The crystal structure of RNA polymerase II in the act of transcription was determined at 3.3 Å resolution. Duplex DNA is seen entering the main cleft of the enzyme and unwinding before the active site. Nine base pairs of DNA-RNA hybrid extend from the active center at nearly right angles to the entering DNA, with the 3′ end of the RNA in the nucleotide addition site. The 3′ end is positioned above a pore, through which nucleotides may enter and through which RNA may be extruded during back-tracking. The 5′-most residue of the RNA is close to the point of entry to an exit groove. Changes in protein structure between the transcribing complex and free enzyme include closure of a clamp over the DNA and RNA and ordering of a series of “switches” at the base of the clamp to create a binding site complementary to the DNA-RNA hybrid. Protein–nucleic acid contacts help explain DNA and RNA strand separation, the specificity of RNA synthesis, “abortive cycling” during transcription initiation, and RNA and DNA translocation during transcription elongation.

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Summary Most human genes are loaded with promoter proximally paused RNA polymerase II (Pol II) molecules that are poised for release into productive elongation by P-TEFb. We present evidence that Gdown1, a protein that renders Pol II responsive to mediator, is involved in Pol II elongation control. During in vitro transcription assays Gdown1 specifically blocked elongation stimulation by TFIIF, inhibited the termination activity of TTF2, and influenced pausing factors NELF and DSIF, but did not affect the function of TFIIS or the mRNA capping enzyme. Without P-TEFb, Gdown1 led to the production of stably paused polymerases in the presence of nuclear extract. Supporting these mechanistic insights, ChIP-Seq demonstrated that Gdown1 mapped over essentially all poised polymerases across the human genome. Our results establish that Gdown1 increases the stability of poised polymerases while maintaining their responsiveness to P-TEFb and suggest that mediator overcomes a Gdown1-mediated block of initiation by allowing TFIIF function.

Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by off target effects. Development of antiviral ribonucleosides for treatment of hepatitis C virus (HCV) infection has been hampered by appearance of toxicity during clinical trials that evaded detection during preclinical studies. It is well established that the human mitochondrial DNA polymerase is an off target for deoxyribonucleoside reverse transcriptase inhibitors. Here we test the hypothesis that triphosphorylated metabolites of therapeutic ribonucleoside analogues are substrates for cellular RNA polymerases. We have used ribonucleoside analogues with activity against HCV as model compounds for therapeutic ribonucleosides. We have included ribonucleoside analogues containing 2′-C-methyl, 4′-methyl and 4′-azido substituents that are non-obligate chain terminators of the HCV RNA polymerase. We show that all of the anti-HCV ribonucleoside analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Unexpectedly, analogues containing 2′-C-methyl, 4′-methyl and 4′-azido substituents were inhibitors of POLRMT and Pol II. Importantly, the proofreading activity of TFIIS was capable of excising these analogues from Pol II transcripts. Evaluation of transcription in cells confirmed sensitivity of POLRMT to antiviral ribonucleosides, while Pol II remained predominantly refractory. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity.

RNA polymerase II (Pol II), whose 12 subunits are conserved across eukaryotes, is at the heart of the machinery responsible for transcription of mRNA. Although associated general transcription factors impart promoter specificity, responsiveness to gene-and tissue-selective activators additionally depends on the multiprotein Mediator coactivator complex. We have isolated from tissue extracts a distinct and abundant mammalian Pol II subpopulation that contains an additional tightly associated polypeptide, Gdown1. Our results establish that Gdown1-containing Pol II, designated Pol II(G), is selectively dependent on and responsive to Mediator. Thus, in an in vitro assay with general transcription factors, Pol II lacking Gdown1 displays unfettered levels of activator-dependent transcription in the presence or absence of Mediator. In contrast, Pol II(G) is dramatically less efficient in responding to activators in the absence of Mediator yet is highly and efficiently responsive to activators in the presence of Mediator. Our results reveal a transcriptional control mechanism in which Mediatordependent regulation is enforced by means of Gdown1, which likely restricts Pol II function only to be reversed by Mediator.activator ͉ transcription ͉ repression ͉ negative regulator