Structure and Function of RNA Polymerase II

STRUCTURE AND FUNCTION OF RNA POLYMERASE II By PATRICK CRAMER Institute of Biochemistry and Gene Center, University of Munich, 81377 Munich, Germany

I. Perspective . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . II. Structure of RNA Polymerase II . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . A. Overview of Structure Determinations. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . B. Ten-Subunit Core Polymerase.. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . C. Rpb4/7 Complex.. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . D. Complete 12-Subunit Polymerase. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . E. Polymerase-TFIIS Complex . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . III. Function of RNA Polymerase II. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . A. Overview of the Transcription Cycle . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . B. Initiation Complex Assembly and Promoter DNA Loading . . . . . . . . . . . .. . . . . . . . C. Initiation-Elongation Transition . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . D. Elongation, Processivity, and Transcription Bubble Maintenance. . . . .. . . . . . . . E. Catalysis, Fidelity, Specificity, and Translocation. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . F. Backtracking, Pausing, Arrest, and Proofreading . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . G. Coupling to RNA Processing and Other Nuclear Events . . . . . . . . . . . . . . .. . . . . . . . H. Termination, Polymerase Recycling, Reinitiation, and Regulation. . . .. . . . . . . . IV. Comparison with Other Polymerases . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . A. Eukaryotic RNA Polymerases I and III. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . B. Bacterial and Archaeal RNA Polymerases . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . C. Single-Subunit DNA and RNA Polymerases. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . V. Conclusions . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .

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I. Perspective RNA polymerase II (Pol II) is the central enzyme that catalyses DNAdirected mRNA synthesis during the transcription of protein-coding genes. Pol II consists of a 10-subunit catalytic core, which alone is capable of elongating the RNA transcript, and a complex of two subunits, Rpb4/7, that is required for transcription initiation. Structures of individual Pol II subunits and subunit domains have been determined by nuclear magnetic resonance and X-ray analysis (Table I), and various forms and complexes of Pol II have been studied by electron microscopy (Asturias et al., 1997; Darst et al., 1991a; Jensen et al., 1998; Leuther et al., 1996). Here, however, I will concentrate on high-resolution structures of the 10-subunit Pol II core (Bushnell et al., 2002; Cramer et al., 2000; Cramer et al., 2001; Gnatt et al., 2001), an archaeal counterpart of Rpb4/7 (Todone et al., 2001), and x-ray crystallographic backbone models of the complete 12-subunit Pol II 1 ADVANCES IN PROTEIN CHEMISTRY, Vol. 67

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Table I High-Resolution Structural Studies of RNA Polymerase II Structure Rpb5

Organism

Method X-ray

Rpb6

Saccharomyces cerevisiae Human

Rpb8 Rpb9 C-terminal domain Rpb 10 homolog

Resolution PDB* [A˚] code

Reference

1.9

1dzf

NMR*



1qk1

S. cerevisiae

NMR



1ald

Thermococcus celer

NMR



1qyp

Methanobacterium NMR thermoautotrophicum S. cerevisiae X-ray



1ef4

3.1

1i3q

S. cerevisiae

X-ray

2.8

1i50

S. cerevisiae

X-ray

3.3

1i6h

S. cerevisiae

X-ray

2.8

1k83

Bushnell et al., 2002

X-ray

1.75

1go3

Pol II

Methanococcus jannaschii S. cerevisiae

X-ray

4.2

1nt9

Pol II

S. cerevisiae

X-ray

4.1

1nik

Pol II-TFIIS complex

S. cerevisiae

X-ray

3.8

1pqv

Todone et al., 2001 Armache et al., 2003 Bushnell and Kornberg, 2003 Kettenberger et al., 2003

Pol II core1 form 1 Pol II core1 form 2 Pol II core1 tailed-template elongation complex Pol II core1 -amanitin complex Rpb4/7 complex

Todone et al., 2000 del Rio-Portilla et al., 1999 Krapp et al., 1998 Wang et al., 1998 Mackereth et al., 2000 Cramer et al., 2000 Cramer et al., 2001 Gnatt et al., 2001

1 Pol II core comprises 10 subunits, Rpb1, Rpb2, Rpb3, Rpb4, Rpb5, Rpb6, Rpb7, Rpb8, Rpb9, Rpb10, Rpb11, Rpb12 and lacks the Rpb4/7 complex. *PDB: protein data bank; NMR: nuclear magnetic resonance.

(Armache et al., 2003; Bushnell and Kornberg, 2003), and of Pol II in complex with the elongation factor TFIIS (Kettenberger, et al., 2003) (Table I). These structures were published over the last 3 years and will be described in Chapter 2. Interpretation of the structures alongside biochemical and genetic data has provided valuable insights into many aspects of the transcription mechanism and will be discussed in Chapter 3. In Chapter 4, the conservation of the Pol II structure throughout species, its use as a model

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for RNA polymerases I and III, and the consequences for understanding other polymerases are described.

II. Structure of RNA Polymerase II A. Overview of Structure Determinations Pol II is an asymmetric and large multiprotein complex with a total molecular weight of 0.5 MDa. High-resolution structural studies of Pol II by x-ray crystallography required large amounts of pure protein that cannot be obtained by overexpression because of the complexity of the enzyme. These difficulties have so far limited crystallographic studies of Pol II to the endogenous enzyme from Saccharomyces cerevisiae, which can be purified in milligram quantitites from yeast culture. Yeast Pol II preparations, however, contain substoichiometric amounts of the Rpb4/7 complex, giving rise to heterogeneity that impedes crystallization. This problem was overcome with the use of a rpb4 deletion strain of yeast. Purification from this strain yields the Pol II core, lacking both Rpb4 and Rpb7 (Darst et al., 1991b; Edwards et al., 1990). Initial studies of Pol II by electron microscopy (Asturias et al., 1997; Darst et al., 1991a; Jensen et al., 1998; Leuther et al., 1996) laid the ground for structural studies at high resolution, but several experimental difficulties had to be overcome first. Three-dimensional crystals were obtained (Fu et al., 1998, 1999) and were improved by induced crystal shrinkage (Cramer et al., 2000). Phase determination relied on heavy atom clusters (Cramer et al., 2000; Fu et al., 1999) and nonstandard heavy-metal compounds (Cramer et al., 2000). Interpretation of the experimental electron density maps was facilitated by placement of subunit structures that had been determined previously (Table I). Map interpretation also relied on phase combination and on the use of sequence markers (Cramer et al., 2000), including partially incorporated selenomethionine (Bushnell et al., 2001). These efforts first resulted in a backbone model of the Pol II core, which revealed the subunit architecture of the enzyme and functional elements (Cramer et al., 2000). Nucleic acids could also be placed on the Pol II backbone model (Cramer et al., 2000) with the use of electron microscopy data, which had earlier revealed the location of downstream DNA (Poglitsch et al., 1999). One year later, refined atomic structures of the Pol II core were reported in two crystal forms at 2.8- and 3.1-A˚ resolution (Cramer et al., 2001). The atomic core structures then enabled structure determination by molecular replacement of a minimal elongation complex of the yeast Pol II core (Gnatt et al., 1997; Gnatt et al.,

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2001) and a complex of the Pol II core with the mushroom toxin -amanitin (Bushnell et al., 2002). Lacking from the Pol II core structures was the Rpb4/7 complex. The structure of an archaeal counterpart of an isolated Rpb4/7 complex was, however, determined (Todone et al., 2001), and the location of the Rpb4/7 complex on the Pol II core surface was revealed by cryo-electron microscopy (Craighead et al., 2002). Recently, backbone models of the complete 12-subunit Pol II were derived by two groups independently, with the use of x-ray crystallographic data to around 4-A˚ resolution (Armache et al., 2003; Bushnell and Kornberg, 2003), and a model for the complex of the complete Pol II with the elongation factor TFIIS at 3.8-A˚ resolution was also reported (Kettenberger et al., 2003). The described structural studies of yeast Pol II are directly relevant for the Pol II enzymes in higher organisms, since the Pol II subunits are very well conserved in sequence and function. Approximately half of the amino acid residues in the twelve Pol II subunits are identical between yeast and human sequences. Furthermore, most yeast subunits can functionally replace their human counterparts (Woychik, 1998). The human Rpb4/7 complex can also functionally replace its yeast counterpart (Khazak et al., 1995), indicating that the core-Rpb4/7 interface is conserved.

