Nucleosomes uniquely positioned on high-affinity DNA sequences present a polar barrier to transcription by human and yeast RNA polymerase II (Pol II). In one transcriptional orientation, these nucleosomes provide a strong, factor- and salt-insensitive barrier at the entry into the H3/H4 tetramer that can be recapitulated without H2A/H2B dimers. The same nucleosomes transcribed in the opposite orientation form a weaker, more diffuse barrier that is largely relieved by higher salt, TFIIS, or FACT. Barrier properties are therefore dictated by both the local nucleosome structure (influenced by the strength of the histone-DNA interactions) and the location of the high-affinity DNA region within the nucleosome. Pol II transcribes DNA sequences at the entry into the tetramer much less efficiently than the same sequences located distal to the nucleosome dyad. Thus, entry into the tetramer by Pol II facilitates further transcription, perhaps due to partial unfolding of the tetramer from DNA.

Transcription factors TFIIB and TFIIF are both required for RNA polymerase II preinitiation complex (PIC) assembly, but their roles at and downstream of initiation are not clear. We now show that TFIIF phosphorylated by casein kinase 2 remains competent to support PIC assembly but is not stably retained in the PIC. PICs completely lacking TFIIF are not defective in initiation or subsequent promoter clearance, demonstrating that TFIIF is not required for initiation or clearance. Lack of TFIIF in the PIC reduces transcription levels at some promoters, coincident with reduced retention of TFIIB. TFIIB is normally associated with the early elongation complex and is only destabilized at +12 to +13. However, if TFIIF is not retained in the PIC, TFIIB can be lost immediately after initiation. TFIIF therefore has an important role in stabilizing TFIIB within the PIC and after transcription initiates.general transcription factors | transcript initiation P romoter-directed transcript initiation by RNA polymerase II (pol II) on double-stranded templates requires at minimum the general transcription factors TBP (TATA box-binding protein), TFIIB, TFIIF, TFIIE, and TFIIH. Genetic and biochemical evidence indicates that TFIIB can direct transcription start site selection (1-4) with the assistance of TFIIF (5-8). When TFIIB is in complex with pol II, a TFIIB domain referred to as the finger (9) or reader (10) enters pol II and approaches the catalytic center. Structural investigations of the yeast preinitiation complex (PIC) revealed a segment of TFIIF localized near the TFIIB domain that presumably directs transcription start site selection (7,11,12). It was suggested that this interaction with TFIIF is important in stabilizing TFIIB within the PIC (12). Release of TFIIB from the elongation complex (EC) is reported to begin 7 to 16 bases downstream of transcription start (13). TFIIF should remain associated with the EC to facilitate rapid and effective transcript elongation (14, 15), but this factor may be transiently lost after initiation (16).The functional roles of TFIIB and TFIIF within the PIC are not fully understood. Transcription from a preformed transcription bubble required only TFIIB and TBP, although TFIIF was strongly stimulatory in that system (17). RNA synthesis from bubble templates in the presence of the complete set of general factors was also very strongly dependent on TFIIF (18). These results raise an important question: Do TFIIB and TFIIF participate in the formation of initial bonds, or are they simply structural components that direct pol II to the correct start site? An important related issue concerns the continued residence of these factors within the EC: Is the proposed loss of TFIIF after initiation linked to the expected release of TFIIB?In the present paper we take advantage of our recent observation that phosphorylation of TFIIF with casein kinase 2 (CK2) abolishes the ability of TFIIF to associate with pol II in ECs and stimulate elongation effectively (19), while leaving TFIIF's support of tra...

Nucleosome traversal by RNA polymerase II (pol II) and recovery of chromatin structure after transcription are essential for proper gene expression. In this paper we show that nucleosomes assembled with Sin mutant histones present a much weaker barrier to traversal by pol II and are less likely to survive transcription. Increases in traversal from incorporation of Sin mutant histones and histones lacking H2A/H2B amino-terminal tails were in most cases additive, indicating that traversal can be facilitated by distinct mechanisms. We had identified a key intermediate in traversal, the zero (+)-loop, which mediates nucleosome survival during transcription. Sin mutations probably destabilize these intermediates and thus increase the likelihood of nucleosome disassociation.

