Pausing and premature termination of human RNA polymerase II during transcription of adenovirus in vivo and in vitro.

Abstract

The major late transcriptional unit of adenovirus type 2 has served as a model for studying transcription in eukaryotes. We report that pausing and premature termination are intrinsic to the transcription of this transcriptional unit by RNA polymerase II. In vivo and in isolated nuclei, transcription pauses at discrete sites proximal to the initiation site and can prematurely terminate at nucleotide 175 and possibly also at nucleotide 120. The prematurely terminated RNAs are not associated with the transcription complexes and accumulate in the cell nucleus in vivo, whereas paused RNAs remain associated with the transcription complexes and elongate into full-length transcripts. Pausing is also reproduced in the transcription complexes in a soluble system. 5,6-Dichloro-1-1&D-ribofuranosylbenzimidazole enhances pausing but not premature termination, and its action is reversible. The proposed premature termination site at nucleotide 175 in adenovirus type 2 bears sequence homology to the tRI site in coliphage A.In eukaryotes, the expression of both cellular and viral genes appears to be regulated primarily at the transcriptional level (1). In prokaryotes, transcription is regulated not only at the level of initiation but also by attenuation-the termination of transcription shortly following initiation (2). Little is known about the mechanism and regulation of transcription initiation in eukaryotes, and virtually nothing is understood about the events associated with elongation and termination. Adenovirus type 2 (Ad-2)-infected HeLa cells contain small RNA species complementary to the promoter-proximal region of the major late transcription unit; these are represented in extramolarity compared to other regions of the unit at steady state in vivo (3) and in isolated nuclei (4). Synthesis of these RNAs persists in the presence of 5,6-dichloro-1-,B3D-ribofuranosylbenzimidazole (DRB), which apparently reduces the synthesis of productive transcripts (5). These results were interpreted to imply premature termination of transcription in adenovirus. Premature termination was also suggested to occur in Chinese hamster cells (6) and in globinproducing cells (7), where DRB had a similar effect. However, the critical experiments to ascertain the fate of these small transcripts have not been performed. Although promoter-proximal "attenuation" has been suggested to occur in simian virus 40 due to the appearance of a small RNA during transcription in vitro (8), the existence of this small RNA in simian virus 40-infected cells has not been demonstrated.Although pausing during transcription has not been observed in the transcription of eukaryotic genes in vivo, it is a common occurrence in the transcription of bacterial and phage genes (9). Kadesch and Chamberlain (10) reported that polymerase II from calf thymus pauses during transcription of a T7 bacteriophage template in vitro. Coppola et al.(11) found that in a soluble system under limited substrate concentration, the human polymerase II pauses within 30 nucleotides (nt)...

RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells.

“…In vitro studies of the lacUVS promoter reveal another type of incomplete transcription in which E. coli RNA polymerase can cycle producing short (10 base) RNA chains reiteratively without leaving the promoter (5). Several in vitro and in vivo studies have indicated that premature termination may limit the number of full-length transcripts produced from eucaryotic genes as well (1,21,31 (4,28). The organization of the essential components of the hsp7O promoter is similar to many eucaryotic promoters.…”

The RNA polymerase II ternary complex cleaves the nascent transcript in a 3'----5' direction in the presence of elongation factor SII.

“…To study the effect of SII at the two well-documented sites within the first intron of the Ad2 ML transcription unit (Maderious and Chen Kiang 1984), we produced Sarkosyl-rinsed early elongation complexes paused at position + 12 {see Izban and Luse 1991) downstream of the Ad2 ML promoter on the pSmaF-1 plasmid. We then restarted elongation using either 1 mM NTPs {Fig.…”

“…A number of DNA sequences that serve as blocks to elongation in vivo have been characterized using in vitro transcription systems (e.g., see Maderious and Chen Kiang 1984;Kerppola and Kane 1990). These sequences have been termed intrinsic termination sites, although a significant portion of "core" RNA polymerase II ternary complexes (polymerases devoid of the elongation factors mentioned above) can elongate through these regions in vitro.…”

The block to transcriptional elongation within the human c-myc gene is determined in the promoter-proximal region.

“…Viral transcription units, including SV40, adenovirus, and the murine minute virus provided the first examples of premature termination or attenuation in eukaryotes (Hay et al 1982;Ben-Aser and Aloni 1984;Maderious and Chen-Kiang 1984). Control at the level of elongation has also been observed in several eukaryotic cellular genes, including c-myc, c-myb, c-fos, ADA, 1-myc, and histone 3.3, and in the human retroviruses HIV-1 and HIV-2 (Spencer and Groudine 1990).…”