B. Ten-Subunit Core Polymerase Five Pol II subunits, Rpb1, Rpb2, Rpb3, Rpb6, and Rpb11, show sequence and structural similarity in all cellular RNA polymerases and are referred to as the ‘‘core’’ subunits (Table II). One of the core subunits, Rpb6, and four other subunits, Rpb5, Rpb8, Rpb10, and Rpb12, are shared between the three eukaryotic RNA polymerases I, II, and III, and are referred to as the ‘‘common’’ subunits. The 10-subunit Pol II core comprises the core and common subunits and in addition, subunit Rpb9. The Pol II core structures show that the two large subunits, Rpb1 and Rpb2, form the central mass of the enzyme and opposite sides of a positively charged ‘‘cleft’’ that contains the active center (Fig. 1). The two large subunits are bridged on one side by a module of subunits Rpb3, Rpb10, Rpb11, and Rpb12. Around the periphery of the enzyme, Rpb5, Rpb6, and Rpb8 assemble with Rpb1, and Rpb9 binds to both Rpb1 and Rpb2. Subunits can be divided into domain-like regions, to aid interpretation of genetic and biochemical data and to facilitate the design of mutagenesis experiments (Cramer et al., 2001). Subunits Rpb1 and Rpb9 each bind two zinc ions, and subunits Rpb2, Rpb3, Rpb10, and Rpb12 each bind one zinc ion. All eight zinc ions are near the Pol II surface, apparently stabilizing the enzyme.

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STRUCTURE AND FUNCTION OF RNA POLYMERASE II

Table II RNA Polymerase Subunits Eukaryotes Bacteria

Class1

A0 + A00 B (B0 + B00 ) D L K

0    !

H — N P X F E þ1 other

— — — — — — — —

Core Core Core Core Core and common Common Common Common Common Unclear Rpb4/7 Rpb4/7 Specific Specific Specific

Pol I

Pol II

Pol III

Archaea

A190 A135 AC40 AC19 Rpb6

Rpb1 Rpb2 Rpb3 Rpb11 Rpb6

C160 C128 AC40 AC19 Rpb6

Rpb5 Rpb8 Rpb10 Rpb12 A12.2 A14 A43 A34.5 A49

Rpb5 Rpb8 Rpb10 Rpb12 Rpb9 Rpb4 Rpb7 —

Rpb5 Rpb8 Rpb10 Rpb12 C11 C17 C25 C82 C34 C31

1

Core: Sequence partially homologous in all RNA polymerases. Common: shared by all eukaryotic RNA polymerases, Rpb4/7: Rpb4/7 heterodimer and its structural counterparts. Unclear: It is unclear if A12.2 and C11 are true Rpb9 homologs. It appears that the C-terminal domain of the Pol II subunit C11 is functionally and structurally homologous to the Pol II transcript cleavage factor TFIIS.

Structural elements of Pol II have been given generic names if they appeared to be functionally relevant (Table III). The Rpb1 side of the cleft is formed by a mobile ‘‘clamp,’’ whereas the Rpb2 side consists of two domains, termed ‘‘lobe’’ and ‘‘protrusion.’’ The entrance to the cleft is formed between the ‘‘upper jaw’’ and the ‘‘lower jaw’’ of Pol II, which include subunits Rpb9 and Rpb5, respectively. The end of the cleft is blocked by a protein ‘‘wall.’’ The active center is formed by the floor of the cleft at its end and is located between the protrusion, the wall, and the clamp. Before the active center and opposite of the wall, a long ‘‘bridge’’ helix spans the cleft. The bridge partially lines a ‘‘pore’’ in the active center, which widens toward the other side of the enzyme, creating an inverted ‘‘funnel.’’ The rim of the pore also includes the highly conserved ‘‘aspartate loop’’ of Rpb1 that forms part of the active site. This loop comprises three invariant aspartate residues that stably bind a Mg2þ ion, termed ‘‘metal A.’’ The aspartate loop was identified as part of the active site by site-specific hydroxy radical cleavage (Zaychikov et al., 1996).

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Table III Structural Elements of RNA Polymerase II Pol II element and subelement

Subunit

Homology region

Rpb1 Rpb1 Rpb2 Rpb1

D D F F

Clamp

Rpb1 Rpb2 Rpb1 Rpb2 Rpb2, Rpb1 Rpb1 Rpb1

A, B, C, H H, C I I, H

Rpb1

A

Rpb1 Rpb9

G

Switch 1, 2 Switch 3 Switch 4,5 Rudder Lid Zipper Jaws Rpb1/9 jaw (upper jaw)

B

Catalysis (Catalysis) (NTP binding) Positioning of nascent base pair, stabilization of twist between bases in the template strand, maintenance of downstream end of the bubble, -amanitin-binding, (translocation) Processivity, template strand binding, hybrid retention, bubble maintenance, (initiation factor binding) Template strand binding, clamp mobility, processivity Template strand binding, processivity Clamp mobility Stabilization of the elongation complex (maintenance of upstream end of hybrid, creation of RNA exit tunnel) (maintenance of upstream edge of bubble) TFIIS binding, (interaction with downstream DNA during initiation and elongation)

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Active center Metal A site Metal B site Bridge

Function (proposed if in parenthesis)

Mobile modules Jaw-lobe Shelf Trigger loop Pocket Tip

Rpb5 Rpb2 Rpb2 Rpb1 Rpb2 Rpb1, Rpb6, Rpb11 Rpb1, Rpb8 Rpb1 Rpb1 Rpb2 Rpb2 Rpb1 Rpb2 Rpb1

Rpb1, Rpb2, Rpb9 Rpb1, Rpb5 Rpb1 Rpb1, Rpb2, Rpb6 Rpb7

Interaction with downstream DNA during elongation G, H

Hybrid binding, (maintenance of upstream end of the bubble) (initiation factor interaction, RNA exit tunnel formation), (RNA exit) (RNA exit)

I (Alternative RNA exit routes beyond the saddle) F, G F

C H

G Rpb1 H, Rpb2 I

-amanitin-binding, TFIIS binding, (NTP entry) TFIIS binding, (NTP entry, RNA exit during backtracking and arrest) TFIIS binding, crevice opening triggers conformational changes

(Maintenance of downstream end of bubble) (Initiation factor interactions) (Interaction with downstream DNA) CTD flexibility, Rpb7 binding modulation of Pol II activity throughout the transcription cycle, binding of Mediator and RNA processing factors

STRUCTURE AND FUNCTION OF RNA POLYMERASE II

Rpb5 jaw (lower jaw) Wall (flap) Flap loop Saddle between wall and clamp RNA exit grooves 1, 2 Funnel Pore Crevice Fork Fork loop 1 Fork loop 2 Dock domain Lobe Linker CTD

TFIIS binding, (translocation) Binds the Rpb7 tip (allosteric regulation of clamp) Binds into the pocket below the clamp of the Pol II core

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A second metal ion, ‘‘metal B,’’ is weakly bound further in the pore, between the Rpb1 aspartate loop and one or two conserved acidic residues in Rpb2 (Cramer et al., 2001). Low occupancy of this second metal binding site indicates that the metal may be exchangeable. Both metal ions are accessible from one side. Two adjacent metal binding sites were also observed in a high-resolution structure of a bacterial RNA polymerase holoenzyme (Vassylyev et al., 2002). The clamp is a mobile domain that was suggested to retain nucleic acids in the cleft (Cramer et al., 2000; Fu et al., 1999). The clamp is trapped in two different open states in the free core structures (Cramer et al., 2001) but is rotated and closed in the structure of the core elongation complex (Gnatt et al., 2001). In the elongation complex structure, the clamp binds the DNA template strand before and within the DNA-RNA hybrid (Fig. 2A). Template strand binding involves three out of five ‘‘switch’’ regions. The switch regions form the base of the clamp that connects the clamp to the remainder of Pol II. On clamp closure, the switches change conformation or undergo folding transitions. The closed conformation of the clamp is also observed in electron microscopic images of the 12-subunit Pol II (Craighead et al., 2002). The clamp is formed by two regions in Rpb1, located at the N terminus and near the C terminus, and the C-terminal region of Rpb2. Three zinc ions stabilize the unique clamp fold. The C-terminal region of Rpb1 protrudes from the base of the clamp on the outside of Pol II and gets disordered after a few residues. These last ordered residues of Rpb1 constitute the beginning of a ‘‘linker’’ that connects to the C-terminal repeat domain (CTD) of Rpb1. The linker comprises about 100 and 150 residues in yeast and human, respectively, and is not conserved. The CTD is a unique feature of Pol II and consists of repeats of a heptapeptide with the consensus sequence Tyr-Ser-ProThr-Ser-Pro-Ser. A total of 26 and 52 CTD repeats are found in yeast and human Rpb1, respectively. The CTD and most of the linker are not ordered in the Pol II crystal structures. Nuclear magnetic resonance and circular dichroism studies of CTD peptides in solution revealed little residual structure (Cagas and Corden, 1995). If the linker and CTD would adopt a fully extended conformation, the C-terminus of Rpb1 could extend almost 1000 A˚ from the Pol II surface, about seven times the diameter of Pol II. Thus, the CTD could in principle reach anywhere on the Pol II surface. However, it is likely that the unphosphorylated CTD adopts a compacted state near the beginning of the linker on the Pol II surface (Cramer et al., 2001). A compacted weak protein density was detected near the Pol II core by electron microscopy (Meredith et al., 1996).

