The eukaryotic RNA polymerases Pol I, Pol II, and Pol III are the central multiprotein machines that synthesize ribosomal, messenger, and transfer RNA, respectively. Here we provide a catalog of available structural information for these three enzymes. Most structural data have been accumulated for Pol II and its functional complexes. These studies have provided insights into many aspects of the transcription mechanism, including initiation at promoter DNA, elongation of the mRNA chain, tunability of the polymerase active site, which supports RNA synthesis and cleavage, and the response of Pol II to DNA lesions. Detailed structural studies of Pol I and Pol III were reported recently and showed that the active center region and core enzymes are similar to Pol II and that strong structural differences on the surfaces account for gene class-specific functions.

We show that RNA polymerase (Pol) II prevents erroneous transcription in vitro with different strategies that depend on the type of DNARNA base mismatch. Certain mismatches are efficiently formed but impair RNA extension. Other mismatches allow for RNA extension but are inefficiently formed and efficiently proofread by RNA cleavage. X-ray analysis reveals that a TU mismatch impairs RNA extension by forming a wobble base pair at the Pol II active center that dissociates the catalytic metal ion and misaligns the RNA 3' end. The mismatch can also stabilize a paused state of Pol II with a frayed RNA 3' nucleotide. The frayed nucleotide binds in the Pol II pore either parallel or perpendicular to the DNA-RNA hybrid axis (fraying sites I and II, respectively) and overlaps the nucleoside triphosphate (NTP) site, explaining how it halts transcription during proofreading, before backtracking and RNA cleavage.

Spt5 is the only known RNA polymerase-associated factor that is conserved in all three domains of life. We have solved the structure of the Methanococcus jannaschii Spt4/5 complex by X-ray crystallography, and characterized its function and interaction with the archaeal RNAP in a wholly recombinant in vitro transcription system. Archaeal Spt4 and Spt5 form a stable complex that associates with RNAP independently of the DNA–RNA scaffold of the elongation complex. The association of Spt4/5 with RNAP results in a stimulation of transcription processivity, both in the absence and the presence of the non-template strand. A domain deletion analysis reveals the molecular anatomy of Spt4/5—the Spt5 Nus-G N-terminal (NGN) domain is the effector domain of the complex that both mediates the interaction with RNAP and is essential for its elongation activity. Using a mutagenesis approach, we have identified a hydrophobic pocket on the Spt5 NGN domain as binding site for RNAP, and reciprocally the RNAP clamp coiled-coil motif as binding site for Spt4/5.

The anticancer drug cisplatin forms 1,2-d(GpG) DNA intrastrand cross-links (cisplatin lesions) that stall RNA polymerase II (Pol II) and trigger transcription-coupled DNA repair. Here we present a structure-function analysis of Pol II stalling at a cisplatin lesion in the DNA template. Pol II stalling results from a translocation barrier that prevents delivery of the lesion to the active site. AMP misincorporation occurs at the barrier and also at an abasic site, suggesting that it arises from nontemplated synthesis according to an 'A-rule' known for DNA polymerases. Pol II can bypass a cisplatin lesion that is artificially placed beyond the translocation barrier, even in the presence of a G . A mismatch. Thus, the barrier prevents transcriptional mutagenesis. The stalling mechanism differs from that of Pol II stalling at a photolesion, which involves delivery of the lesion to the active site and lesion-templated misincorporation that blocks transcription.Cisplatin (cis-diamminedichloroplatinum(II)) is a widely used anticancer drug that forms DNA adducts that interfere with replication and transcription 1 . The most frequent cisplatin DNA adducts are 1,2-d(GpG) intrastrand cross-links, or cisplatin lesions, in which platinum coordinates the N7 atoms of adjacent guanosines in a DNA strand 2 (Fig. 1a). A cisplatin lesion in the DNA template strand blocks transcription elongation by the single-subunit RNA polymerase from phage T7 (ref.3) and by Pol II 4-6 , and leads to stable polymerase stalling 7 . The stalled Pol II elongation complex can be bound by the elongation factor TFIIS, which stimulates polymerase back-tracking and 3¢ RNA cleavage 6 . A small fraction of polymerases can apparently read through a cisplatin lesion 6 .The detailed molecular mechanisms of cisplatin DNA adduct processing by nucleic acid polymerases are not understood. Here we have used a combination of X-ray crystallography and RNA-extension assays to derive the molecular mechanism of Saccharomyces cerevisiae Pol II stalling at a cisplatin lesion. Comparison of the results with our previous analysis of Pol II stalling at a DNA photolesion, a TT cyclobutane pyrimidine dimer 8 (CPD, Fig. 1a), reveals that the two types of dinucleotide lesions trigger transcriptional stalling by different mechanisms. RESULTS Structure of a cisplatin-damaged Pol II elongation complexTo elucidate recognition of cisplatin-induced DNA damage by transcribing Pol II, we carried out a structure-function analysis of elongation complexes containing a cisplatin lesion in the template DNA strand. Elongation complexes were reconstituted from the 12-subunit S. cerevisiae Pol II and nucleic acid scaffolds as described 8,9 .A cisplatin lesion was first incorporated at registers +2/+3 of the template strand, directly downstream of the NTP-binding site at register +1 (scaffold A, Fig. 1b). The crystal structure of the resulting cisplatin-damaged elongation complex (complex A) was determined at 3.8-Å resolution (Fig. 1c,d and Methods). A very strong peak in the anomalous differe...

