Structural basis of Dot1L stimulation by histone H2B lysine 120 ubiquitination. Valencia-Sanchez et al.

Original gels and membranes images shown in Figures 3, 4 and 7, which were used in EMSA gels images and Western Blots images, respectively. The images were used to calculate app Kd constants of Dot1L to Nucleosomes, and to monitor H3K79 histone methylation by Dot1L.THIS DATASET IS ARCHIVED AT DANS/EASY, BUT NOT ACCESSIBLE HERE. TO VIEW A LIST OF FILES AND ACCESS THE FILES IN THIS DATASET CLICK ON THE DOI-LINK ABOV

The dynamic machinery of mRNA elongation.

Two complementary X-ray studies of the interaction between RNA polymerase II and nucleic acids have improved our understanding of mRNA elongation. These studies suggest how RNA polymerase II unwinds DNA, how it separates the RNA product from the DNA template and how it incorporates nucleoside triphosphate (NTP) substrates into the growing RNA chain. The tunable polymerase active center apparently allows repositioning of a catalytic metal ion, rotation of NTPs before their incorporation, RNA repositioning by a transcript cleavage factor, and modulation of enzyme activity by a bacterial small molecule regulator and its protein cofactor

Architecture of the RNA polymerase II-TFIIS complex and implications for mRNA cleavage.

The transcription elongation factor TFIIS induces mRNA cleavage by enhancing the intrinsic nuclease activity of RNA polymerase (Pol) II. We have diffused TFIIS into Pol II crystals and derived a model of the Pol II-TFIIS complex from X-ray diffraction data to 3.8 Å resolution. TFIIS extends from the polymerase surface via a pore to the internal active site, spanning a distance of 100 Å. Two essential and invariant acidic residues in a TFIIS loop complement the Pol II active site and could position a metal ion and a water molecule for hydrolytic RNA cleavage. TFIIS also induces extensive structural changes in Pol II that would realign nucleic acids in the active center. Our results support the idea that Pol II contains a single tunable active site for RNA polymerization and cleavage, in contrast to DNA polymerases with two separate active sites for DNA polymerization and cleavage

Architecture of initiation-competent 12-subunit RNA polymerase II

RNA polymerase (Pol) II consists of a 10-polypeptide catalytic core and the two-subunit Rpb47 complex that is required for transcription initiation. Previous structures of the Pol II core revealed a ‘‘clamp,’’ which binds the DNA template strand via three ‘‘switch regions,’’ and a flexible ‘‘linker’’ to the C-terminal repeat domain (CTD). Here we derived a model of the complete Pol II by fitting structures of the core and Rpb47 to a 4.2-Å crystallographic electron density map. Rpb47 protrudes from the polymerase ‘‘upstream face,’’ on which initiation factors assemble for promoter DNA loading. Rpb7 forms a wedge between the clamp and the linker, restricting the clamp to a closed position. The wedge allosterically prevents entry of the promoter DNA duplex into the active center cleft and induces in two switch regions a conformation poised for template-strand binding. Interaction of Rpb47 with the linker explains Rpb4-mediated recruitment of the CTD phosphatase to the CTD during Pol II recycling. The core–Rpb7 interaction and some functions of Rpb47 are apparently conserved in all eukaryotic and archaeal RNA polymerases but not in the bacterial enzyme

Structures of complete RNA polymerase II and its subcomplex, Rpb4/7.

We determined the x-ray structure of the RNA polymerase (Pol) II subcomplex Rpb4/7 at 2.3 Å resolution, combined it with a previous structure of the 10-subunit polymerase core, and refined an atomic model of the complete 12-subunit Pol II at 3.8-Å resolution. Comparison of the complete Pol II structure with structures of the Pol II core and free Rpb4/7 shows that the core-Rpb4/7 interaction goes along with formation of an α-helix in the linker region of the largest Pol II subunit and with folding of the conserved Rpb7 tip loop. Details of the core-Rpb4/7 interface explain facilitated Rpb4/7 dissociation in a temperature-sensitive Pol II mutant and specific assembly of Pol I with its Rpb4/7 counterpart, A43/14. The refined atomic model of Pol II serves as the new reference structure for analysis of the transcription mechanism and enables structure solution of complexes of the complete enzyme with additional factors and nucleic acids by molecular replacement

Structural biology of RNA polymerase III: Subcomplex C17/25 X-ray structure and 11 subunit enzyme model.

We obtained an 11 subunit model of RNA polymerase (Pol) III by combining a homology model of the nine subunit core enzyme with a new X-ray structure of the subcomplex C17/25. Compared to Pol II, Pol III shows a conserved active center for RNA synthesis but a structurally different upstream face for specific initiation complex assembly during promoter selection. The Pol III upstream face includes a HRDC domain in subunit C17 that is translated by 35 Å and rotated by 150° compared to its Pol II counterpart. The HRDC domain is essential in vivo, folds independently in vitro, and, unlike other HRDC domains, shows no indication of nucleic acid binding. Thus, the HRDC domain is a functional module that could account for the role of C17 in Pol III promoter-specific initiation. During elongation, C17/25 may bind Pol III transcripts emerging from the adjacent exit pore, because the subcomplex binds to tRNA in vitro

RNA polymerase II–TFIIB structure and mechanism of transcription initiation

To initiate gene transcription, RNA polymerase II (Pol II) requires the transcription factor IIB (B). Here we present the crystal structure of the complete Pol II–B complex at 4.3 Å resolution, and complementary functional data. The results indicate the mechanism of transcription initiation, including the transition to RNA elongation. Promoter DNA is positioned over the Pol II active centre cleft with the 'B-core' domain that binds the wall at the end of the cleft. DNA is then opened with the help of the 'B-linker' that binds the Pol II rudder and clamp coiled-coil at the edge of the cleft. The DNA template strand slips into the cleft and is scanned for the transcription start site with the help of the 'B-reader' that approaches the active site. Synthesis of the RNA chain and rewinding of upstream DNA displace the B-reader and B-linker, respectively, to trigger B release and elongation complex formation