STRUCTURE AND FUNCTION OF RNA POLYMERASE II

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C. Rpb4/7 Complex The core structures lacked subunits Rpb4 and Rpb7, which form a stable heterodimer that can dissociate from the yeast Pol II core under mild denaturing conditions and on ion exchange chromatography (Edwards et al., 1991). Whereas Rpb7 is well conserved in sequence, Rpb4 shows only weak sequence conservation. Rpb7 is an essential protein (McKune et al., 1993). Rpb4 is not essential in S. cerevisiae (Woychik and Young, 1989), whereas it is required for viability of the fission yeast Saccharomyces pombe (Sakurai et al., 1999). Counterparts of the Rpb4/7 complex exist in the eukaryotic RNA polymerases Pol I and Pol III (Hu et al., 2002; Peyroche et al., 2002; Sadhale and Woychik, 1994; Shematorova and Shpakovski, 1999; Siaut et al., 2003) and in the archaeal enzymes (Werner et al., 2000). The structure of an archaeal Rpb4/7 counterpart revealed that Rpb7 spans an elongated complex and is organized in two domains, an N-terminal ribonucleoprotein (RNP)-like domain and a C-terminal domain that includes an oligonucleotide/oligosaccharide-binding fold (Todone et al., 2001). The Rpb4 homolog binds at the connection between the two Rpb7 domains and forms a conserved hydrophobic interface with the Rpb7 homolog. Conservation of the interface is demonstrated by the formation of chimeric heterodimers with Rpb4 and Rpb7 from various species (Guilfoyle and Larkin, 1998; Sakurai et al., 1999; Werner et al., 2000). Mutagenesis and surface conservation indicate a potential nucleic acid binding face of the Rpb4/7 complex that could account for binding of single-stranded nucleic acids in vitro (Orlicky et al., 2001; Todone et al., 2001). Cryo-electron microscopy of the 12-subunit yeast Pol II revealed an additional density on the outside of the core that was interpreted as the Rpb4/7 complex (Craighead et al., 2002). This density coincides with a stalk of protein protruding from the core of Pol I in electron microscopy images (Bischler et al., 2002). With the use of immunolabeling, the stalk in Pol I was shown to contain counterparts of Rpb4 and Rpb7 (Bischler et al., 2002). In the electron microscopic reconstructions, most of the Rpb4/7 surface appears to be exposed and easily accessible for interactions with other proteins or nucleic acids. Electron microscopy in solution further revealed that the clamp adopts a closed state in the 12-subunit Pol II that includes the Rpb4/7 complex (Craighead et al., 2002).

D. Complete 12-Subunit Polymerase The above findings and proposals about the location and function of the Rpb4/7 complex were generally confirmed and extended by recent crystallographic backbone models of the complete Pol II that includes the

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Rpb4/7 complex. These models were derived independently by two groups (Armache et al., 2003; Bushnell and Kornberg, 2003) and show that Rpb4/7 protrudes from the polymerase surface near the base of the clamp (Fig. 1). The Rpb4/7 complex interacts with the Pol II core through Rpb7, which binds to regions of Rpb1, Rpb2, and Rpb6. Most of the Rpb4/7 surface is exposed and accessible for interactions with proteins or nucleic

Fig. 1. Two views of the complete yeast Pol II (Armache et al., 2003). The 12 protein subunits are shown as ribbon diagrams in different colors, as indicated in the schematic diagram. The active site metal ion A is depicted as a pink sphere. Zinc ions are shown as cyan spheres. A highly similar model was reported by Bushnell and Kornberg, 2003. CTD, C-terminal repeat domain.

STRUCTURE AND FUNCTION OF RNA POLYMERASE II

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acids. Rpb4/7 binds to the Pol II core with the N-terminal RNP-like domain of Rpb7: termed the tip. Consistent with the core-Rpb7 interaction, Rpb7 alone can bind to core (Sheffer et al., 1999), and Rpb7 is essential for yeast growth (McKune et al., 1993), whereas Rpb4 is not (Woychik and Young, 1989). Deletion of the rpb4 gene in yeast facilitates dissociation of Rpb7 from core (Edwards et al., 1991). The models indicate that loss of the Rpb4-Rpb7 interface on Rpb4 deletion destabilizes Rpb7 and facilitates Rpb7 dissociation. The Rpb4/7 complex forms a wedge between the clamp and the linker, apparently restricting the clamp to a closed position. In particular, the Rpb7 tip partially fills a surface ‘‘pocket’’ formed between the clamp, the linker, and the core subunit Rpb6. The pocket is lined by five protein regions: three in Rpb1 and one each in Rpb2 and Rpb6. Rpb4/7 binding to the pocket thus holds together three subunits and may stabilize the Pol II subunit assembly.

E. Polymerase-TFIIS Complex Very recently, a backbone model for the complex of the complete Pol II with the elongation factor TFIIS (or SII) was reported at 3.8-A˚ resolution (Kettenberger et al., 2003). To obtain this structure, recombinant TFIIS comprising domains II and III of the three-domain factor was soaked into harvested crystals of the complete Pol II. Successful protein soaking was enabled by the very large solvent channels of the crystals and the fact that the TFIIS-binding site on Pol II is not obstructed by crystal contacts. The resulting 13-polypeptide asymmetric complex has a molecular weight of 536 kDa. The crystal lattice accommodated extensive structural changes induced by TFIIS around the active site of Pol II and in the periphery of the enzyme. The structure shows that TFIIS extends along the Pol II surface, spanning a distance of 100 A˚ (Fig. 2B). TFIIS domain II docks to the exposed Rpb1 jaw domain of Pol II. The TFIIS interdomain linker extends from domain II along the Pol II surface into the funnel. Domain III inserts into the Pol II pore, and approaches the polymerase active site from the bottom face of the enzyme as predicted (Cramer et al., 2000). TFIIS domain III reaches the Pol II active site with the highly conserved loop of the protruding  hairpin. The domain II hairpin complements the polymerase active site with acidic groups that are essential for TFIIS function. Two invariant acidic residues in this loop, D290 and E291, are in close proximity of the Pol II catalytic metal ion A and are essential for TFIIS activity ( Jeon et al., 1994).

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Fig. 2. Structure of the Pol II core elongation complex and the Pol II-TFIIS complex. (A) Schematic cut-away view of the tailed-template yeast Pol II core complex (Gnatt et al., 2001). The view is related to the one on the bottom of Figure 1 by a 90-degrees rotation around a vertical axis. The DNA template and nontemplate strands are shown in blue and green, respectively, and the RNA in red. Four bases in the template strand are highlighted as sticks protruding from the DNA backbone. The yellow oval indicates the presumed location of the binding site for the incoming NTP. During polymerization, Pol II moves to the right. (B) Backbone model of the complete 12-subunit Pol II (grey) in complex with the elongation factor TFIIS (orange, Kettenberger et al., 2003). Parts of Pol II are omitted for clarity. DNA and RNA have been modeled according to the structure in (A). During backtracking, Pol II moves to the left.

In addition to the active site complementation, TFIIS induces structural changes in the Pol II active center. Binding of TFIIS domain III induces folding of the Rpb1 ‘‘trigger loop’’ (Vassylyev et al., 2002) and shifts the bridge helix. These changes probably result in a repositioning of nucleic

STRUCTURE AND FUNCTION OF RNA POLYMERASE II

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acids in the active center. TFIIS further induces a coordinated repositioning of about one-third of the polymerase mass, which includes the jaws, the clamp, and the Rpb1 cleft and foot domains and corresponds essentially to three mobile polymerase modules (Cramer et al., 2001). The repositioning seems to be caused by insertion of TFIIS into the Pol II funnel and pore, where it opens an additional crevice.