Structure-function analysis has revealed the mechanism of yeast RNA polymerase II transcription at 8-oxoguanine (8-oxoG), the major DNA lesion resulting from oxidative stress. When polymerase II encounters 8-oxoG in the DNA template strand, it can misincorporate adenine, which forms a Hoogsteen bp with 8-oxoG at the active center. This requires rotation of the 8-oxoG base from the standard anti- to an uncommon syn-conformation, which likely occurs during 8-oxoG loading into the active site. The misincorporated adenine escapes intrinsic proofreading, resulting in transcriptional mutagenesis that is observed directly by mass spectrometric RNA analysis.

ChitO (chito-oligosaccharide oxidase) from Fusarium graminearum catalyses the regioselective oxidation of N-acetylated oligosaccharides. The enzyme harbours an FAD cofactor that is covalently attached to His94 and Cys154. The functional role of this unusual bi-covalent flavin-protein linkage was studied by site-directed mutagenesis. The double mutant (H94A/C154A) was not expressed, which suggests that a covalent flavin-protein bond is needed for protein stability. The single mutants H94A and C154A were expressed as FAD-containing enzymes in which one of the covalent FAD-protein bonds was disrupted relative to the wild-type enzyme. Both mutants were poorly active, as the k(cat) decreased (8.3- and 3-fold respectively) and the K(m) increased drastically (34- and 75-fold respectively) when using GlcNac as the substrate. Pre-steady-state analysis revealed that the rate of reduction in the mutant enzymes is decreased by 3 orders of magnitude when compared with wild-type ChitO (k(red)=750 s(-1)) and thereby limits the turnover rate. Spectroelectrochemical titrations revealed that wild-type ChitO exhibits a relatively high redox potential (+131 mV) and the C154A mutant displays a lower potential (+70 mV), while the H94A mutant displays a relatively high potential of approximately +164 mV. The results show that a high redox potential is not the only prerequisite to ensure efficient catalysis and that removal of either of the covalent bonds may perturb the geometry of the Michaelis complex. Besides tuning the redox properties, the bi-covalent binding of the FAD cofactor in ChitO is essential for a catalytically competent conformation of the active site.

RNA polymerase II (Pol II) is the eukaryotic enzyme that is responsible for transcribing all protein-coding genes into messenger RNA (mRNA). The mRNA-transcription cycle can be divided into three stages: initiation, elongation and termination. During elongation, Pol II moves along a DNA template and synthesizes a complementary RNA chain in a processive manner. X-ray structural analysis has proved to be a potent tool for elucidating the mechanism of Pol II elongation. Crystallographic snapshots of different functional states of the Pol II elongation complex (EC) have elucidated mechanistic details of nucleotide addition and Pol II translocation. Further structural studies in combination with in vitro transcription experiments led to a mechanistic understanding of various additional features of the EC, including its inhibition by the fungal toxin -amanitin, the tunability of the active site by the elongation factor TFIIS, the recognition of DNA lesions and the use of RNA as a template.

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