The requirements for transcriptional activation by RNA polymerase II were examined using chromatin templates assembled in vitro and a transcription system composed of the human general transcription factors and RNA polymerase II. Activator-induced, energy-dependent chromatin remodeling promoted efficient preinitiation complex formation and transcription initiation, but was not sufficient for productive transcription. Polymerases that initiated transcription on remodeled chromatin templates encountered a block to transcription proximal to the promoter. Entry into productive transcription required an accessory factor present in HeLa cell nuclear extract, FACT (facilitates chromatin transcription), which we have purified. FACT acts subsequent to transcription initiation to release RNA polymerase II from a nucleosome-induced block to productive transcription. The biochemical properties and polypeptide composition of FACT suggest that it is a novel protein factor that facilitates transcript elongation through nucleosomes.

The products of the yeast CDC73 and PAF1 genes were originally identified as RNA polymerase II-associated proteins. Paf1p is a nuclear protein important for cell growth and transcriptional regulation of a subset of yeast genes. In this study we demonstrate that the product of CDC73 is a nuclear protein that interacts directly with purified RNA polymerase II in vitro. Deletion of CDC73 confers a temperature-sensitive phenotype. Combination of the cdc73 mutation with the more severe paf1 mutation does not result in an enhanced phenotype, indicating that the two proteins may function in the same cellular processes. To determine the relationship between Cdc73p and Paf1p and the recently described holoenzyme form of RNA polymerase II, we created yeast strains containing glutathione S-transferase ( A minimal set of transcription factors (RNA polymerase II plus TATA-binding protein [TBP], TFIIB, TFIIE, TFIIF, and TFIIH) are necessary for mRNA promoter-specific transcription initiation in vitro (for reviews, see references 7 and 12). Regulated transcription requires, in addition to these basal factors, many accessory proteins responsible for conveying regulatory signals to the general transcriptional machinery (68). There are at least two different classes of accessory factors that have been well characterized. One class includes the TBPassociated factors (TAFs) (for a review, see reference 58). In vitro reconstitution experiments strongly implicate the TAFs in the process of transcriptional activation (10). Another class of accessory factors exists in the mediator complex associated with the C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II (for a review, see reference 33). In the yeast Saccharomyces cerevisiae, the mediator can associate with RNA polymerase II and several general initiation factors to form a large protein complex termed the holoenzyme (30, 32). Most components of the holoenzyme, including the Srbps, Gal11p, Sin4p, Rgr1p, and Swi/Snfps, were originally identified by mutations that caused transcriptional alterations in yeast (24,27,34,39,43,53,64). Although mutations in some of these gene products affect the expression of only subsets of yeast genes, an analysis of temperature-sensitive mutations of SRB4 and SRB6 revealed transcription defects at all class II promoters assayed (57). Mammalian RNA polymerase II-containing complexes that include Srbp homologs, and many of the general transcription factors as well as DNA-repair factors, have recently been described (36,42).The reported complex forms of RNA polymerase II vary widely in terms of composition. In particular, some of the general initiation factors (TBP, TFIIE, and TFIIH) are present in some complexes but not others (30,32,36,42). In addition, some of the factors, including the Srbps and Gal11p, can be found in dissociable subcomplexes (30,34). Although it is probable that some of these differences reflect the different purification protocols used to isolate these extremely large complexes from widely differing cell type...