III. Function of RNA Polymerase II A. Overview of the Transcription Cycle The transcription cycle may be divided in three major phases: initiation, elongation, and termination. Steps during transcription initiation include promoter DNA binding, DNA melting, and initial synthesis of short RNA transcripts. The transition from initiation to elongation, referred to as ‘‘promoter escape,’’ also occurs in a stepwise fashion. Promoter escape leads to a stable elongation complex that is characterized by an open DNA region, the ‘‘transcription bubble.’’ The incoming and exiting DNA duplex, located before and after the bubble, respectively, is referred to as downstream and upstream DNA. The bubble contains the DNA-RNA hybrid, a heteroduplex of eight to nine base pairs. At one end of the hybrid, the growing RNA 30 -end is engaged with the active site. At the other end of the hybrid, the DNA and RNA strands are separated. After successful RNA chain elongation, transcription terminates and Pol II dissocitates from the template. Some of the steps during the transcription cycle can be carried out by Pol II alone. Pol II can maintain an open transcription bubble, translocate along the template DNA, synthesize RNA from the template, and proofread the nascent RNA. For all other steps during the transcription cycle, however, Pol II requires additional proteins. Several steps of the transcription cycle are accompanied by phosphorylation or dephosphorylation of the Pol II CTD (Dahmus, 1996; O’Brien et al., 1994). During initiation, the CTD gets phosphorylated and the CTD phosphorylation pattern changes during elongation. CTD phosphorylation patterns govern specific interaction with RNA processing factors, thereby coupling transcription to RNA maturation events. Recycling of Pol II after termination involves CTD dephosphorylation, as initiation requires unphosphorylated Pol II. Proteins involved in phosphorylation and dephosphorylation of Pol II and other regulatory proteins influence the transcription cycle at various steps. For each step in the transcription cycle, insights coming from the Pol II structures are discussed below. At several points, supporting biochemical data are included that were

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obtained with bacterial RNA polymerase, for which a large amount of mechanistic information has accumulated (von Hippel, 1998). As discussed in Chapter 4, bacterial and eukaryotic RNA polymerases show a conserved core and share many functional features.

B. Initiation Complex Assembly and Promoter DNA Loading To bind and melt promoter DNA, Pol II requires the general transcription factors TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (Buratowski, 1994; Buratowski et al., 1989; Kornberg, 1999), which in yeast consist of one, 14, two, three, and nine polypeptides, respectively. The general transcription factors assemble with Pol II on promoter DNA and are involved in sequence-specific promoter recognition (TFIIB, TFIID), prevention of nonspecific DNA binding (TFIIF), DNA melting (TFIIE, TFIIH), and phosphorylation of the CTD (TFIIE, TFIIH). Many Pol II promoters contain a TATA box about 25–30 base pairs upstream of the transcription start site. TFIID binds to the TATA box via its TATA box–binding protein (TBP) subunit. According to order-of-addition experiments, stepwise assembly of the initiation complex starts with the formation of a TFIID/TBP-DNA complex, followed by binding of TFIIB to TBP and to a promoter element adjacent to the TATA box, the TFIIB response element BRE (Buratowski et al., 1989; Lagrange et al., 1998). Assembly of TBP and TFIIB on TATA box DNA has been studied biochemically and structurally (Cox et al., 1997; Kim et al., 1993; Kim et al., 1993; Kosa et al., 1997; Littlefield et al., 1999; Nikolov et al., 1995; O’Brien et al., 1998; Sigler and Tsai, 2000). In addition to TBP and TFIIB, loading of promoter DNA onto Pol II minimally requires TFIIF (Killeen et al., 1992), which forms a stable complex with Pol II. An additional factor, TFIIA, can stabilize the TFIID-DNA complex (Pugh, 2000). Other core promoter elements are known, including the initiator element (Smale et al., 1998) and the downstream promoter element (Burke et al., 1998). Depending on the specific promoter structure, there are apparently various routes to the initiation complex. As an alternative to the stepwise assembly of the initiation complex, it has been suggested that a large Pol II ‘‘holoenzyme’’ can be recruited to a promoter in a single step. Such holoenzymes were purified from yeast (Koleske and Young, 1994) and mammalian cells (Ossipow et al., 1995) and comprise Pol II, general transcription factors, and various other proteins (Greenblatt, 1997; Myer and Young, 1998). The position of the general transcription factors with respect to promoter DNA in the initiation complex can be inferred from site-specific protein–DNA crosslinking (Ebright, 1998). The crosslinking data, taken

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together with topological considerations and with structural data, predict that TBP, TFIIB, and TFIIF interact with the ‘‘upstream face’’ of Pol II. The upstream face of the enzyme includes parts of the Rpb4/7 complex, parts of the clamp, the outside of the wall, the ‘‘saddle’’ between the clamp and the wall, and the ‘‘dock’’ domain of the largest Pol II subunit. Biochemical data indicate that Rpb4/7 stabilizes a minimal initiation complex ( Jensen et al., 1998), suggesting that Rpb4/7 interacts with one or more general transcription factors. There is evidence that TFIIB binds adjacent to the Rpb4/7 complex, because Rpb4/7 binds near Rpb6 (Bischler et al., 2002; Craighead et al., 2002) and the archaeal homolog of TFIIB binds the archaeal Rpb6 homolog (Magill et al., 2001). Initiation factors interact with counterparts of the Rpb4/7 complex in the two other eukaryotic RNA polymerases, Pol I and Pol III. In Pol III, the Rpb4 homolog binds to a region corresponding to the linker in Pol II (Siaut et al., 2003) and to the TFIIB-related initiation factor Brf1 (Ferri et al., 2000). The Rpb7 homolog of Pol I also binds an initiation factor, called Rrn3/TIF-IA (Peyroche et al., 2000; Yuan et al., 2002). Thus Rpb4/7 and its counterparts seem to bridge the polymerase core with initiation factors. Differences between Rpb4/7 and its counterparts in other polymerases may contribute to promoter specificity. One function of TBP, TFIIB, and TFIIF is apparently to bring the promoter DNA duplex to a location on the Pol II surface that is appropriate for DNA melting and initiation of RNA synthesis at the transcription start site. There are two prominent possible locations of the initially loaded promoter DNA duplex. The promoter duplex may initially bind above the cleft on the enzyme surface. Alternatively, promoter DNA may be bound inside the Pol II cleft, closer to the active site. The structure of the free Pol II core showed that dramatic opening of the clamp can create sufficient space to allow for loading of duplex DNA into the Pol II cleft (Cramer et al., 2001). However, the Rpb4/7 complex acts as a wedge that prevents entry of the promoter DNA duplex into the active center cleft (Armache et al., 2003; Bushnell and Kornberg, 2003). Because the Rpb4/7 complex is apparently not dissociating rapidly in all species, it is likely that the promoter DNA duplex initially binds outside the cleft far above the active center.

C. Initiation-Elongation Transition After loading of promoter DNA onto Pol II, duplex DNA is melted upstream of the transcription start site (Holstege et al., 1997; Pan and Greenblatt, 1994; Wang et al., 1992). DNA melting requires TFIIH, which

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comprises two ATP-dependent helicase activities that unwind DNA. Two alternative models for DNA opening have been proposed (Fiedler and Marc Timmers, 2000). Crosslinking data indicate that TFIIH interacts with the downstream DNA and acts from a distance (Kim et al., 2000). TFIIE bridges between Pol II and TFIIH and stimulates TFIIH activity (Maxon et al., 1994; Ohkuma, 1997). In Pol I, the initial melted DNA region is about nine base pairs long, and the mature transcription bubble extends over approximately 19 base pairs (Kahl et al., 2000). After RNA synthesis has initiated within the bubble, the bubble size remains flexible during early transcription (Fiedler and Timmers, 2001). If DNA would be melted in the cleft, the DNA nontemplate strand must be expelled from the cleft before the clamp can close. It is, however, possible that DNA is loaded on top of the cleft and remains above the cleft for melting. In this case, the template strand could pass the clamp after DNA melting; it could slip into the cleft and bind to the site formed by switch regions 1–3, as observed in the core elongation complex (Gnatt et al., 2001). Until the nascent transcript is about 15 nucleotides long, the early transcribing complex is functionally unstable. In some cases the transcript can even slip upstream along the DNA template by several bases and can be reextended (Luse and Pal, 2002). Early transcribing Pol II complexes have to undergo three transitions (Dvir, 2002). In the beginning, short RNAs are frequently released and Pol II has to restart transcription (‘‘abortive cycling’’). There is a decline in the level of abortive transcription when the RNA reaches a length of about four nucleotides, and this transition is termed ‘‘escape commitment’’ (Goodrich and Kugel, 2000, 2002). A second barrier has to be overcome when the RNA reaches a length of about 10 nucleotides. A third transition is reflected in the continued requirement for the ATP cofactor and TFIIH until the RNA is about 15 nucleotides long. Successful passage of early Pol II elongation complexes through all three transitions has been referred to as ‘‘promoter clearance.’’ The early initiation-elongation transitions limit the rate of Pol II transcription and can be enhanced by TFIIE, TFIIH, and ATP (Goodrich and Kugel, 1998). Transitions that underlie promoter clearance may be rationalized with the Pol II structures. At the very beginning of transcription, contacts of Pol II with nascent RNA are crucial. To allow for the synthesis of the first phosphodiester bond, nucleoside triphosphates must be held by the protein. The resulting dinucleotide RNA must still be held by protein– RNA contacts, as observed in the core elongation complex structure (Gnatt et al., 2001), as the energy of base-pairing alone is insufficient for its retention. Equally, RNA is still bound by Pol II at the position of the third nucleotide. Despite the observed RNA-Pol II contacts, short RNA

