Initiation of transcription on nucleosomal templates.

Abstract

We describe a simple method that uses curved DNAs to move nucleosomes relative to a sequence of interest.With this method, small changes in the association of a T7 RNA polymerase promoter with a nucleosome are shown to lead to significant changes in transcription efficiency.The assembly of nucleosomes onto promoter sequences in vitro is inhibitory to transcription initiation (1-4). Although the exact reason for this repression is unknown, DNA wound around the histone core is believed to be inaccessible to DNA-binding proteins such as RNA polymerases (5). In contrast, once transcription is initiated, RNA polymerase can progress through a nucleosomal template (2, 6, 7).Evidence from in vivo experiments suggests that precisely positioned nucleosomes may be important in repressing gene transcription (8-11). It is not known precisely which DNA sequences need to be incorporated into a nucleosome to prevent the interaction of other, sequence-specific DNAbinding proteins. Linker DNA, which separates nucleosomes, is accessible to RNA polymerase (12, 13). However, the length oflinker DNA that is required for RNA polymerase to bind and initiate transcription has not been defined.In this study, we have used the preference of nucleosomes to bind to curved DNAs (14,15) to alter the position of a nucleosome relative to the promoter for bacteriophage T7 RNA polymerase. Small changes in the position of promoter sequences with respect to the boundaries of a nucleosome are shown to have large effects on transcription efficiency. MATERIALS AND METHODSSynthetic DNA Curves. Sequence-directed bending of DNA has been detected by electrophoretic and electron microscopic analysis (16)(17)(18). These experimental observations led to the development of structural models for bent DNA, allowing the design and synthesis of extended DNA curves (19,20). Intrinsic bending of DNA molecules results from local structural polymorphism in regions of homopolymeric dA-dT, especially when these sequences phase with the helical periodicity of DNA [i.e., 10.5 base pairs (bp) in solution].The five different DNA double helices listed below were cloned into the BamHI site of a plasmid containing the T7 promoter and a chloramphenicol acetyltransferase (CAT) gene (Fig. 1). This plasmid (T7-CAT) is a derivative of pUC18 (5081-bp total length; (C. Collis, P. L. Molloy, G. W. Both and H.R.D., unpublished work). Clones were selected that had a single orientation with the BamHI site recreated to the right of the curved DNA insert. In each of the DNA molecules an additional two base pairs were inserted that adjusted the periodicity of a repeat (GGCCTAAAT) to 10.5 bp per helical turn of DNA. These base pairs are marked with asterisks. _3Individual fragments have the repeats staggered by two bases relative to each other. This alters the inclination of the curved DNA to the promoter. The distance from the beginning of the curve to the T7 promoter also changes, a fact we make use of later.In order to radiolabel these DNA fragments, 10 jig of plasmid DNA was res...

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

“…The pML5 plasmids have a slightly modified downstream region that allows transcription to proceed to + 15 in the absence of GTP, thereby forming a paused elongation complex termed 15n complex. Some of the constructs include a single copy (pML5-N) or four copies (pML5-4N and -4NR) of a DNA fragment that has been shown to direct nucleosome assembly to one particular location (Wolffe and Drew 1989).…”

“…pML5-N was constructed by removing a 150-bp HindIII-PvuII fragment of the CAT gene expected to contain a nucleosomepositioning sequence from pRSVcat (Wolffe and Drew 1989) …”

A transcriptionally active tRNA gene interferes with nucleosome positioning in vivo.

“…When nucleosome assembly was preceded by incubation of the transcriptional template with essential trans-acting factors, this inhibition was removed, suggesting that competition between these factors and histones determined the ultimate transcriptional competence of the promoter (9,37,75). Other studies have shown that incorporation of specific promoter sequences within a nucleosome is sufficient to inhibit transcription in vitro by SP6 (33) or T7 RNA polymerase (74) or RNA polymerase II (33) or III (38,54,70).…”

Role of Histone H1 as an Architectural Determinant of Chromatin Structure and as a Specific Repressor of Transcription onXenopus Oocyte 5S rRNA Genes

“…The core histones, linker histones, and HMG proteins could also contribute to the assembly of specific regulatory nucleoprotein architectures through structure-and sequence-selective interactions with DNA (87,109,112). Core histones may selectively recognize both DNA structure such as curvature (83,112) and sequence (25,101).…”