At the National Synchrotron Radiation Research Center, a small/wide‐angle X‐ray scattering (SAXS/WAXS) instrument has been installed at the BL23A beamline with a superconducting wiggler insertion device. This beamline is equipped with double Si(111) crystal and double Mo/B4C multilayer monochromators, and an Si‐based plane mirror that can selectively deflect the beam downwards for grazing‐incidence SAXS (GISAXS) studies of air–liquid or liquid–liquid interfaces. The SAXS/WAXS instrument, situated in an experimental hutch, comprises collimation, sample and post‐sample stages. Pinholes and slits have been incorporated into the beam collimation system spanning a distance of ∼5 m. The sample stage can accommodate various sample geometries for air–liquid interfaces, thin films, and solution and solid samples. The post‐sample section consists of a 1 m WAXS section with two linear gas detectors, a vacuum bellows (1–4 m), a two‐beamstop system and the SAXS detector system, all situated on a motorized optical bench for motion in six degrees of freedom. In particular, the vacuum bellows of a large inner diameter (260 mm) provides continuous changes of the sample‐to‐detector distance under vacuum. Synchronized SAXS and WAXS measurements are realized via a data‐acquisition protocol that can integrate the two linear gas detectors for WAXS and the area detector for SAXS (gas type or Mar165 CCD); the protocol also incorporates sample changing and temperature control for programmable data collection. The performance of the instrument is illustrated via several different measurements, including (1) simultaneous SAXS/WAXS and differential scanning calorimetry for polymer crystallization, (2) structural evolution with a large ordering spacing of ∼250 nm in a supramolecular complex, (3) SAXS for polymer blends under in situ drawing, (4) SAXS and anomalous SAXS for unilamellar lipid vesicles and metalloprotein solutions, (5) anomalous GISAXS for oriented membranes of Br‐labeled lipids embedded with peptides, and (6) GISAXS for silicate films formed in situ at the air–water interface.

We have investigated transcript elongation efficiency by RNA polymerase II on chromatin templates in vitro. Circular plasmid DNAs bearing purified RNA polymerase II transcription complexes were assembled into nucleosomes using purified histones and transient exposure to high salt, followed by dilution and dialysis. This approach resulted in nucleosome assembly beginning immediately downstream of the transcription complexes. RNA polymerases on these nucleosomal templates could extend their 15-or 35-nucleotide nascent RNAs by only about 10 nucleotides in 15 min, even in the presence of elongation factors TFIIF and SII. Efficient transcript elongation did occur upon dissociation of nucleosomes with 1% sarkosyl, indicating that the RNA polymerases were not damaged by the high salt reconstitution procedure. Since the elongation complexes were released by sarkosyl but not by SII, these complexes apparently did not enter the arrested conformation when they encountered nucleosomes. Surprisingly, elongation was no more efficient on nucleosomal templates reconstituted only with H3/H4 tetramers, even in the presence of elongation factors and/or competitor DNA at high concentration. Thus, in a purified system lacking nucleosome remodeling factors, not only the core histone octamer but also the H3/H4 tetramer provide an nearly absolute block to transcript elongation by RNA polymerase II, even in the presence of elongation factors.The basic structural unit of the eukaryotic chromosome is the nucleosome, which consists of an octamer of the H2A, H2B, H3, and H4 histone proteins around which 145 bp 1 of DNA are wrapped (recently reviewed in Ref. 1). Despite the apparently tight packaging of transcriptionally active DNA in nucleosomes (see Ref. 2 and references therein), RNA polymerase II achieves high rates (25 nucleotides/s) of RNA chain elongation in the cell (3, 4). However, attempts to elongate RNA polymerase II transcripts in vitro using reconstituted chromatin templates have not reproduced efficient chain elongation (Refs. 5 and 6, but see also Ref. 7). A number of reasons can be envisioned for this failure. It is known, for example, that nucleosomes from transcriptionally active regions have highly acetylated histones (recently reviewed in Refs. 8 and 9); it has also been suggested that such nucleosomes might be deficient in histones H2A and H2B (10). Several molecular complexes have recently been described that can, in the presence of ATP, modify nucleosomes so that their DNA is more accessible for transcription factor binding (11)(12)(13)(14). It is conceivable that these chromatin remodeling activities might act within transcribed regions, making DNA within the gene body more accessible to RNA polymerase (7). Numerous studies using single-subunit bacteriophage RNA polymerases have demonstrated that these enzymes can efficiently elongate nascent RNA on reconstituted chromatin templates (15-22). Interestingly, transcription of mononucleosomal templates by SP6 RNA polymerase has been shown to be accompanied by displacemen...