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

Abstract

We have previously reported that both in vivo and in vitro, RNA polymerase H pauses or prematurely terminates transcription at a specific attenuation site located 142 to 147 nucleotides downstream from the P4 promoter of minute virus of mice (MVM). In this report, we show that an in vitro block to transcription elongation in HeLa whole-cell extract occurs at elevated KCI concentrations (0.2 to 1.5 M) but not at the standard KCI concentration (50 mM). Briefly initiated transcription complexes, devoid of dissociated elongation factors by passage through a Sephacryl S-1000 column at 0.3 M KCI, were allowed to elongate the briefly initiated nascent RNA, and a block to transcription elongation at the attenuation site was observed independently of the KCl concentration at the time of elongation. Moreover, the block to elongation was overcome by the addition, during elongation, to the column of purified complexes of whole-cell extract from EA cells but not from MVM-infected EA cells or HeLa cells. The general transcription factors IIF and UX were also shown to alleviate this block to transcription elongation. On the basis of these results, we suggest that the block to elongation at the MVM attenuation site observed late in MVM infection results, at least in part, from the inactivation of the general transcription elongation factors.Recently, it has been shown that eukaryotic RNA polymerase II, like the prokaryotic polymerase, can pause or terminate both in vivo and in vitro transcription within viral (1,3,16,17,19,24,28,29,32,38) and cellular (4,8,9,15,18) genes and thus modulate downstream gene expression. This mechanism is termed attenuation (39). To date, however, little is known about cis and trans elements involved in the attenuation mechanism in eukaryotes. We have shown that at least in some viral systems, RNA polymerase II can respond to a stem-loop structure in the RNA followed by polyuridines as a signal for the elongation block in vitro (1,3,16,19,28,29,32). In other systems, different sequences constitute the attenuation signal (2,9,31,34,40).One of the model systems in our attenuation studies has been the parvovirus minute virus of mice (MVM). MVM has a genome containing linear single-stranded DNA (5,149 nucleotides [nt]). It is an autonomous virus but is dependent for replication upon cellular functions that are expressed during S phase (10,12,13,36). Transcription initiation on the double-stranded replicative form catalyzed by the host RNA polymerase II has been shown to map to two MVM promoters: P4, located at nt 201 + 5; and P38, located at nt 2005 ± 4 (3, 20, 25).We have previously shown that in vivo and in vitro, RNA polymerase II attenuates transcription at a specific location 142 to 147 nt downstream from the P4 promoter (3, 28). The attenuator RNA was found and mapped in vivo in A9 cells late after infection in both the nuclear and cytoplasmic fractions. Moreover, through the use of in vitro systems, we have shown that the attenuator RNA is a result of pausing or termination and not of processing...

Control of formation of two distinct classes of RNA polymerase II elongation complexes.

“…The reason runoff in the presence of high Sarkosyl did not return to the level in the untreated reactions is that reinitiation is inhibited above 0.025% Sarkosyl (26). Other investigators working on attenuation in synthetic and natural virus sequences have found stimulations with elevated concentration of KCI (6,38). Sarkosyl and heparin have been used to inhibit reinitiation by many investigators (1, 6, 31 (31).…”

“…The second factor, factor 5 from D. melanogaster (or mammalian TFIFF or RAP 30/74), is required for initiation and also stimulates the elongation rate of RNA polymerase II (5,9,19,56,57). The third factor, TFIIX, was identified in HeLa cell extract and stimulates elongation by RNA polymerase 11 (5,38). Finally, a recently identified yeast protein, YES, stimulates the elongation rate of RNA polymerase II (12).…”

In Vitro Analysis of Elongation and Termination by Mutant RNA Polymerases with Altered Termination Behavior

“…The extent of the elongation block correlates with the stability of the RNA hairpin and the length of the T stretch in the DNA (7). Regulation of attenuation at these sites in vivo might be effected through changes in the abundance of elongation factors (like transcription factor IIS) or in their interaction with the transcribing polymerases on the genes with attenuation signals (9,28). RNA pol I from Saccharomyces cerevisiae also terminates in a T-rich region coding for the last 12 nucleotides of the transcript (30).…”

Control of transcription arrest in intron 1 of the murine adenosine deaminase gene.

“…Upon dilution or removal of the disruptive agent by gel filtration, exogenously added SII was able to promote read-through of intron 1 arrested complexes (26). Others have used similar approaches, including high-salt washes of immobilized templates, to study the elongation factors themselves (4,5,25,32). A second and very different effect of the increased postinitiation KCl concentrations was an increase in the overall levels of transcription from the ADA promoter (Fig.…”

“…A number of genes from Drosophila melanogaster as well, including the heat shock genes hsp70 and hsp26, show regulation at the level of transcription elongation (35,44,51). Viral transcription units also show this type of control, including those of human immunodeficiency virus (46, 54), adenovirus (42, 49, 56), polyomavirus (53), and the minute virus of mice (2,32,48). The diversity among these examples may indicate that control of transcription elongation is a widespread gene regulatory phenomenon.…”