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Elongation by RNA Polymerase II: the Short and Long of It

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Elongation by RNA Polymerase II: the Short and Long of It Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Elongation by RNA polymerase II: the short and long of it Robert J. Sims III,2 Rimma Belotserkovskaya,2 and Danny Reinberg1,2,3 1Howard Hughes Medical Institute and 2Division of Nucleic Acids Enzymology, Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA Appreciable advances into the process of transcript elon- with the RNAP II transcript elongation complex (TEC). gation by RNA polymerase II (RNAP II) have identified It also appears that distinct elongation factors function this stage as a dynamic and highly regulated step of the in specific transcriptional contexts; the requirements for transcription cycle. Here, we discuss the many factors these specialized factors are largely unknown and high- that regulate the elongation stage of transcription. Our light the need to better understand elongation in vivo. discussion includes the classical elongation factors that Many molecular and biochemical approaches have been modulate the activity of RNAP II, and the more recently used to quantify the different aspects of elongation, identified factors that facilitate elongation on chromatin many of which are discussed below. It remains impor- templates. Additionally, we discuss the factors that as- tant to assimilate both in vitro and in vivo experimental sociate with RNAP II, but do not modulate its catalytic systems when discussing what constitutes an elongation activity. Elongation is highlighted as a central process factor. An elongation factor can be defined as any mol- that coordinates multiple stages in mRNA biogenesis ecule that affects the activities of or is associated with and maturation. the TEC. We suggest that there are at least two major types of transcript elongation factors: one family, re- The regulation of gene expression is one of the most ferred to as active elongation factors, and a second fam- intensely studied areas in all of science. Differential gene ily, denoted as passive elongation factors. The difference expression in multicellular organisms forms the founda- resides in whether the factor affects the catalytic activity tion of cell-type specificity. Deregulation of the appro- of RNAP II, regardless of whether the factor remains as- priate pattern of gene expression has profound effects on sociated with the TEC. Passive factors refer to those that cellular function and underlies many diseases. Although are associated with the moving polymerase, but do not there are many cellular processes that control gene ex- affect its catalytic activity. Thus, an active elongation pression, the most direct regulation occurs during tran- factor might transitorily associate with the TEC and, scription. The transcription of protein-coding genes in after executing its function, dissociate from the moving eukaryotes is performed by RNA polymerase II (RNAP polymerase. II). Up until recently, the vast majority of studies aimed In this review, we focus mainly on the regulation of at elucidating the molecular mechanisms of transcrip- transcript elongation and how this stage of the transcrip- tion regulation have focused on early stages, such as the tion cycle is central to the integration and coordination formation of a transcription initiation complex (preini- of multiple events during transcription. Our discussion tiation) and initiation (see below). For many years, tran- includes the “classical” elongation factors that modulate script elongation has been thought of as the trivial addi- the activity of RNAP II, and the more recently identified tion of ribonucleoside triphosphates to the growing factors that facilitate transcript elongation on chromatin mRNA chain. It is now apparent that this process is a templates. We end our review discussing the central role dynamic and highly regulated stage of the transcription of elongation in the coordination of events that result in cycle capable of coordinating downstream events. Nu- efficient mRNA production and export to the cytoplasm. merous factors have been identified that specifically tar- get the elongation stage of transcription. Importantly, multiple steps in mRNA maturation, including pre- RNAP II transcription mRNA capping, splicing, 3Ј-end processing, surveil- Transcription cycle lance, and export, are modulated through interactions The generation of a mature mRNA molecule by RNAP II involves multiple processes, some of which occur se- [Keywords: Elongation; transcription; RNA polymerase II; chromatin; CTD] quentially and others in parallel. The primary phases of 3Corresponding author. transcript generation form a so-called transcription cycle E-MAIL [email protected]; FAX (732) 235-5294. