Donal S. Luse

FACT, a Factor that Facilitates Transcript Elongation through Nucleosomes

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 lung-specific CC10 gene is regulated by transcription factors from the AP-1, octamer, and hepatocyte nuclear factor 3 families.

We have shown that a large fragment (-2339 to +57) from the rat CC10 gene directed lung-specific expression of a reporter construct in transgenic animals. Upon transfection, a smaller fragment (-165 to +57) supported reporter gene expression exclusively in the Clara cell-like NCI-H441 cell line, suggesting that a Clara cell-specific transcriptional element resided on this fragment (B. R. Stripp, P. L. Sawaya, D. S. Luse, K. A. Wikenheiser, S. E. Wert, J. A. Huffman, D. L. Lattier, G. Singh, S. L. Katyal, and J. A. Whitsett, J. Biol. Chem. 267:14703-14712, 1992). The interactions of nuclear proteins with a particular segment of the CC10 promoter which extends from 79 to 128 bp upstream of the CC10 transcription initiation site (CC10 region I) have now been studied. This sequence can stimulate both in vitro transcription in H441 nuclear extract and transient expression of reporter constructs in H441 cells. Electrophoretic mobility shift assays using extracts from H441, HeLa, rat liver, and fetal sheep lung cells were used to demonstrate that members of the AP-1, octamer, and HNF-3 families bind to CC10 region I. Transcription factors from H441 cells which are capable of binding to CC10 region I are either absent in HeLa, rat liver, and fetal sheep lung extracts or enriched in H441 extracts relative to extracts from non-Clara cells.

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.

THE H3/H4 TETRAMER BLOCKS TRANSCRIPT ELONGATION BY RNA POLYMERASE II IN VITRO

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.

Mechanism of transcription through a nucleosome by RNA polymerase II.

Efficient maintenance of chromatin structure during passage of RNA polymerase II (Pol II) is critical for cell survival and functioning. Moderate-level transcription of eukaryotic genes by Pol II is accompanied by nucleosome survival, extensive exchange of histones H2A/H2B and minimal exchange of histones H3/H4. Complementary in vitro studies have shown that transcription through chromatin by single Pol II complexes is uniquely coupled with nucleosome survival via formation of a small intranucleosomal DNA loop (Ø-loop) containing the transcribing enzyme. In contrast, transient displacement and exchange of all core histones are observed during intense transcription. Indeed, multiple transcribing Pol II complexes can efficiently overcome the high nucleosomal barrier and displace the entire histone octamer in vitro. Thus, various Pol II complexes can remodel chromatin to different extents. The mechanisms of nucleosome survival and displacement during transcription and the role of DNA-histone interactions and various factors during this process are discussed. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.

Transcription initiation by RNA polymerase II in vitro. At least two nucleotides must be added to form a stable ternary complex.

Publisher Summary Euglena gracilis 2 contain NADH (EC 1.6.99.8)- and NADPH (EC 1.6.99.11)-linked aquacobalamin reductases. The Euglena enzyme is located only in the soluble fraction of the mitochondria. The enzyme has been purified to homogeneity only from Euglena gracilis. This chapter summarizes NADPH-linked aquacobalamin reductase from Euglena gracilis, a photosynthetic protozoan. The aquacobalamin reductase activity is assayed by spectrophotometric estimation of the amount of aquacobalamin converted to cob(II)alamin. Mitochondria (986 mg of protein) of Euglena gracilis are suspended in 33 ml of 10 m M Tris–acetate buffer, pH 7.0, containing 1 mM EDTA and 1 μ M dithiothreitol (DTT), disrupted by sonic oscillation (10 kHz, four 20-sec pulses), and centrifuged at 100,000 g for 60 rain to remove the membrane fraction. The Euglena aquacobalamin reductase reduced approximately 2 mol of aquacobalamin/mol of NADPH. The enzyme was specific for aquacobalamin but not for cyanocobalamin, and NADH could not replace NADPH. The enzyme did not require FAD or FMN as cofactors for the reduction of aquacobalamin. The enzyme reduced 2,6-dinitrophenolindophenol, potassium ferricyanide, and cytochrome c, as well as aquacobalamin under aerobic conditions; and under anaerobic conditions, it reduced FAD, FMN, methyl viologen, and benzyl viologen. Cyanocobalamin was not reduced by the purified enzyme under anaerobic conditions. The results indicate that the Euglena enzyme has an NADPH-diaphorase-like activity.

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.

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.

Promoter clearance by RNA polymerase II is an extended, multistep process strongly affected by sequence.

ABSTRACT We have characterized RNA polymerase II complexes halted from +16 to +49 on two templates which differ in the initial 20 nucleotides (nt) of the transcribed region. On a template with a purine-rich initial transcript, most complexes halted between +20 and +32 become arrested and cannot resume RNA synthesis without the SII elongation factor. These arrested complexes all translocate upstream to the same location, such that about 12 to 13 bases of RNA remain in each of the complexes after SII-mediated transcript cleavage. Much less arrest is observed over this same region with a second template in which the initially transcribed region is pyrimidine rich, but those complexes which do arrest on the second template also translocate upstream to the same location observed with the first template. Complexes stalled at +16 to +18 on either template do not become arrested. Complexes stalled at several locations downstream of +35 become partially arrested, but these more promoter-distal arrested complexes translocate upstream by less than 10 nt; that is, they do not translocate to a common, far-upstream location. Kinetic studies with nonlimiting levels of nucleoside triphosphates reveal strong pausing between +20 and +30 on both templates. These results indicate that promoter clearance by RNA polymerase II is at least a two-step process: a preclearance escape phase extending up to about +18 followed by an unstable clearance phase which extends over the formation of 9 to 17 more bonds. Polymerases halted during the clearance phase translocate upstream to the preclearance location and arrest in at least one sequence context.

Structural Changes in the RNA Polymerase II Transcription Complex during Transition from Initiation to Elongation

ABSTRACT We obtained exonuclease III (exoIII) footprints for a series of RNA polymerase II transcription complexes stalled between positions +20 to +51. Downstream advance of the exoIII footprint is normally tightly coordinated with RNA synthesis. However, arrested RNA polymerases slide back along the template, as indicated by exoIII footprints in which the last transcribed base is abnormally close to the downstream edge of the footprint. None of the polymerase II complexes stalled between +20 and +51 were arrested. Nevertheless, the exoIII footprints of complexes with 20-, 23-, or 25-nucleotide RNAs resembled those of arrested complexes, with the last transcribed base very close to the footprint’s front edge. The exoIII footprint of the +27 complex was displaced downstream by 17 bp compared to the footprint of the +25 complex. Many complexes between +27 and +42 also showed evidence of sliding back along the template. We compared the effects of template sequence and transcript length by constructing a new template in which the initial transcribed sequence was duplicated beginning at +98. The exoIII footprints of transcription complexes stalled between +122 to +130 on this DNA did not resemble those of arrested complexes, in contrast to the footprints of analogous complexes stalled over the same DNA sequences early in transcription. Our results indicate that the RNA polymerase II transcription complex passes through a major, sequence-independent structural transition about 25 bases downstream of the starting point of transcription. The fully mature form of the elongation complex may not appear until more than 40 bonds have been made.