Transcription on nucleosomal templates by RNA polymerase II in vitro: inhibition of elongation with enhancement of sequence-specific pausing.

Abstract

The process by which RNA polymerase II elongates RNA chains in vivo, where the template is at least partially in a nucleosomal configuration, remains poorly understood. To approach this question we have partially purified RNA polymerase lI transcription complexes paused early in elongation. These complexes were then used as substrates for chromatin reconstitution. Elongation of the nascent RNA chains on these nucleosomal templates is severely inhibited relative to elongation on naked DNA templates. Elongation on the nucleosomal templates results in a reproducible template-specific pattern of transcripts generated by RNA polymerase pausing. The RNA polymerases are not terminated because the large majority will resume elongation upon the addition of Sarkosyl or 400 mM KCI. The effectiveness of RNA polymerase II pause/termination sites is enhanced by the presence of nucleosomes. For example, a pause site similar in sequence to the c-myc gene exon 1 terminator is used four to seven times more effectively in reconstituted templates. A comparison of elongation on templates bearing phased nucleosomes and on reconstituted templates that show no predominant phasing pattern indicates that the locations of pause sites are not related to the positions of the nucleosomes. Rather, the major determinant of RNA polymerase pausing on the nucleosomal templates appears to be the underlying DNA sequence.[Key Words: RNA polymerase II; nucleosome; chromatin] Received November 8, 1990; revised version accepted February 5, 1991.A number of studies have indicated that transcriptionally active regions may continue to be packaged in nucleosomes while transcription occurs (Nacheva et al. 1989;Chen et al. 1990;Ericsson et al. 1990;Pederson and Morse 1990). This raises the question of how RNA polymerase II can effectively elongate RNA chains on these nucleoprotein templates. Studies with model systems using bacteriophage RNA polymerases and reconstituted templates indicate that nucleosomes need not be insurmountable barriers to RNA synthesis. These single-subunit RNA polymerases can efficiently elongate on both short fragments bearing one or two nucleosomes (Lorch et al. 1987;Losa and Brown 1987;Morse 1989) and on templates with extended arrays of nucleosomes (Pfaffle et al. 1990). RNA polymerase II will efficiently traverse a single nucleosome (Lorch et al. 1987), but the effect of long nucleosomal arrays on polymerase II elongation has not been established.The necessity for approaching this question biochem-ically has been emphasized by the growing realization that control of elongation by RNA polymerase II is an important aspect of gene regulation. Premature termination or pausing by RNA polymerase II has been observed in vivo for a number of genes, such as the proto-oncogenes c-myc and c-myb, the adenosine deaminase gene, and the hsp70 gene of Drosophila melanogaster (for review, see Spencer and Groudine 1990 and references therein). In vitro systems have been employed that reproduce some of the transcriptional pausing/termination events...

Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II

“…1C). We also acquired F-v data in the presence of 100 mM ammonium ion, which is known to increase elongation rates in vitro to levels closely resembling transcription rates estimated in vivo (see below) (20)(21)(22).…”

“…A stimulatory effect of ammonium on the overall rate of transcription has been previously reported (20)(21)(22), but the mechanism for its action is still unknown. To assess the role of ammonium on individual steps in the NAC, we added 100 mM ammonium chloride to the buffer (in addition to the 130 mM KCl already present) for transcription reactions of WT RNAPII and its TL mutants.…”

Regulation of eukaryotic gene expression by transcriptional attenuation.

“…It was suggested that a structural element causing a bend in the DNA may be part of the signal for termination in this system. Transcriptional pausing at specific DNA sequences was greater in a system in which nascent RNA chains were elongated on purified RNA polymerase II transcription complexes after chromatin re-662 constitution (Izban and Luse, 1991). Premature transcriptional termination on c-myc templates was also observed in in vitro transcription reactions using HeLa cell nuclear extracts (London et al, 1991), with the degree of termination depending on the growth conditions of the HeLa cells from which the extracts were prepared.…”

Nucleosomal arrangement affects single-molecule transcription dynamics

“…In vitro experiments have revealed that a single nucleosome exhibits a significant mechanical barrier to the transcribing Pol II (3)(4)(5)(6)(7). Using optical tweezers, it was shown that a nucleosome both decreases the rate of forward translocation and increases polymerase pausing and backtracking (3,5).…”