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dinucleotides and trinucleotides are often lost, RNA synthesis must restart, and repetitive RNA loss and transcription initiation results in abortive cycling. RNA that has grown to a length of at least four nucleotides is generally not contacted by Pol II any more and is apparently held in the elongation complex solely by base pairing with the DNA template strand. This change in RNA interactions reflects the first transition in stability of the early transcribing complex that occurs at a transcript length of four residues, beyond which the RNA is generally retained. Maybe the Pol II–RNA contacts are limited to the crucial contacts of the first few nucleotides, to facilitate RNA mobility and translocation of nucleic acids. In the bacterial RNA polymerase, a portion of the  initiation factor apparently interferes with the path of the early transcript, inducing abortive cycling (Murakami et al., 2002). It is possible that in the Pol II system, one of the general transcription factors is located similarly and plays a similar role. The Pol II structures also provide an explanation for the second transition in stability of the transcribing complex that occurs at an RNA length of around 10 nucleotides. The 50 -end of a 10-residue RNA would be located just beyond the DNA-RNA hybrid, after its removal from the DNA template strand. At this point, the RNA is apparently redirected to the Pol II ‘‘saddle’’ and an exit tunnel (compare section D). Threading of RNA into the exit tunnel and binding of RNA to its exit groove may underlie the second transition in elongation complex stability. The third transition, which occurs when the RNA is about 15 nucleotides long, may reflect successful positioning of all bubble-maintaining structural elements of Pol II with respect to the bubble and detachment of RNA from the Pol II surface. Two possible RNA exit grooves have been suggested beyond the saddle, and binding of RNA to the saddle and to one of the exit grooves could account for an additional gain in stability of the elongation complex. In addition, RNA may bind to a nearby potential nucleic-acid binding face of the Rpb4/7 complex. The described transitions may involve reshaping of protein–nucleic acid contacts and may require slight changes in the clamp position. Because downstream DNA contributes to the stability of early transcribing complexes (Wang et al., 2003), it is likely that the downstream DNA contacts Pol II during initiation and promoter escape. A candidate subunit for such interaction is Rpb9, as it is located at an appropriate position and as mutations in Rpb9 lead to changes in the position of the transcription start site (Furter-Graves et al., 1994; Hull et al., 1995). Indeed, a domain in the bacterial enzyme at an approximately corresponding location contacts downstream DNA (Ederth et al., 2002). After successful promoter clearance, the early elongation complex can pause in a promoter proximal position (Albert et al., 1997; Li et al., 1996; Raschke et al., 1999). This promoter-proximal pausing of polymerase

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provides a means of rapid response to stimulatory signals. Exonuclease III footprinting indicates another transition from initiation to elongation that occurs around 25 bases downstream of the transcription start site (Luse and Samkurashvili, 1998). In other studies it was found that the transition to full elongation competence is dependent on the synthesis of even longer RNAs of a length of 50 nucleotides (Ujvari et al., 2002). This late transition is reversible by shortening the nascent RNA. The structural basis of these transitions is unclear. The production of a fully competent elongation complex is referred to as ‘‘promoter escape.’’ In addition to the structural changes described, the initiationelongation transition involves phosphorylation of the Pol II CTD. Elongationally competent polymerases show a phosphorylated CTD (Cadena and Dahmus, 1987; O’Brien et al., 1994) that adopts a far more extended structure than the unphosphorylated CTD (Corden and Zhang, 1991). There is a temporal relationship between CTD phosphorylation and the progression of Pol II through the transcription cycle (Dahmus, 1996). Both initiation and elongation are regulated by phosphorylation/dephosphorylation events (Greenblatt and Kobor, 2002). Several kinases (Prelich, 2002) and at least one phosphatase, Fcp1 (Kobor et al., 1999), control the phosphorylation state of the Pol II CTD. In addition, several general transcription factors and elongation factors are phosphoproteins (Greenblatt and Kobor, 2002). Five out of the seven amino acids in the CTD consensus repeat may in principle be phosphorylated. During initiation, the CTD is phosphorylated mainly at serine 5, a reaction catalyzed by the kinase Cdk7/Kin28 within TFIIH. Serine 5 phosphorylation is detected primarily at promoter regions and serine 2 is phosphorylated in coding regions (Komarnitsky et al., 2000), indicating that a change in the phosphorylation pattern accompanies the transition from initiation to elongation. This change apparently plays a role in the first RNA processing event, 50 -RNA capping (cf. section III.G). Substitution of serines 2 or 5 to alanine is lethal in yeast (Corden and West, 1995). In addition, changing tyrosine 1 to phenylalanine is lethal (Corden and West, 1995), indicating that tyrosine 1 is also a target for phosphorylation. Indeed, tyrosine 1 is phosphorylated in mammalian cells by the Abl kinase (Baskaran et al., 1997). The significance of this CTD modification is, however, unclear. The CTD is also a target for the modulation by peptidyl prolyl isomerases that catalyzes isomerization of prolines (Hunter, 1998; Shaw, 2002).

D. Elongation, Processivity, and Transcription Bubble Maintenance A functional model of the elongation complex was derived for bacterial RNA polymerase from biochemical data (Korzheva et al., 1998; Nudler, 1999). X-ray crystallographic data and site-specific protein–nucleic acid

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crosslinking provided a three-dimensional model of the bacterial RNA polymerase elongation complex (Korzheva et al., 2000). The location of nucleic acids in the Pol II elongation complex was modeled on the basis of electron crystallographic analysis (Cramer et al., 2000; Poglitsch et al., 1999) and site-specific polymerase–nucleic acid crosslinking data (Burgess and Wooddell, 2000). The later X-ray structure of a Pol II elongation complex allowed direct observation of the course of the nucleic acid strands in the DNA-RNA hybrid and of the DNA template strand just before the hybrid (Gnatt et al., 2001). The crystallized complex was formed by transcription of a DNA with a single-strand extension, a ‘‘tailed template,’’ in the presence of only three nucleoside triphosphates, leading to pausing at a discrete site (Gnatt, 2002; Gnatt et al., 1997; Gnatt et al., 2001). From these studies has emerged the following view of the elongation complex: Downstream DNA enters Pol II near two mobile ‘‘jaws’’ and extends through the cleft toward the active site. Beyond the active site, the DNA-RNA hybrid extends upward, toward the wall. The axis of the downstream DNA duplex and the DNA-RNA hybrid heteroduplex enclose an angle of almost 90 degrees. The growing RNA 30 -end is located above the pore, which allows entry of nucleoside triphosphates from below during RNA synthesis. In the crystal structure of the Pol II elongation complex, the incoming DNA duplex is mobile and badly ordered. However, three nucleotides before the active site, the DNA template strand becomes well ordered by binding to the bridge helix and to two ‘‘switch’’ regions at the base of the clamp, switches 1 and 2. A 90-degree twist between subsequent nucleotides orients a DNA base toward the active site for base pairing with an incoming RNA nucleotide. This base pair is the first of nine base pairs of the DNA-RNA hybrid that emanate from the active site. The hybrid length agrees with the length observed biochemically (Kireeva et al., 2000; Nudler et al., 1997). The DNA template strand within the hybrid is partly bound by switch region 3. The DNA nontemplate strand is disordered in the Pol II core elongation complex structure, maybe because the complex lacks the upstream DNA duplex and a complete bubble. The location of the nontemplate strand and the upstream DNA duplex during Pol II elongation is still unclear and may change during transcription. The property of the polymerase to stay attached to the template, even during transcription of long genes, is often referred to as processivity. The major cause of processivity is believed to be the high stability of the Pol II elongation complex. Elongation complex stability is caused by tight binding of the DNA-RNA hybrid to RNA polymerase (Kireeva et al., 2000; Sidorenkov et al., 1998). This stability can be accounted for by a highly complementary hybrid-binding site, in which the hybrid is imbedded. Enclosure of the hybrid results in protection of the RNA from digestion

