Structural basis of archaeal RNA polymerase transcription elongation and Spt4/5 recruitment.

Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, only Spt5 is found in all domains of life, but Spt5 has been shaped during evolution, which is also reflected in the heterodimerization of Spt5 with Spt4 in Archaea and Eukaryotes. To unravel the mechanistic basis of Spt4/5 function in Archaea, we performed structure-function analyses using the archaeal transcriptional machinery of Pyrococcus furiosus (Pfu).

The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans.

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth, and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk, and in vitro transcription indicates a reduced proofreading activity.

Preparation of RNA Polymerase Complexes for Their Analysis by Single-Particle Cryo-Electron Microscopy.

Recent technological progress revealed new prospects of high-resolution structure determination of macromolecular complexes using cryo-electron microscopy (cryo-EM) . In the field of RNA polymerase (Pol) I research, a number of cryo-EM studies contributed to understanding the highly specialized mechanisms underlying the transcription of ribosomal RNA genes . Despite a broad applicability of the cryo-EM method itself, preparation of samples for high-resolution data collection can be challenging.

Structural Studies of Eukaryotic RNA Polymerase I Using Cryo-Electron Microscopy.

Technical advances have pushed the resolution limit of single-particle cryo-electron microscopy (cryo-EM) throughout the past decade and made the technique accessible to a wide range of samples. Among them, multisubunit DNA-dependent RNA polymerases (Pols) are a prominent example. This review aims at briefly summarizing the architecture and structural adaptations of Pol I, highlighting the importance of cryo-electron microscopy in determining the structures of transcription complexes.

Specialization of RNA Polymerase I in Comparison to Other Nuclear RNA Polymerases of Saccharomyces cerevisiae.

In archaea and bacteria the major classes of RNAs are synthesized by one DNA-dependent RNA polymerase (RNAP). In contrast, most eukaryotes have three highly specialized RNAPs to transcribe the nuclear genome. RNAP I synthesizes almost exclusively ribosomal (r)RNA, RNAP II synthesizes mRNA as well as many noncoding RNAs involved in RNA processing or RNA silencing pathways and RNAP III synthesizes mainly tRNA and 5S rRNA.

Analysis of Yeast RNAP I Transcription of Nucleosomal Templates In Vitro.

Nuclear eukaryotic RNA polymerases (RNAPs) transcribe a chromatin template in vivo. Since the basic unit of chromatin, the nucleosome, renders the DNA largely inaccessible, RNAPs have to overcome the nucleosomal barrier for efficient RNA synthesis. Gaining mechanistical insights in the transcription of chromatin templates will be essential to understand the complex process of eukaryotic gene expression.

Establishment and Maintenance of Open Ribosomal RNA Gene Chromatin States in Eukaryotes.

In growing eukaryotic cells, nuclear ribosomal (r)RNA synthesis by RNA polymerase (RNAP) I accounts for the vast majority of cellular transcription. This high output is achieved by the presence of multiple copies of rRNA genes in eukaryotic genomes transcribed at a high rate. In contrast to most of the other transcribed genomic loci, actively transcribed rRNA genes are largely devoid of nucleosomes adapting a characteristic "open" chromatin state, whereas a significant fraction of rRNA genes resides in a transcriptionally inactive nucleosomal "closed" chromatin state.

Structure of the NLRP3 decamer bound to the cytokine release inhibitor CRID3.

NLRP3 is an intracellular sensor protein that when activated by a broad spectrum of exogenous and endogenous stimuli leads to inflammasome formation and pyroptosis. The conformational states of NLRP3 and the way antagonistic small molecules act at the molecular level remain poorly understood. Here we report the cryo-electron microscopy structures of full-length human NLRP3 in its native form and complexed with the inhibitor CRID3 (also named MCC950).

Analysis of subunit folding contribution of three yeast large ribosomal subunit proteins required for stabilisation and processing of intermediate nuclear rRNA precursors.

In yeast and human cells many of the ribosomal proteins (r-proteins) are required for the stabilisation and productive processing of rRNA precursors. Functional coupling of r-protein assembly with the stabilisation and maturation of subunit precursors potentially promotes the production of ribosomes with defined composition. To further decipher mechanisms of such an intrinsic quality control pathway we analysed here the contribution of three yeast large ribosomal subunit r-proteins rpL2 (uL2), rpL25 (uL23) and rpL34 (eL34) for intermediate nuclear subunit folding steps.

Structure of human RNA polymerase III.

In eukaryotes, RNA Polymerase (Pol) III is specialized for the transcription of tRNAs and other short, untranslated RNAs. Pol III is a determinant of cellular growth and lifespan across eukaryotes. Upregulation of Pol III transcription is observed in cancer and causative Pol III mutations have been described in neurodevelopmental disorders and hypersensitivity to viral infection.

Structural basis of RNA polymerase I pre-initiation complex formation and promoter melting.

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a prerequisite for the biosynthesis of ribosomes in eukaryotes. Compared to Pols II and III, the mechanisms underlying promoter recognition, initiation complex formation and DNA melting by Pol I substantially diverge. Here, we report the high-resolution cryo-EM reconstruction of a Pol I early initiation intermediate assembled on a double-stranded promoter scaffold that prevents the establishment of downstream DNA contacts.

RNA polymerase I (Pol I) passage through nucleosomes depends on Pol I subunits binding its lobe structure.

RNA polymerase I (Pol I) is a highly efficient enzyme specialized in synthesizing most ribosomal RNAs. After nucleosome deposition at each round of rDNA replication, the Pol I transcription machinery has to deal with nucleosomal barriers. It has been suggested that Pol I-associated factors facilitate chromatin transcription, but it is unknown whether Pol I has an intrinsic capacity to transcribe through nucleosomes.

Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I.

Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I.

The C-terminal region of Net1 is an activator of RNA polymerase I transcription with conserved features from yeast to human.

RNA polymerase I (Pol I) synthesizes ribosomal RNA (rRNA) in all eukaryotes, accounting for the major part of transcriptional activity in proliferating cells. Although basal Pol I transcription factors have been characterized in diverse organisms, the molecular basis of the robust rRNA production in vivo remains largely unknown. In S.

Analysis of S. cerevisiae RNA Polymerase I Transcription In Vitro.

RNA polymerase I (Pol I) activity is crucial to provide cells with sufficient amounts of ribosomal RNA (rRNA). Synthesis of rRNA takes place in the nucleolus, is tightly regulated and is coordinated with synthesis and assembly of ribosomal proteins, finally resulting in the formation of mature ribosomes. Many studies on Pol I mechanisms and regulation in the model organism S.

Structure of the initiation-competent RNA polymerase I and its implication for transcription.

Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level rRNA production relies on efficient binding of initiation factors to the rRNA gene promoter and recruitment of Pol I complexes containing initiation factor Rrn3. Here, we determine the cryo-EM structure of the Pol I-Rrn3 complex at 7.

Binding of the termination factor Nsi1 to its cognate DNA site is sufficient to terminate RNA polymerase I transcription in vitro and to induce termination in vivo.

Different models have been proposed explaining how eukaryotic gene transcription is terminated. Recently, Nsi1, a factor involved in silencing of ribosomal DNA (rDNA), was shown to be required for efficient termination of rDNA transcription by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae. Nsi1 contains Myb-like DNA binding domains and associates in vivo near the 3' end of rRNA genes to rDNA, but information about which and how DNA sequences might influence Nsi1-dependent termination is lacking.