The nucleosome is generally found to be a strong barrier to transcript elongation by RNA polymerase II (pol II) in vitro. The elongation factors TFIIF and TFIIS have been shown to cooperate in maintaining pol II in the catalytically competent state on pure DNA templates. We now show that although TFIIF or TFIIS alone is modestly stimulatory for nucleosome traversal, both factors together increase transcription through nucleosomes in a synergistic manner. We also studied the effect of TFIIF and TFIIS on transcription of nucleosomes containing a Sin mutant histone. The Sin point mutations reduce critical histone-DNA contacts near the center of the nucleosome. Significantly, we found that nucleosomes with a Sin mutant histone are traversed to the same extent and at nearly the same rate as equivalent pure DNA templates if both TFIIS and TFIIF are present. Thus, the nucleosome is not necessarily an insurmountable barrier to transcript elongation by pol II. If unfolding of template DNA from the nucleosome surface is facilitated and the tendency of pol II to retreat from barriers is countered, transcription of nucleosomal templates can be rapid and efficient.It is now appreciated that control of transcript elongation is an important aspect of gene regulation (recently reviewed in Ref. 1). A major checkpoint occurs as RNA polymerase II (pol II) 2 passes from initiation into productive transcript elongation, ϳ50 bp downstream of the transcription start site in many genes (2-6). This checkpoint is roughly coincident with the initial contact between pol II and the first nucleosome of the transcription unit (7-9). Once pol II crosses this initial checkpoint, it can elongate transcripts efficiently over hundreds of kilobases of predominantly nucleosomal template (10). The rate of transcript elongation in vivo (10) slightly exceeds the maximum rates reported for transcript elongation by pol II on pure DNA templates in vitro (11,12). To understand both the initial elongation checkpoint and the exceptional efficiency of transcript elongation downstream of that point, it is essential to develop in vitro systems that duplicate these processes. Progress toward this goal has been somewhat limited because even single nucleosomes have generally proven to be strong barriers to transcribing pol II in vitro (11,(13)(14)(15)(16)(17).Given the tight association of all 146 bp of nucleosomal DNA with the histone octamer surface, one might initially imagine that the nucleosome presents an essentially continuous barrier to transcript elongation by pol II. However, studies with templates bearing single, precisely positioned nucleosomes indicated that the transcriptional barrier for pol II is more discrete. The strongest pauses for human pol II typically occur 45-55 bp within the nucleosome. After pol II crosses this barrier and the nucleosome dyad, transcription continues largely uninhibited through the remainder of the nucleosome (13). This suggests that traversal is controlled primarily by the unfolding of the template DNA from the octamer ...

We determined the effect of the N-terminal histone tails on nucleosome traversal by yeast and human RNA polymerase II (pol II). Removal of H2A/H2B tails, H3/H4 tails, or all tails increased complete traversal of the nucleosome by human pol II, although the increase varied considerably depending on the template and on which tails were removed. Human pol II achieved >80% traversal of one nucleosomal template lacking the H2A/H2B tails, but even in those reactions, the transcript elongation rate was lower than the rate on pure DNA templates. For yeast pol II, transcription proceeded much farther into the nucleosome in the absence of tails, but complete read-through was not substantially increased by tail removal. Transcription factor IIS provided roughly the same level of read-through stimulation for transcript elongation in the presence or absence of tails. FACT also stimulated elongation on nucleosomal templates, and this effect was similar regardless of the presence of tails. For both polymerases, removal of the H2A/H2B tails reduced pausing throughout the nucleosome, suggesting that histone tails affect a common step at most points during nucleosome traversal. We conclude that histone tails provide a significant part of the nucleosomal barrier to pol II transcript elongation.It has long been appreciated that nucleosomes form a strong blockade to transcript elongation by pol II in vitro (1, 2). It has not been established what role, if any, the N-terminal tails of the histones play in this blockade. The core structure of the nucleosome does not depend on the tails (3-7). However, the tails are strongly positively charged, and they could associate nonspecifically with the DNA, thereby impeding polymerase access to the template (8 -10). The tails could influence traversal in other ways, e.g. by affecting the ability of the H2A/H2B dimer to exchange, which is likely to be involved in at least some traversal events (2). The N-terminal tails could also affect more complex unfolding transitions in the nucleosome, which would facilitate traversal (11, 12). Higher order chromatin structure, which could affect the efficiency of transcription through chromatin, can be influenced by the N-terminal tails (13-16). Covalent modifications of the tails, especially of the H3 tail of promoter-proximal nucleosomes, are well known to be correlated with transcriptional activity in vivo (reviewed in . A recent survey of transcriptionally active human genes revealed that most of these genes contain a high level of RNA polymerase II (pol II) 3 immediately downstream of the transcription start and a strongly positioned nucleosome with a promoter-proximal edge at about position ϩ40 (20). This suggests that the ability of pol II to cross the first nucleosome it encounters after transcript initiation is a major and general control point for gene expression. In light of all of these findings, we decided to explore directly the effect of the histone tails on the ability of pol II to traverse single nucleosomes. As an initial test of the effec...