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by RNAses (Komissarova and Kashlev, 1998). The complementary hybridbinding site is partially created upon clamp closure and folding of switches 1–3, which interact with the DNA template strand. Overextended hybrids have a negative effect on elongation complex stability (Kireeva et al., 2000). Interaction of the hybrid with its binding site ensures that the stability of the elongation complex, and thus processive transcription, is coupled with the presence of RNA. Because the Pol II core alone is sufficient to maintain the transcription bubble and the DNA-RNA hybrid during RNA chain elongation, there must be exposed elements on the enzyme surface that keep the nucleic acid strands apart. Protein elements are needed to separate the DNA strands downstream of the active site and to separate the RNA from the DNA template strand at the upstream end of the hybrid. On the basis of their location with respect to nucleic acids, several Pol II structural elements are predicted to maintain the bubble and the hybrid. These proposals are currently tested by site-directed mutagenesis. Separation of the DNA strands at the downstream edge of the bubble may be attributed to binding of the DNA template strand by switch regions 1 and 2 and to blocking of the path of the nontemplate strand by ‘‘fork loop 2.’’ In the Pol II-TFIIS complex structure, fork loop 2 is ordered and restricts the cleft to a diameter of 15 A˚ , consistent with the proposal that this loop removes the DNA nontemplate strand from the template strand before the active site. Maintenance of the upstream end of the hybrid and the bubble may involve three loops protruding from the edge of the clamp into the cleft. The two lower loops, called ‘‘rudder’’ and ‘‘lid,’’ are close to the upstream end of the hybrid. Mutagenesis of the rudder in bacterial RNA polymerase showed that this element stabilizes the elongation complex but that it is not involved in maintaining the hybrid length (Kuznedelov et al., 2002). The lid may be involved in separating RNA from DNA at the upstream end of the hybrid. The upper loop, called the ‘‘zipper,’’ could help maintain the upstream end of the transcription bubble. All three loops show some mobility and are present in all cellular RNA polymerases. The lid in bacterial polymerase interacts with the  factor (Murakami et al., 2002; Vassylyev, 2002), indicating that the lid in Pol II could contact a general transcription factor. The lid approaches another loop that protrudes from the opposite side, from the top of the wall (‘‘flap loop’’). The saddle, lip, and flap loop create a putative RNA exit tunnel. The flap loop in bacterial RNA polymerase binds to nascent RNA hairpins that pause or terminate transcription (Landick, 2001; Toulokhonov et al., 2001).

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E. Catalysis, Fidelity, Specificity, and Translocation The location of metal ions A and B is generally consistent with the geometry of substrate binding observed in the Pol II core elongation complex structure and in x-ray structures of nucleic acid complexes of single-subunit DNA polymerases (Doublie et al., 1998; Franklin et al., 2001; Pelletier et al., 1994; Sawaya et al., 1997), although the metal ions in the free enzyme may not be observed at the exact same location where they would be found during catalysis. On this basis, a working model for the nucleotide addition cycle during RNA chain elongation by Pol II was suggested. According to this model, the cycle starts with entry of the nucleoside triphosphate (NTP) substrate together with metal B, and its binding between the bridge helix and the end of the hybrid, to form a base pair with the ‘‘coding’’ DNA base. The NTP binding site of Pol II has not been defined, but it can be inferred from the site observed in structures of single-subunit DNA polymerases. Correct orientation of the substrates and metal ions would lead to synthesis of a new phosphodiester bond and to release of pyrophosphate, maybe together with metal ion B. The resulting complex adopts the pretranslocation state, which was apparently trapped in the core Pol II elongation complex structure, with the RNA 30 -terminal nucleotide occupying the NTP binding site (Gnatt et al., 2001). Subsequent translocation of nucleic acids would align the new RNA 30 end with metal A and would free the NTP binding site, preparing Pol II for another cycle of nucleotide addition. Fidelity of transcription may be defined as the property of Pol II that generally ensures incorporation of the correct nucleotide complementary to the base in the template strand. Fidelity must rely on correct positioning of the incoming NTP to optimize Watson–Crick base pairing between the NTP and the coding base in the DNA template strand, which together form the nascent base pair. Understanding the mechanistic basis for Pol II fidelity would require a structure of Pol II with bound DNA, RNA, and incoming NTP, which is currently not available. However, it is likely that fidelity relies in part on binding and positioning of the nascent base pair from the minor groove side, as observed in single-subunit polymerases (Chapter IV,C). Another important property of Pol II is its specificity for RNA synthesis rather than DNA synthesis. Specificity for synthesizing RNA may be achieved by at least three mechanisms. First, the discriminating 20 -OH group of the incoming NTP may be hydrogen-bonded by a conserved Pol II residue (Cramer et al., 2001; Gnatt et al., 2001). Second, 20 -OH groups of the last few nucleotides that were incorporated into the growing RNA are

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directly hydrogen-bonded by Pol II residues, such that accidentally incorporated deoxyribonucleotides would destabilize the elongation complex, resulting in a proofreading reaction (see following). Finally, the active center of Pol II is complementary to the resulting DNA-RNA hybrid duplex that adopts a specific conformation intermediary between canonical A-forms and B-forms. DNA synthesis would lead to canonical B-form DNA that would not fit into the hybrid binding site. Pol II apparently binds DNA and RNA tightly to create a stable and processive elongation complex. At the same time, Pol II allows for precise translocation of nucleic acids over its surface and moves along the DNA template with a considerable speed of several hundred nucleotides per minute. The question of how rapid translocation and tight nucleic acid binding can be achieved at the same time is a central mystery of the Pol II mechanism and of the mechanism of other nucleic acid metabolizing enzymes. Hints for understanding translocation are provided by the Pol II structures. First, nucleic acids are only contacted via their backbones, and base interactions that would impede translocation are not observed. Second, there are many positively charged protein groups that form a ‘‘second shell’’ around the nucleic acids, at a distance of up to 8 A˚ from the nucleic acid backbones. Such long-range electrostatic interactions may enable tight binding of nucleic acids without restricting their movement. Finally, translocation may be accompanied by conformational changes in Pol II regions around the nucleic acids. Such conformational changes could maintain some of the protein–nucleic acid contacts, resulting in a lowering of the energy barrier between pretranslation and posttranslocation states. One such conformational change may be bending of the bridge helix, as observed indirectly by a comparison of structures of Pol II and the bacterial RNA polymerase (Cramer et al., 2001; Darst, 2001; Gnatt et al., 2001). A corresponding ‘‘O-helix’’ in singlesubunit polymerases also stacks against the template-product nucleic acid duplex and can also change its conformation (Li et al., 1998). A highresolution structure of a bacterial RNA polymerase holoenzyme revealed a ‘‘trigger’’ loop that may cooperate with the bridge helix (Vassylyev et al., 2002). Indeed, a corresponding trigger loop in Pol II is mobile but becomes ordered on TFIIS binding (Kettenberger et al., 2003). In addition to the bridge helix, conformational changes in other Pol II structural elements may accompany translocation of nucleic acids, such as relative movements of mobile modules that surround incoming DNA (Cramer et al., 2001). Pol II elongation may be inhibited by binding of the cyclic octapeptide -amanitin, the toxin of the ‘‘death cap’’ mushroom. -amanitin does not greatly influence NTP binding (Chafin et al., 1995), and a phosphodiester

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bond can still be formed when the toxin is added to an elongation complex (Gu et al., 1993). However, the rate of transcription is dramatically reduced, such that only several nucleotides are incorporated per minute. These biochemical observations are consistent with the structure of a Pol II core--amanitin complex. In this structure, -amanitin is seen binding to the bridge helix from below. Thus, -amanitin cannot interfere with access of nucleic acids to the cleft or with entry of NTP substrates through the pore. Instead, -amanitin could possibly restrain the bridge helix movement and thereby block conformational changes that are important for translocation. However, given the speculative nature of the conformational change of the bridge helix during translocation, understanding the exact mechanism of Pol II inhibition by -amanitin requires further study. The binding sites for -amanitin and domain III of TFIIS overlap, explaining why the toxin interferes with TFIIS activity (Izban and Luse, 1992; Weilbaecher et al., 2003).

F. Backtracking, Pausing, Arrest, and Proofreading Pol II does not move along the DNA template in a unidirectional manner. The polymerase, rather, oscillates between forward and backward movements. Reverse movement of Pol II along DNA and RNA is referred to as ‘‘backtracking.’’ As a result of backtracking, RNA polymerase elongation complexes can adopt different conformational states (Erie, 2002). Oscillation back and forth along DNA and RNA was demonstrated for bacterial RNA polymerase (Kashlev and Komissarova, 1997a). Oscillatory movement of the polymerase can explain DNA and RNA footprints that are irregular in length. Shorter and longer footprints are apparently reflections of mixed populations of the elongation complex in productive and backtracked states. Before the concept of oscillatory movement, the irregular footprints were interpreted as an ‘‘inchworming’’ motion of the polymerase, with the enzyme contracting and expanding along the template. Inchworming requires independent movement of two flexibly linked parts of Pol II and is, thus, inconsistant with the Pol II structure. During backtracking, a Pol II structural element must keep the two DNA strands at the upstream end of the bubble separated, but it is unclear which Pol II element this is. The bridge helix apparently removes the RNA 30 -end from the DNA template strand during backtracking. The backtracked RNA is apparently extruded through the pore into the funnel. Backtracking of Pol II during the elongation phase can lead to transcriptional pausing and arrest. Pausing and arrest are blocks to transcription that

