Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression
ReviewElongation by RNA polymerase II: structure–function relationship
Introduction
Transcription is the crucial point in the regulation of gene expression. Considering that the blueprint of the human genome has recently been published [1], [2], the disclosure of the mechanisms involved in the “reading” of the genome acquires an ever-increasing importance. Transcription by RNA polymerase II is the mechanism by which the eukaryotic genome is “read” to generate mRNA. Regulation of the process underlies fundamental biological processes such as development, morphogenesis and oncogenesis. In the case of cancer, a clear link between transcription and oncogenesis exists. For example, tumor suppressor proteins associated with breast cancer such as P53 and those encoded by the BRCA1 gene function as regulators of the transcription apparatus [3], [4], [5], [6], [7], [8]. Other oncoproteins, such as EWS/Fli1, the prime candidate involved in Ewing tumors, have also been found to interact with the human transcription machinery [9].
Recent evidence indicates the stage of RNA chain elongation as a key point in the regulation of transcription and as playing a role in human disease [10]. With this in mind, structural and functional studies of the motors of transcription, the RNA polymerases, are crucial for revealing mechanisms underlying cellular and physiological function in the normal and diseased states. Although the focus of this review is the structural and functional relationships of RNAP during the process of transcription elongation, the steps taken to arrive at the RNAP elongation complex structure could be applied to most large macromolecular complexes as well.
Section snippets
The transcription motors
At the heart of eukaryotic transcription lie three multisubunit RNA polymerases, RNA polymerases I, II and III, which are targets of complicated mechanisms of regulation. Subunits of the three eukaryotic RNA polymerases either are identical and shared or are homologous [11], [12]. Eukaryotic polymerases consist of a dozen or so polypeptides with total masses of over 500 kDa. Of the three forms of RNA polymerases found in eukaryotes, RNA polymerase II (RNAP) alone is responsible for generating
RNAP transcript elongation
Transcription consists of several distinct stages including initiation at a promoter, elongation of the RNA chain and the last stage of termination where RNAP completes the synthesis of RNA. Initiation is the first step prior to transcript elongation and follows promoter binding in which RNAP, with the aid of additional general transcription factors (GTFs), recognizes a promoter. Global regulation of promoter binding and initiation is a highly modulated process and can involve tens to hundreds
Choice of model for structural analysis
The wealth of biochemical data raises many questions as to the mechanisms underlying transcription elongation and its regulation by RNAP such as how the transcription bubble is generated and maintained, how DNA and RNA are separated, and what affects elongation complex stability. The improper regulation of transcription, a fundamental element of the cancer cell, further inspired the need for determining the structure of RNAP alone, as an elongation complex and in complex with regulatory
Subunit structure of RNA polymerase II
yRNAP contains 12 subunits, RNA polymerase Beta 1–12 (RPB1–12), with a molecular mass of over 500 kDa. The two largest subunits, RPB1 and RPB2, together account for over half the mass of RNAP and contain all the necessary biochemical elements for synthesizing RNA from a template. All subunits besides Rpb4 and Rpb7 are found as a single copy within the enzyme.
Two subunits, RPB4 and RPB9, are not essential for cell survival as has been shown by the generation of deletions of these subunits in
Low-resolution structure of RNAPII
The first low-resolution structures of RNAPII were accomplished by generating two-dimensional RNAP crystals on lipid layers, which were subjected to electron microscope (EM) structural techniques [63] and revealed the molecules' topography at 16 Å. The structure contained a cleft and several grooves that could accommodate nucleic acids. The successful structural determination had additional important implications for the high-resolution work, which was to follow. Through these studies, it was
Generation of elongation complexes of RNAPII
With the low-resolution EM structure at hand, an efficient means of generating homogeneous complexes of RNAP engaged in transcription was needed prior to its structural determination. Although as mentioned above, RNA polymerase II requires a set of additional protein factors to start transcription at a promoter, the polymerase alone is capable of initiation on a single strand protruding from the 3′-end of duplex DNA (Fig. 2) [65]. Such a tailed template may be viewed as half of an unwound
Low-resolution EM structure of RNAP in the act of transcription
Once relatively homogeneous and stable elongation complexes were at hand, a three-dimensional reconstructed low-resolution EM structure of RNAP in the act of transcription was successfully achieved [70]. Initially it was not clear that at low-resolution DNA could be observed. For this reason, the downstream region of the DNA was linked through biotin to a streptavidin molecule whose bulkier density should clearly be visible. As it turned out, the streptavidin and some of the DNA were visible.
