Arik Dvir

Control of elongation by RNA polymerase II

The elongation stage of eukaryotic mRNA synthesis can be regulated by transcription factors that interact directly with the RNA polymerase II (pol II) elongation complex and by activities that modulate the structure of its chromatin template. Recent studies have revealed new elongation factors and have implicated the general initiation factors TFIIE, TFIIF and TFIIH, as well as the C-terminal domain (CTD) of the largest subunit of pol II, in elongation. The recently reported high-resolution crystal structure of RNA polymerase II, which provides insight into the architecture of the elongation complex, marks a new era of investigation into transcription elongation.

Mechanism of transcription initiation and promoter escape by RNA polymerase II.

Recently, key advances in biochemical and structural studies of RNA polymerase II (pol II) and the basal transcriptional machinery have shed considerable light on the basic mechanisms underlying the initiation stage of eukaryotic mRNA synthesis. The development of methods for obtaining crystal structures of pol II and its complexes has revolutionized transcriptional studies and holds promise that aspects of initiation will soon be understood at atomic resolution; crosslinking studies have revealed intriguing features of the topology of the pol II initiation complex and provided working models for dynamic steps of initiation; and mechanistic studies have identified promoter escape as a critical step during initiation and brought to light novel roles for the general initiation factors TFIIE, TFIIF, and TFIIH in this process.

Promoter escape by RNA polymerase II.

Transcription of protein-coding genes is one of the most fundamental processes that underlies all life and is a primary mechanism of biological regulation. In eukaryotic cells, transcription depends on the formation of a complex at the promoter region of the gene that minimally includes RNA polymerase II and several auxiliary proteins known as the general transcription factors. Transcription initiation follows at the promoter site given the availability of nucleoside triphosphates and ATP. Soon after the polymerase begins the synthesis of the nascent mRNA chain, it enters a critical stage, referred to as promoter escape, that is characterized by physical and functional instability of the transcription complex. These include formation of abortive transcripts, strong dependence on ATP cofactor, the general transcription factor TFIIH and downstream template. These criteria are no longer in effect when the nascent RNA reaches a length of 14-15 nucleotides. Towards the end of promoter escape, disruption or adjustment of protein-protein and protein-DNA interactions, including the release of some of the general transcription factors from the early transcription complex is to be expected, allowing the transition to the elongation stage of transcription. In this review, we examine the experimental evidence that defines promoter escape as a distinct stage in transcription, and point out areas where critical information is missing.

TFIIH action in transcription initiation and promoter escape requires distinct regions of downstream promoter DNA.

TFIIH is a multifunctional RNA polymerase II general initiation factor that includes two DNA helicases encoded by the Xeroderma pigmentosum complementation group B (XPB) and D (XPD) genes and a cyclin-dependent protein kinase encoded by the CDK7 gene. Previous studies have shown that the TFIIH XPB DNA helicase plays critical roles not only in transcription initiation, where it catalyzes ATP-dependent formation of the open complex, but also in efficient promoter escape, where it suppresses arrest of very early RNA polymerase II elongation intermediates. In this report, we present evidence that ATP-dependent TFIIH action in transcription initiation and promoter escape requires distinct regions of the DNA template; these regions are well separated from the promoter region unwound by the XPB DNA helicase and extend, respectively, ≈23–39 and ≈39–50 bp downstream from the transcriptional start site. Taken together, our findings bring to light a role for promoter DNA in TFIIH action and are consistent with the model that TFIIH translocates along promoter DNA ahead of the RNA polymerase II elongation complex until polymerase has escaped the promoter.