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ and include preinitiation, initiation, promoter clearance, gad.1235904. elongation, and termination. The transcription cycle GENES & DEVELOPMENT 18:2437–2468 © 2004 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/04; www.genesdev.org 2437 Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Sims et al. starts with preinitiation complex (PIC) assembly at the gation, it is difficult to distinguish these as separable promoter. The PIC includes the general transcription stages in many experimental systems (Uptain et al. factors (GTFs) IID, IIB, IIE, IIF, and IIH and RNAP II, as 1997). The earliest stages of transcription are marked by well as several additional cofactors (Orphanides et al. instability of the transcription complex and a measur- 1996). Formation of an open complex between RNAP II able tendency to release the RNA. Appearance of the and the DNA template is a prerequisite for transcription stalled RNAP II–DNA complex and short RNA products initiation. Melting of the double-stranded DNA into a during promoter clearance indicates that immediately single-stranded bubble is an ATP-dependent process and after transcription starts, a stable TEC is not yet formed, requires the action of two GTFs, IIE and IIH (Goodrich and RNAP II is viewed to be in a mode of abortive ini- and Tjian 1994; Holstege et al. 1996; Kim et al. 2000). tiation. Newly initiated RNAP II has a tendency to slip Once the open complex is established, transcription ini- laterally (Pal and Luse 2002). A recent study has demon- tiation occurs upon addition of the two initiating nucleo- strated that a decrease in slippage is a two-step process: side triphosphates (NTPs) dictated by the DNA sequence first, slippage is greatly reduced after the length of the and formation of the first phosphodiester bond. The pres- RNA–DNA hybrid reaches 8–9 nt, and then it becomes ence of all NTPs allows RNAP II to clear the promoter, undetectable after synthesis of a 23-nt-long RNA (Pal although ATP appears to be particularly important for and Luse 2003). The first step in the increase of RNAP II the transition from initiation to elongation (Dvir et al. stability can be explained by previous work. Biochemical 1996). Before RNAP II becomes engaged into productive studies have determined that RNAP II TECs are unstable transcript elongation, it must pass through a stage before the RNA–DNA hybrid reaches 8 nt in length known as promoter clearance (see below). During this (Kireeva et al. 2000). In agreement with this result, the stage, the PIC is partially disassembled: a subset of GTFs high-resolution crystal structure of the yeast RNAP II remains at the promoter, serving as a scaffold for the TEC containing a 14-nt RNA shows that the RNA–DNA formation of the next transcription initiation complex hybrid within the transcription bubble is 9 nt long (Zawel et al. 1995; Yudkovsky et al. 2000). The GTF (Gnatt et al. 2001). At present, it is not understood how TFIIH plays an integral role in facilitating promoter a 23-nt-long transcript stabilizes the elongating RNAP II clearance, in part by preventing premature arrest (Good- (Pal and Luse 2003). One possibility is that the interac- rich and Tjian 1994; Dvir et al. 1997; Kumar et al. 1998). tion of the extended transcript with the RNA exit chan- However, of all the GTFs, only TFIIF can be found in the nel promotes additional changes in the conformation of RNAP II TEC (Zawel et al. 1995). Once the promoter is the enzyme, which results in further stabilization. Struc- cleared, the next round of transcription can be reiniti- tural studies of the RNAP II TEC containing a RNA tran- ated. Reinitiation of transcription has the potential to be script longer than 14 nt would provide a more definitive a much faster process relative to the initial round, and is answer to this question. The recent cocrystal of RNAP responsible for the bulk of transcription in the cell (Jiang II–TFIIB identified that TFIIB might impede the exit path and Gralla 1993; Ranish et al. 1999; Orphanides and of the newly formed RNA (Bushnell et al. 2004). The Reinberg 2000). The final step in the cycle is transcript authors suggest that the continued presence of TFIIB termination. At this stage, the mRNA is cleaved, poly- could result in abortive initiation, and subsequent re- adenylated, and transported to the cytoplasm, where it moval of TFIIB would allow for promoter escape. Many will be translated (Proudfoot et al. 2002). Interestingly, of the details surrounding promoter clearance await the transcript cleavage-polyadenylation specificity factor characterization, and although complex, understanding (CPSF) can be recruited by TFIID apparently
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