High-resolution structure of RNAPII and a two metal ion active site
As a point of interest, determining the high-resolution structure of large complex macromolecules is currently a challenging task, which can take over 10 years [71], [72]. Firstly, large amounts of pure, homogeneous and intact material need to be generated. In the case of RNAP, the protein was found to be prone to protease degradation, especially the CTD of Rpb1. Indeed, the generation and use of a CTD-specific antibody was essential for production of intact homogeneous enzyme [73]. Large
High-resolution structure of the elongation complex
The structures of yRNAP lack subunits 4 and 7, as these subunits were prohibitive to crystallization. The general features of yRNAP are displayed in Fig. 3 below. Looking down at the top of the enzyme, three border regions of a large cleft exist, with a single open end as depicted in Fig. 3. The first border domain was termed the clamp and is discussed separately below. Most of the first and second borders of the cleft are composed of Rpb1 and Rpb2 linked through a “bridging” helix of Rpb1. In
The clamp domain
RNAP structures revealed that the clamp domain of the first cleft border plays crucial roles in the process of transcription. As mentioned above, the lower resolution 5 Å X-ray structure was compared to the EM 16 Å structure and only one domain, the clamp domain, was observed to have shifted. In the X-ray structure it appeared as if the clamp was open, whereas in the EM structure it appeared closed. This was indeed confirmed in the higher resolution X-ray structures of the enzyme at rest, which
The hybrid
The RNA/DNA hybrid of the elongation complex was observed as nine bases precisely matching earlier biochemical studies and is highly underwound compared to a free DNA–RNA duplex. Indeed, it appears that biochemical findings of a three-and nine-base hybrid were justified. The first three bases of the RNA are well buried and protected in the active site and are held in place by both DNA and protein interactions. Although the DNA interacts with protein residues all along its length, the five
Translocation on the template
For transcription to succeed, RNAP must move across the template or vice-versa. The distinction may be relative to the position of observation. If we were “standing” on the DNA, we would see the RNAP moving and vice-versa. Interestingly, there exists evidence suggesting that from the vantage point of the nucleus, the DNA may be moving. Transcription appears to occur in discrete factories with RNAP fixed to the nuclear skeleton [84], [85]. Results suggest DNA template translocation as a result
RNA/DNA exit from the molecule
In the elongation complex structure, nucleotides upstream of the hybrid were not observed despite their presence. The RNA strand should be between 12 and 15 bases, though RNA and DNA beyond the hybrid are disordered and not visible. Density of the hybrid ended near polypeptide loops that were proposed to separate the RNA and DNA strands. Two of three of these loops, the rudder and the lid, were disordered in the elongation complex. The third loop, the rudder, remained ordered. In the bacterial
Position of the CTD
In the section on transcript elongation above, it was noted that the CTD of Rpb1 contains a heptapeptide repeat that may be heavily phosphorylated. The CTD is a key regulatory domain of RNAP, which binds mediator, elongation factors, capping enzyme, splicing factors as well as termination factors. Although its structure is disordered and not observed in these X-ray studies, the position of the CTD can be inferred from the lack of density in the tightly packed RNAP crystals [55]. One region
Elongation and transcript stability
As discussed in Section 3, two stages of elongation are completed before elongation commitment and promoter clearance is achieved. In the first stage, a transition occurs from an RNA length of three to four bases accompanied by an increase in stability. Such a transition may involve movement of amino acid side chains that could interact, either short- or long-range, with the RNA. This would result in the fine positioning of the three bases of RNA/DNA hybrid nearest to the active site magnesium.
TFIIS, RNAP and arrested transcription
Arrested transcription occurs when the 3′ hydroxyl of the RNA strand loses contact with the active site after RNAP backtracks on the DNA template (Fig. 1). The structure of the elongation complex clearly shows the 3′ hydroxyl of the RNA lying immediately above the funnel-shaped pore (Fig. 5). This is highly suggestive of a mechanism whereby the RNA slides down the pore causing the loss of contact of the 3′ hydroxyl by the active site. Indeed, some weak electron density (personal observation) is
Other RNA polymerases
Studies of RNA polymerases from bacteria not only have allowed for a wealth of biochemical information but also have been successfully employed for structural determination [87], [94]. Of prime importance is the structure of the α2ββ′ subunit RNA polymerase from the Thermus aquaticus (tRNAP) [87]. The Rpb3–Rpb11 heterodimer of yeast RNAP is structurally homologous to the bacterial α2 subunit. Rpb1 and Rpb2 in yRNAP are also structurally homologous to the bacterial β′ and β subunits,
Rifampicin inhibition of RNA polymerase
Tuberculosis (TB) caused by the Mycobacterium tuberculosis is known to reach epidemic proportions in some regions and lead to death. Rifampicin is one of the key antibiotics in TB treatment. Rifampicin inhibits bacterial DNA-dependent RNA polymerases by binding to the β subunit and preventing transcript elongation during the earliest stages of RNA synthesis [97]. The affinity of rifampicin to eukaryotic polymerases is much lower than that of the bacterial counterpart, making it a very potent
Future prospects
Transcription elongation by RNA polymerase is a complex process that is regulated by many elongation factors. Structural studies of the bacterial RNA polymerase and yeast RNAP shed light on these mechanisms including hybrid strand separation, DNA template translocation on RNA polymerase, abortive initiation, transcript arrest, and processivity [98]. Structural studies relating to RNA polymerase elongation also have importance in the development of treatments for cancer, AIDS and bacterial
Acknowledgements
I am most appreciative of Dr. Roger Kornberg, Stanford University in his granting me the wonderful opportunity to achieve the yeast RNAP elongation complex structure in his laboratory and for his guidance. I am further indebted to all co-workers whose names are not mentioned here, lest one is omitted. Work by the author towards the structure of RNAP elongation complex was sponsored by the USAMRC Breast Cancer Initiative DAMD17-97-7099 and does not necessarily reflect the policy of the
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2011, Mutation Research - Fundamental and Molecular Mechanisms of MutagenesisCitation Excerpt :Collectively, these results once again fail to support a role for TFIIS in TC-NER. TFIIS is thought to aid RNA polymerase to bypass arrest sites [24,25,47]. In yeast, TFIIS can extend into the active site of an arrested RNA polymerase II where it can modify its catalytic activity [26,27].