Structure of RNA polymerase I transcribing ribosomal DNA genes.

RNA polymerase I (Pol I) is a highly processive enzyme that transcribes ribosomal DNA (rDNA) and regulates growth of eukaryotic cells. Crystal structures of free Pol I from the yeast Saccharomyces cerevisiae have revealed dimers of the enzyme stabilized by a 'connector' element and an expanded cleft containing the active centre in an inactive conformation. The central bridge helix was unfolded and a Pol-I-specific 'expander' element occupied the DNA-template-binding site. The structure of Pol I in its active transcribing conformation has yet to be determined, whereas structures of Pol II and Pol III have been solved with bound DNA template and RNA transcript. Here we report structures of active transcribing Pol I from yeast solved by two different cryo-electron microscopy approaches. A single-particle structure at 3.8 Å resolution reveals a contracted active centre cleft with bound DNA and RNA, and a narrowed pore beneath the active site that no longer holds the RNA-cleavage-stimulating domain of subunit A12.2. A structure at 29 Å resolution that was determined from cryo-electron tomograms of Pol I enzymes transcribing cellular rDNA confirms contraction of the cleft and reveals that incoming and exiting rDNA enclose an angle of around 150°. The structures suggest a model for the regulation of transcription elongation in which contracted and expanded polymerase conformations are associated with active and inactive states, respectively.

The Structural Basis of Transcription: 10†Years After the Nobel Prize in Chemistry.

Transcription is the first step in the expression of genetic information in all living cells. The regulation of transcription underlies cell differentiation, organism development, and the responses of living systems to changes in the environment. During transcription, the enzyme RNA polymerase uses DNA as a template to synthesize a complementary RNA copy from a gene. Herein, we summarize the progress in our understanding of the structural basis of eukaryotic gene transcription that has been made in the ten years since the Nobel Prize in Chemistry was given to Roger Kornberg in 2006. The basis for transcription initiation and RNA chain elongation is emerging, but the intricate mechanisms of transcription regulation remain to be elucidated. The field has also developed hybrid methods for structural biology that combine several techniques to determine the three-dimensional architecture of large and transient macromolecular assemblies.

Conserved RNA polymerase II initiation complex structure.

Recent cryo-electron microscopic studies have arrived at atomic models of the core transcription initiation complex comprising RNA polymerase (Pol) II and the basal transcription factors TBP, TFIIA, TFIIB, TFIIE, and TFIIF. A detailed comparison of two independently derived yeast and human core initiation complex structures reveals that they are virtually identical, demonstrating the conservation of the basic transcription machinery amongst eukaryotes. The additional factors TFIID, TFIIH, and Mediator have been located on the periphery of the core initiation complex, providing the topology of the entire initiation assembly, which comprises approximately 70 polypeptides with a molecular weight of ∼4 Megadalton.

Conserved architecture of the core RNA polymerase II initiation complex.

During transcription initiation at promoters of protein-coding genes, RNA polymerase (Pol) II assembles with TBP, TFIIB and TFIIF into a conserved core initiation complex that recruits additional factors. The core complex stabilizes open DNA and initiates RNA synthesis, and it is conserved in the Pol I and Pol III transcription systems. Here, we derive the domain architecture of the yeast core pol II initiation complex during transcription initiation. The yeast complex resembles the human initiation complex and reveals that the TFIIF Tfg2 winged helix domain swings over promoter DNA. An 'arm' and a 'charged helix' in TFIIF function in transcription start site selection and initial RNA synthesis, respectively, and apparently extend into the active centre cleft. Our model provides the basis for further structure-function analysis of the entire transcription initiation complex.