coding genes are polyadenylated [poly(A)+] (29). Therefore, the strong possibility exists that the study of 3'-end formation of an mRNA in yeast cells will shed light on the actual transcription termination event. Termination is known to be important in the regulation of a variety of bacterial operons (reviewed in references 17 and 24) and appears to be involved in the regulation of at least one eucaryotic gene (9, 10, 28). We have been investigating the sequence determinants of transcription termination on a DNA segment in S. cerev,isiae. This segment is derived from Drosophila melanogaster and complements a yeast adenine-8 mutation (15). We showed previously that mRNA 3' ends actually occur 50 to 90 base pairs (bp) downstream from a control signal, the 3' boundary of which we mapped precisely (13). Here we show that this signal is no more than 21 bp long. We also present evidence suggesting that an 8-bp portion of this signal promotes partial termination. It