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can be signaled intrinsically, by certain DNA sequences, or extrinsically, through additional protein factors (Uptain et al., 1997). Pausing is defined as a temporary block to elongation, from which Pol II can escape by itself, without the need for accessory factors. Pol II that has paused at certain DNA sites has generally backtracked by several nucleotides. The DNA-RNA hybrid normally prevents backtracking of Pol II and maintains the register of transcription (Nudler et al., 1997). However, destabilization of the hybrid at specific DNA sites leads to backtracking (Nudler et al., 1997) and appears to be the primary determinant for pausing (Landick and Palangat, 2001). Mutations that affect pausing are found in homology block F of the largest Pol II subunit (Thuillier et al., 1996), a region that lines the funnel into which RNA is extruded during backtracking. A single-molecule study showed that pausing is a reversible intermediary state between arrest and normal elongation (Davenport et al., 2000). Single-molecule analysis further revealed uniform elongation kinetics, but differences in the frequency and duration of pausing (Adelman et al., 2002). In addition to pausing that involves backtracking, another type of pausing has been observed for the bacterial polymerase, in which the RNA 30 -end disengages from the active site by hypertranslocation of the enzyme (Artsimovitch and Landick, 2000). During transcriptional arrest, RNA polymerase also translocates backwards, leaving the RNA 30 -end intact and extruded (Kashlev and Komissarova, 1997b). RNA polymerase cannot be rescued from arrest by mechanical force (Forde et al., 2002). The need for backtracking and extrusion of the RNA explains why blocking translocation is not sufficient to cause arrest (Samkurashvili and Luse, 1996). Arrest goes along with an increased accomodation of RNA in Pol II (Gu et al., 1996). During normal elongation, about 18 nucleotides of RNA are protected from ribonuclease cleavage, whereas at an arrest site up to 27 nucleotides are protected. The major difference between transcriptional pausing and arrest is that arrested Pol II, in contrast to the paused enzyme, cannot escape without transcript cleavage and the help of an extrinsic protein, the transcript cleavage factor TFIIS. Pol II has a weak intrinsic 30 ! 50 nuclease activity that is greatly stimulated by TFIIS. Bacterial RNA polymerase has been shown to have an intrinsic transcript cleavage activity (Orlova et al., 1995). In the presence of TFIIS, Pol II can cleave the RNA from its 30 -end primarily in dinucleotide increments, although mononucleotides and longer oligonucleotides are also observed (Gu and Reines, 1995; Izban and Luse, 1992, 1993a, 1993b; Hawley and Wang, 1993). Dinucleotides and 7–9-mer oligonucleotides are released from paused and arrested complexes, respectively (Gu and Reines, 1995), showing that arrest involves more extensive backtracking than pausing. TFIIS contacts the 30 -end of the RNA in the Pol II elongation complex (Powell et al., 1996).

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The recent Pol II-TFIIS structure provides a detailed picture of how TFIIS gains access to the Pol II active center via the funnel and pore from below and how TFIIS complements the active site with two functionally essential acidic residues (Kettenberger et al., 2003). TFIIS, however, does not fill the entire pore, it only restricts it. There is enough space for simultaneous binding of backtracked RNA and TFIIS in the pore, as required during TFIIS-mediated rescue of an arrested Pol II elongation complex. Structural and biochemical data indicate that the mechanism of TFIIS-induced RNA cleavage by Pol II involves positioning and activation of a nucleophilic water molecule with the help of a metal ion, to allow for an in-line attack of the phosphodiester bond to be cleaved (Sosunov et al., 2003; Kettenberger et al., 2003). In vitro, TFIIS can also stimulate ‘‘proofreading’’ of the nascent transcript (Agarwal and Jeon, 1996; Thomas et al., 1998), an activity that removes incorrectly incorporated nucleotides from the growing RNA. The following view of the mechanism of mRNA proofreading has emerged from many biochemical observations and the available Pol II structures. Incorporation of the correct nucleotide drives rapid forward translocation (Nedialkov et al., 2003). However, misincorporation of a nucleotide leads to slow forward translocation (Thomas et al., 1998), opening a time window for hydrolytic RNA cleavage and removal of the misincorporated nucleotide. Because a misincorporated nucleotide and the resulting mismatch base pair destabilizes the DNA-RNA hybrid and the elongation complex, misincorporation can also trigger backtracking (Nudler et al., 1997). Backtracking by one nucleotide would lead to cleavage of an RNA dinucleotide. Cleavage of mononucleotides (from the pretranslocation state) and of dinucleotides (from a backtracked state) would both result in a new RNA 30 -end at the active site, from which polymerization can continue. The Pol II-TFIIS complex structure provides evidence that Pol II contains a single tunable active site for both RNA polymerization and cleavage/proofreading, instead of two catalytic sites with distinct locations (Kettenberger et al., 2003). It had been suggested previously that the active sites for RNA polymerization and cleavage are close together or even identical (Powell et al., 1996; Rudd et al., 1994; Hawley and Wang, 1993). In addition to TFIIS, several other proteins influence Pol II elongation, pausing, and arrest, and some of these factors are involved in disease (Conaway and Conaway, 1999; Shilatifard, 1998a, 1998b). Deregulation of Pol II elongation can lead to certain types of cancer (Groudine and Krumm, 1995).

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G. Coupling to RNA Processing and to Other Nuclear Events In addition to the Pol II machinery, expression of protein-coding genes requires multicomponent machines in the nucleus that carry out various steps of mRNA processing, RNA export, and RNA surveillance. Over the last years, a large number of experimental observations showed that there is extensive coupling between these nuclear gene expression machines (Bentley, 2002; Hirose and Manley, 2000; Maniatis and Reed, 2002; Orphanides and Reinberg, 2002). The physical basis for coupling between transcription and mRNA processing appears to be the interaction of RNA processing factors with the phosphorylated Pol II CTD (Hirose and Manley, 2000; Proudfoot, 2000; Steinmetz, 1997). The CTD is flexibly linked to a region beyond the saddle, from which RNA exits, consistent with its role in coupling transcription to mRNA processing (Cramer et al., 2001). There is a tight coupling between transcription and the first RNA processing event, 50 -RNA capping. Capping occurs already when the nascent RNA has reached a length of 25–30 nucleotides and, thus, must take place near the Pol II surface. There is accumulating evidence for the existence of an early elongation checkpoint that ensures that the nascent RNA has received its 50 -cap structure that protects it from degradation (Orphanides and Reinberg, 2002). Other RNA processing events, splicing and 30 -end formation, also occur in a transcription-coupled manner. Pol II transcription elongation is further coupled to events of chromatin remodeling and modification (Orphanides and Reinberg, 2000). Recently it was also found that Pol II elongation is coupled to the export of mRNA out of the nucleus (Hammell et al., 2002; Strasser et al., 2002). Taken together, it now appears that Pol II stands at the heart of a giant mRNA factory that comprises several coupled multicomponent machines (Cook, 1999; Sawadogo and Szentirmay, 2000). Details of these coupling phenomena are beyond the scope of this review.

H. Termination, Polymerase Recycling, Reinitiation, and Regulation Transcription termination occurs in a reaction coupled to RNA 30 -end processing. Most eukaryotic mRNA precursors are cleaved in a site-specific manner in the 30 -untranslated region followed by polyadenylation of the upstream cleavage product. A large number of proteins is involved in these reactions, which are beyond the scope of this review (Barabino and Keller, 1999; Proudfoot et al., 2002; Manley and Shatkin, 2000). The exact mechanism of coupling between 30 -end processing and transcription termination remains unclear. Termination goes along with dephosphorylation of the Pol II CTD, but the exact time point of Pol II dephosphorylation is also unclear. Dephosphorylation is required for

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the reinitiation of transcription, as Pol II can only join an initiation complex in its unphosphorylated form. The CTD phosphatase Fcp1 plays a key role in Pol II dephosphorylation and recycling (Cho et al., 1999; Kobor et al., 1999). Fcp 1 binds to Pol II via Rpb4 (Kimura et al., 2002). Rpb4 apparently recruits Fcp 1 to the vicinity of the CTD, as the Rpb4/7 complex binds to the beginning of the linker that connects the Pol II core to the CTD (Armache et al., 2003; Bushnell and Kornberg, 2003; Craighead et al., 2002). Reinitiation of Pol II transcription apparently occurs by a mechanism different from initiation (Hahn, 1998). After initiation, a subset of the transcription machinery remains at the promoter, forming a scaffold for assembly of a new initiation complex. This scaffold comprises TFIIA, TFIID, TFIIE, TFIIH, and the multisubunit Mediator complex and can be stabilized by transcriptional activators (Yudkovsky et al., 2000). Reinitiation as well as initiation are important targets for Pol II regulation. High levels of transcription may rely on rapid initiation and on reinititation of polymerases that have terminated. The transcription elongation phase is also subject to regulation, and Pol II elongation can be stimulated by transcriptional activators (Yankulov et al., 1994). The many levels of Pol II regulation befit the central role of Pol II as the end point of signal transduction pathways. In higher eukaryotes, hundreds of transcription factors use Pol II as a regulatory target to induce changes in gene expression. These regulatory proteins generally affect Pol II indirectly, via so-called coactivator complexes, which include the generally required and conserved Mediator complex. The Mediator complex can physically bridge between Pol II and transcriptional activator and repressor proteins. A recent electron microscopic reconstruction of the Pol II–Mediator complex revealed that the interface between Mediator and Pol II includes the polymerase subunit Rpb3 (Davis et al., 2002). Interestingly, two amino acid substitutions on the Rpb3 surface cause a defect in activated transcription (Tan et al., 2000a). Bacterial RNA polymerase contains a target for transcription activation at a similar location on the enzyme surface (Ebright, 2000; Tan et al., 2000b). Mediator is the subject of another review in this volume.