Promoter Escape by RNA Polymerase II FORMATION OF AN ESCAPE-COMPETENT TRANSCRIPTIONAL INTERMEDIATE IS A PREREQUISITE FOR EXIT OF POLYMERASE FROM THE PROMOTER

Shortly after initiating promoter-specific transcription in vitro, mammalian RNA polymerase II becomes highly susceptible to arrest in a promoter-proximal region 9–13 base pairs downstream of the transcriptional start site (Dvir, A., Conaway, R. C., and Conaway, J. W. (1996) J. Biol. Chem. 271, 23352–23356). Arrest by polymerase in this region is suppressed by TFIIH in an ATP-dependent reaction (Dvir, A., Conaway, R. C., and Conaway, J. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9006–9010). In this report, we present evidence that, in addition to TFIIH and an ATP cofactor, efficient transcription by RNA polymerase II through this promoter-proximal region requires formation of an “escape-competent” transcriptional intermediate. Formation of this intermediate requires template DNA 40–50 base pairs downstream of the transcriptional start site. This requirement for downstream DNA is transient, since template DNA downstream of +40 is dispensable for assembly of the preinitiation complex, for initiation and synthesis of the first 10–12 phosphodiester bonds of nascent transcripts and for further extension of transcripts longer than ∼14 nucleotides. Thus, promoter escape requires that the RNA polymerase II transcription complex undergoes a critical structural transition, likely driven by interaction of one or more components of the transcriptional machinery with template DNA 40–50 base pairs downstream of the transcriptional start site.

The Identification and Purification of a Mammalian-Like Protein Kinase C in the Yeast Saccharomyces cerevisiae

We have purified a yeast protein kinase that is phospholipid-dependent and activated by Diacylglycerol (DAG) in the presence of Ca2+ or by the tumour-promoting agent tetradecanoyl-phorbol acetate (TPA). The properties of this enzyme are similar to those of the mammalian protein kinase C (PKC). The enzyme was purified using chromatography on DEAE-cellulose followed by hydroxylapatite. The latter chromatography separated the activity to three distinguishable sub-species, analogous to the mammalian PKC isoenzymes. The fractions enriched in PKC activity contain proteins that specifically bind TPA, are specifically phosphorylated in the presence of DAG and recognized by anti-mammalian PKC antibodies.

Eukaryotic Gene Transcription

Gene transcription is the most significant mechanism by which cells control the specific expression of their genes. In this review we will focus on the transcription of protein coding genes in eukaryotic cells. The transcription of these genes is catalyzed by the enzyme RNA polymerase II, and, in addition, involves a host of auxiliary and regulatory components. The expression of these genes is controlled by local chromatin structure, promoter elements, and interactions of stimulatory and inhibitory protein factors that act either specifically at the gene level or more broadly at the genome level. We describe not only the critical elements of promoter structure and how they allow efficient recruitment of the transcriptional machinery to the physical site of the gene, but also the kinetic and functional processes that take place once transcription has begun. Recent concepts regarding rate-limiting steps such as promoter escape, elongation, and termination are discussed; as well as recently-discovered enzymes that make part of the transcription process, such as ATP-dependent chromatin remodeling factors, histone acetylases, and Mediator complexes. The identification of the various elements of gene transcription and how they affect transcription is key to understanding how signal transduction networks control the expression of specific genes.

Assays for investigating the mechanism of promoter escape by RNA polymerase II.

Publisher Summary This chapter describes the methods and approaches that have proven useful in investigations of the mechanism of promoter escape by RNA polymerase II, and should be valuable for future efforts to define the mechanism of important transcriptional stage in detail. It discusses that transcription initiation by RNA polymerase II from its promoters is a complex process that requires at minimum the five general initiation factors TFIIB, TBP, TFIIE, TFIIF, and TFIIH and an ATP (dATP) cofactor. Biochemical studies of transcription initiation by RNA polymerase II, in this minimal enzyme system, has revealed that initiation occurs by a multistep mechanism begins with the assembly of polymerase. All five general initiation factor in a stable preinitiation complex at the promoter and proceeds with ATP(dATP)-dependent unwinding of promoter DNA surrounding the transcriptional start site by the TFIIH XPB DNA helicase to form the open complex, synthesis of the first few phosphodiester bonds of nascent transcripts, and escape of polymerase from the promoter. Thus, efficient promoter escape by RNA polymerase II requires that the early elongation complex undergo a critical ATP (dATP)-dependent structural transition that most likely depends on the interaction of polymerase.