The Subunit Positions within RNA Polymerase Holoenzyme Determined by Triangulation of Centre-to-Centre Distances (original) (raw)
Related papers
The Core Subunit Structure in RNA Polymerase Holoenzyme Determined by Neutron Small-Angle Scattering
European Journal of Biochemistry, 1980
The core subunit arrangement a2-P-P' within DNA-dependent RNA polymerase holoenzyme a2PP'0 from Escherichia coli was investigated by neutron small-angle scattering using label triangulation. The quaternary structure of multisubunit biomolecules can be studied by this new method if total reconstitution works in a quantitative way and if extensive replacement of C-bound hydrogen (H) by deuterium (2H) is possible. A substitution of the selected subunits by their fully deuterated analogues was used for the analysis of the overall shapes of the core subunits a2, P and fl' in situ and for the determination of the intersubunit centre-to-centre distances. The contrast between the buffer and the remaining 'hydrogenated' enzyme vanishes if the buffer contains 42 % 2 H 2 0 (matching of scattering length densities). The isotopic hybridization of the enzyme fulfils the conditions of isomorphous replacement as required : molecular functions, like enzyme activity, were completely preserved.
Cell, 2002
to this multistep process: bacterial RNAP core enzyme (Zhang et al., 1999; subunit composition Ј//␣ I /␣ II /), eukaryotic RNAP II core enzyme ⌬4/7 (Cramer et al., 2001; subunit composition 1/2/3/5/6/8/9/10/11/12, where 1, 2, 3, 11, and 6 are homologs of bacterial RNAP and ; Ebright, 2000), and a eukaryotic RNAP II tailed-template elongation complex .
Journal of Applied Crystallography, 1979
In all organisms the multi-subunit enzyme DNA-dependent RNA polymerase catalyses the first basic step in gene expression, the transcription of DNA into RNA. The neutron small-angle scattering effects were studied in the Guinier region by applying the contrastmatching technique. Isotopic labelling was realised by culturing fully deuterated E. coli cells in a heavy-water medium containing deuterated substrates. One or two completely deuterated components (subunits) of polymerase holoenzyme (composition formula ~2/3/3'~, molecular weight 497 000) were recombined with the remaining natural ('hydrogenated') subunits for these neutron measurements. Measurements are presented of radii of gyration R, overall shapes, and pair distances d, of three components (subunits ~2,/3,fl') in RNA polymerase of E. coli. The subunits /3', /3 and ~2 are obviously rather elongated in situ and exhibit (as scattering-equivalent prolate ellipsoids) axial ratios of about 7"1 up to 9:1. The intersubunit centre of gravity distances were analysed to be 7-3_+08 nm for/3-/3', 8.2 +_ 1.2 nm for ~2-/3' and 7-0+ 1-7 nm for ~.2-/3.
Escherichia coli RNA polymerase core and holoenzyme structures
The EMBO Journal, 2000
Multisubunit RNA polymerase is an essential enzyme for regulated gene expression. Here we report two Escherichia coli RNA polymerase structures: an 11.0 A Ê structure of the core RNA polymerase and a 9.5 A Ê structure of the s 70 holoenzyme. Both structures were obtained by cryo-electron microscopy and angular reconstitution. Core RNA polymerase exists in an open conformation. Extensive conformational changes occur between the core and the holoenzyme forms of the RNA polymerase, which are largely associated with movements in b¢. All common RNA polymerase subunits (a 2 , b, b¢) could be localized in both structures, thus suggesting the position of s 70 in the holoenzyme.
RNA polymerase holoenzyme: structure, function and biological implications
Current Opinion in Microbiology, 2003
The past three years have marked the breakthrough in our understanding of the structural and functional organization of RNA polymerase. The latest major advance was the highresolution structures of bacterial RNA polymerase holoenzyme and the holoenzyme in complex with promoter DNA. Together with an array of genetic, biochemical and biophysical data accumulated to date, the structures provide a comprehensive view of dynamic interactions between the major components of transcription machinery during the early stages of the transcription cycle. They include the binding of sigma factor to the core enzyme, and the recognition of promoter sequences and DNA melting by holoenzyme, transcription initiation and promoter clearance.
A general view: Structure and function of the subunits of E. coli RNA polymerase
Journal of Cell and Molecular Biology, 2003
The DNA-dependent RNA polymerases are widespread throughout nature. E. coli RNA polymerase, one of the most well characterized polymerase, consists of two major forms, core enzyme with subunit stoichiometry of α 2 ββ' and holoenzyme which contains an additional σ subunit to core enzyme. E. coli RNA polymerase plays a central role in transcription. While the core enzyme catalyses the elongation and termination of transcription, to initiate core enzyme needs to combine with σ subunit. The three dimensional structure of this multimeric enzyme revealed a thumb-like projection. Using the electron microscope, Tichelar and Heel (1990) proposed a model that is in agreement with both β and β' together constituting a V-like structure and α dimer associates at the short ends, while σ is positioned within the concave side of the core, next to the dimer. In this review, the structure and related functions of the subunits of E. coli DNA-dependent RNA polymerase is presented based on several researches and reviews. Considering biochemical and genetic studies on the RNA polymerase of E. coli, a genetic walk on the subunits is summarized.
1 H n.m.r. of the DNA-dependent RNA polymerase from Escherichia coli
Biochemical Journal, 1982
The1H n.m.r. study of the DNA-dependent RNA polymerase from Escherichia coli has revealed that the holoenzyme (ββ′α2σ) displays two mobile regions: one, observable also in the core enzyme (ββ′α2), is characterized by basic amino acids and its appearance and form depend on ionic strength; the other, specific to the holoenzyme, is characterized by threonine residues and its appearance does not depend on ionic strength.
Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6Å resolution
Nature, 2002
In bacteria, the binding of a single protein, the initiation factor j, to a multi-subunit RNA polymerase core enzyme results in the formation of a holoenzyme, the active form of RNA polymerase essential for transcription initiation. Here we report the crystal structure of a bacterial RNA polymerase holoenzyme from Thermus thermophilus at 2.6 Å resolution. In the structure, two aminoterminal domains of the j subunit form a V-shaped structure near the opening of the upstream DNA-binding channel of the active site cleft. The carboxy-terminal domain of j is near the outlet of the RNA-exit channel, about 57 Å from the N-terminal domains. The extended linker domain forms a hairpin protruding into the active site cleft, then stretching through the RNA-exit channel to connect the N-and C-terminal domains. The holoenzyme structure provides insight into the structural organization of transcription intermediate complexes and into the mechanism of transcription initiation.