IV. Comparison with Other Polymerases A. Eukaryotic RNA Polymerases I and III Pol II belongs to the family of multisubunit RNA polymerases, which also comprises the two other eukaryotic RNA polymerases, Pol I and Pol III. Pol I and Pol III are mainly responsible for synthesis of ribosomal RNA

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and transfer RNA, respectively. All three eukaryotic RNA polymerases share the five common subunits Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12 (Table II). Four core subunits of Pol II, Rpb1, Rpb2, Rpb3, and Rpb11, all have close homologues in Pol I and Pol III (Table II). The largest subunits of Pol I and Pol III, however, lack a C-terminal repeat domain. Recent studies show that the Rpb4/7 complex of Pol II also has structural and functional counterparts in Pol I and Pol III (Hu et al., 2002; Peyroche et al., 2002; Sadhale and Woychik, 1994; Shematorova and Shpakovski, 1999) and in the archaeal RNA polymerase (Werner et al., 2000). Indeed, the Pol II core-Rpb7 interaction is apparently conserved in all eukaryotic and archaeal RNA polymerases, but not in the bacterial enzyme (Kettenberger et al., 2003). In conclusion, the 12 subunits of Pol II are either identical or homologous in all three eukaryotic enzymes, and Pol II is thus a good model for all eukaryotic RNA polymerases. There are, however, minor differences on the enzymes’ surfaces caused by amino acid insertions and deletions. These differences are most likely responsible for conferring specificity toward the interaction with factors specific for Pol I, II, and III. In addition to the 12 subunits that are either identical or homologous, Pol I contains two specific subunits, A34.5 and A49, and Pol III contains a subcomplex of three specific subunits, called C82, C34, and C31, in yeast. The location of the two Pol I–specific subunits has been determined by electron microscopy and immunolabeling (Bischler et al., 2002). The Pol I subunit A49 binds to the top of the clamp, and subunit A34.5 is located near the jaws. The location of the specific C82/C34/C31 complex of Pol III can be inferred from subunit– subunit interaction studies (Ferri et al., 2000; Flores et al., 1999). These studies indicate that the specific subcomplex is located between the largest polymerase subunit and the Rpb4/7 complex counterpart C17/C25. The C11 subunit of Pol III contains a C-terminal domain that apparently corresponds structurally and functionally to domain III of TFIIS (Chedin et al., 1998; Kettenberger et al., 2003), which inserts into the polymerase pore. Thus, in Pol III, the RNA cleavage stimulatory activity is incorporated into a polymerase subunit, in contrast to Pol II, where it is provided by the additional factor TFIIS.

B. Bacterial and Archaeal RNA Polymerases Bacteria and archaea contain a single multisubunit RNA polymerase. X-ray crystallographic structures were determined of a bacterial RNA polymerase from Thermus aquaticus at 3.3-A˚ resolution (Darst, 2001; Zhang et al., 1999). Comparison of this bacterial RNA polymerase structure

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with the structure of yeast Pol II revealed that five ‘‘core’’ subunits underlie a general RNA polymerase architecture with an active center cleft (Cramer, 2002b). The core subunits show a total of 22 regions of sequence homology ( Jokerst et al., 1989; Minakhin et al., 2001; Sweetser et al., 1987; Darst and Zhang, 1998). These homology regions cluster around the active site and generally adopt the same structure in the bacterial and yeast RNA polymerases. Many additional regions share the same structure, although they differ in sequence between the bacterial and yeast polymerases. Thus structure is conserved better than sequence. The structurally conserved core includes the functional elements of the active center, indicating that all multisubunit RNA polymerases share common mechanistic features. Bacteria do not have a homolog of TFIIS, but the transcript cleavage factors GreA and GreB appear to function essentially like TFIIS, as revealed in an electron microscopic study recently (Opalka et al., 2003). A coiled coil of GreB binds in the secondary channel of bacterial polymerase, which corresponds to the Pol II pore, and reaches the active site with an acidic tip. These findings demonstrate in a powerful way the conserved strategies employed for RNA cleavage stimulation by the structurally unrelated bacterial and eukaryotic RNA polymerase cleavage factors. Bacterial RNA polymerase consists of the five core subunits only. In eukaryotic RNA polymerases, up to 10 additional subunits are found around the periphery of the enzymes (Table II). Archeael RNA polymerases comprise between five and seven subunits in addition to the five core subunits (Darcy et al., 1999; Langer et al., 1995). For all Pol II subunits except Rpb8, homologues have been reported in archaeal RNA polymerases. Thus, the overall structure of archaeal RNA polymerases must be very similar to the yeast Pol II structure. Although the archaeal enzymes lack some external domains, they are apparently closely related to the eukaryotic Pol II. The similarity between archaeal RNA polymerases and Pol II extends to the initiation complex. Archaea contain homologues of three Pol II general transcription factors, TBP, TFIIB, and TFIIE (Bell and Jackson, 2001). The archaeal RNA polymerase machinery is thus more closely related to the eukaryotic machinery than to the bacterial system. Indeed, an archaeal TFIIS homologue, TFS, has also been described (Hausner et al., 2000).

C. Single-Subunit DNA and RNA Polymerases Structures of multisubunit RNA polymerases are strikingly different from structures of polymerases of other families, such as the many single-subunit DNA and RNA polymerases. X-ray crystallography of

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single-subunit DNA and RNA polymerases revealed a great structural diversity (Beard and Wilson, 2001; Brautigam and Steitz, 1998; Doublie et al., 1999; Ellenberger and Silvian, 2001; Jager and Pata, 1999; Loeb and Patel, 2001; Steitz, 1999). Nevertheless, most single-subunit polymerases show a similar overall architecture and considerable structural conservation of the active center. Representative structures of the diverse singlesubunit polymerases were compared to Pol II by overlaying corresponding nucleic acids in functional complexes (Cramer, 2002a). In functional complexes of these diverse enzymes, nucleic acids take a similar course through the active center. In all cases studied, the entering DNA duplex encloses an angle of almost 90 degrees with the exiting template-product duplex. At the location of the bend, subsequent DNA template bases are twisted. This twist aligns the ‘‘coding’’ base with the binding site for the incoming nucleoside triphosphate substrate. The nucleoside triphosphate enters through an opening that is found in all polymerases. The nucleotide substrate often binds between an -helix and two catalytic metal ions. The exiting template-product duplex is bound from the minor groove side in all polymerases. Conformational changes on nucleic acid binding have been detected for several different polymerases, but the nature of this ‘‘induced fit’’ differs. Recent structures of elongation complexes of RNA polymerase from bacteriophage T7 have revealed dramatic changes in the conformation of the N-terminal domain on transition from initiation to elongation (Tahirov et al., 2002; Yin and Steitz, 2002). In the Pol II system, corresponding changes remain to be discovered but may be predominantly found in the general transcription factors rather than the polymerase itself. Structural and functional analysis of Pol II supports the idea that DNA and RNA polymerases follow different strategies for nucleic acid cleavage and proofreading. In the Klenow DNA polymerase, the growing DNA shuttles between widely separated active sites for DNA synthesis and cleavage, whereas in Pol II the growing RNA appears to remain at a single tunable active site that switches between RNA synthesis and cleavage modes, with the latter being dramatically enhanced by TFIIS. Despite this difference in strategy, both classes of polymerases may use the same general two–metal ion mechanism for both polymerization and cleavage of nucleic acids.

VI. Conclusions Detailed structures are now available for the Pol II core enzyme in free form, in the form of a minimal elongation complex with bound nucleic acids, and in an inhibited form with bound -amanitin. In addition,

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backbone models of the complete initiation-competent Pol II, including the Rpb4/7 complex, and of the complete Pol II with bound elongation factor TFIIS have recently been described. The structures together with many functional studies have given many insights into the mechanism of mRNA transcription. Structural and functional studies of bacterial RNA polymerase allow for interesting comparisons and evolutionary considerations. The Pol II structures now guide mutagenesis experiments aimed at a dissection of the transcription mechanism. In the future, further structures of Pol II complexes with transcription factors will provide more mechanistic details of the mRNA transcription cycle.

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