Transcription factor TFIIF is not required for initiation by RNA polymerase II, but it is essential to stabilize transcription factor TFIIB in early elongation complexes.

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.

Efficient and rapid nucleosome traversal by RNA polymerase II depends on a combination of transcript elongation factors.

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.

The functions of TFIIF during initiation and transcript elongation are differentially affected by phosphorylation by casein kinase 2.

The RNA polymerase II (pol II) initiation and elongation factor elongation factor TFIIF can be extensively phosphorylated in vivo, although the significance of this modification has not been clear. We now show that phosphorylation of recombinant TFIIF by casein kinase 2 (CK2) reduces or eliminates some of the functions of TFIIF while paradoxically leaving others intact. Phospho-IIF is fully functional in binding to free pol II and is able to support the initiation of transcription. However, the phosphorylated factor does not bind to stalled elongation complexes as measured in a gel mobility shift assay. Significantly, phosphorylation strongly reduces (or for some truncated versions of RAP74, eliminates) stimulation of transcript elongation by TFIIF. Thus, although TFIIF must participate at the initiation of transcription, its ability to continue its association with pol II and stimulate transcript elongation can be specifically regulated by CK2. This is particularly interesting because CK2 is required for initiation at a subset of pol II promoters. Modulation of TFIIF function could be important in controlling promoter-proximal pausing by pol II during the early stage of transcript elongation.

Histone Sin mutations promote nucleosome traversal and histone displacement by RNA polymerase II.

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 (slashed circle)-loop, which mediates nucleosome survival during transcription. Sin mutations probably destabilize these intermediates and thus increase the likelihood of nucleosome disassociation.

RNA emerging from the active site of RNA polymerase II interacts with the Rpb7 subunit.

Structural studies of RNA polymerase II have suggested two possible exit paths for the nascent RNA: groove 1, which points toward the subcomplex of subunits Rpb4 and Rpb7, and groove 2, which points toward Rpb8. These alternatives could not be distinguished previously because less than 10 nucleotides (nt) of transcript were resolved in the structures. We have approached this question by UV cross-linking nascent RNA to components of the transcription complex through uridine analogs located within the first six nucleotides of the RNA. We find that the emerging transcript cross-links to the Rpb7 subunit of RNA polymerase II in various complexes containing 26- to 32-nt transcripts. This interaction is greatly reduced in complexes with 41- or 43-nt RNAs and absent when the transcript is 125 nt. Our results are consistent with groove 1 being the exit path for nascent RNA.

Characterization of a novel RNA polymerase II arrest site which lacks a weak 3' RNA-DNA hybrid.

Transcript elongation by RNA polymerase II is blocked at DNA sequences called arrest sites. An exceptionally weak RNA-DNA hybrid is often thought to be necessary at the point of arrest. We have identified an arrest site from the tyrosine hydroxylase (TH) gene which does not fit this pattern. Transcription of many sequence variants of this site shows that the RNA-DNA hybrid over the three bases immediately preceding the major arrest point may be strong (i.e. C:G) without interfering with arrest. However, arrest at the TH site requires the presence of a pyrimidine at the 3' end and arrest increases when the 3'-most segment is pyrimidine rich. We also demonstrated that arrest at the TH site is completely dependent on the presence of a purine-rich element immediately upstream of the RNA-DNA hybrid. Thus, the RNA polymerase II arrest element from the TH gene has several unanticipated characteristics: arrest is independent of a weak RNA-DNA hybrid at the 3' end of the transcript, but it requires both a pyrimidine at the 3' end and a polypurine element upstream of the RNA-DNA hybrid.

Histone N-terminal tails interfere with nucleosome traversal by RNA polymerase II.

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.

Nucleosomes can form a polar barrier to transcript elongation by RNA polymerase II.

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.

Newly Initiated RNA encounters a factor involved in splicing immediately upon emerging from within RNA polymerase II.

We employed RNA-protein cross-linking to map the path of the nascent RNA as it emerges from within RNA polymerase II. A UV-cross-linkable uridine analog was incorporated at two positions within the first five nucleotides of the transcript. Only the two largest subunits of RNA polymerase II cross-linked to the transcript in complexes containing 17-24-nucleotide (nt) RNAs. Extension of the RNA to 26 or 28 nt revealed an additional strong cross-link to the splicing factor U2AF65. In U17 complexes, in which the RNA is still contained within the polymerase, U2AF65 is tightly bound. In contrast, U2AF65 is more loosely bound in C28 transcription complexes, in which about 10 nt of transcript have emerged from the RNA polymerase. Cross-linking of U2AF65 to RNA in a C28 complex was eliminated by the addition of an excess of an RNA oligonucleotide containing the consensus U2AF65 binding site, but U2AF65 was not displaced by a nonconsensus RNA. These findings indicate that U2AF65 shifts from protein-protein to protein-RNA interactions as the RNA emerges from the polymerase. During transcription of one particular template at low UTP concentration, RNA polymerase II pauses just after synthesizing a transcript segment that is a U2AF65 binding site. Dwell time of the polymerase at this pause site was significantly and specifically reduced by the addition of recombinant U2AF65 to the transcription reaction. Therefore, the association of U2AF65 with RNA polymerase II may function not only to deliver U2AF65 to the nascent transcript but also to modulate efficient transcript elongation.

RNA polymerase II transcription complexes may become arrested if the nascent RNA is shortened to less than 50 nucleotides.

A significant fraction of RNA polymerase II transcription complexes become arrested when halted within a particular initially transcribed region after the synthesis of 23-32-nucleotide RNAs. If polymerases are halted within the same sequence at a promoter-distal location, they remain elongation-competent. However, when the RNAs within these promoter-distal complexes are truncated to between 21 and 48 nucleotides, many of the polymerases become arrested. The degree of the arrest correlates very well with the length of the RNA in both the promoter-proximal and -distal complexes. This effect is also observed when comparing promoter-proximal and promoter-distal complexes halted over a completely different sequence. The unusual propensity of many promoter-proximal RNA polymerase II complexes to arrest may therefore be recreated in promoter-distal complexes simply by shortening the nascent RNA. Thus, the transition to full elongation competence by RNA polymerase II is dependent on the synthesis of about 50 nt of RNA, and this transition is reversible. We also found that arrest is facilitated in promoter-distal complexes by the hybridization of oligonucleotides to the transcript between 30 and 45 bases upstream of the 3'-end.