Crystal of bacterial core RNA polymerase and methods of use thereof
a technology of bacterial core and crystal structure, applied in the field of crystal structure of bacterial core rna polymerase, can solve the problem that so-called “miracle drugs” are not sufficient to accomplish the task of rna polymeras
Inactive Publication Date: 2006-03-09
AFFINIUM PHARMA
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Benefits of technology
The present invention provides crystals of the bacterial core RNA polymerase, which can be used to determine the atomic coordinates of the enzyme. The crystals can be used to design drugs that inhibit the growth of bacteria. The invention also provides methods for identifying and improving inhibitors of the RNA polymerase. The crystals can be grown using vapor diffusion and can diffract X-rays to better than 5.0 Angstroms. The invention also includes isolated nucleic acids and expression vectors that encode the RNA polymerase subunits. The invention also includes methods of using the crystals to grow a crystal of the RNA polymerase.
Problems solved by technology
Although, there was initial optimism in the middle of this century that diseases caused by bacteria would be quickly eradicated, it has become evident that the so-called “miracle drugs” are not sufficient to accomplish this task.
Unfortunately, such identification has heretofore relied on serendipity and / or systematic screening of large numbers of natural and synthetic compounds.
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Crystal Structure of Thermus Aquaticus Core RNA polymerase At 3.3 Å Resolution
Introduction
[0150] To provide a more detailed framework to interpret the existing genetic, biochemical, and biophysical information, as well as to guide further studies aimed at understanding the transcription process and its regulation, the three-dimensional structure of a bacterial core RNAP by X-ray crystallography at 3.3 Å resolution has been determined as detailed below.
Methods
[0151] Purification and crystallization: The preparative procedure for T. aquaticus core RNAP is similar to the preparation of E. coli core RNAP [Polyakov et al., Cell, 83:365-373 (1995)]. Briefly, approximately 200 g wet cell paste is thawed and lysed using a continuous-flow French press. After a low-speed spin, the soluble fraction is precipitated with 0.6% Polymin-P. RNAP is eluted from the Polymin-P pellet with TGED buffer (10 mM Tris -HCl, pH 8, 5% glycerol, 1 mM EDTA, 1 mM DTT) plus 1 M NaCl, then precipitated by addin...
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Abstract
A detailed three-dimensional structure for a bacterial core RNA polymerase is provided. Crystals of the bacterial core RNA polymerase are also included in the invention. The present invention further provides procedures for identifying agents that can inhibit bacterial proliferation through the use of rational drug design predicated on the crystals and crystallographic data disclosed.
Description
FIELD OF THE INVENTION [0001] The present invention provides a crystal of a bacterial core RNA polymerase from Thermus aquaticus. The three-dimensional structural information is included in the invention. The present invention provides procedures for identifying agents that can inhibit bacterial cell growth through the use of rational drug design predicated on the crystallographic data. BACKGROUND OF THE INVENTION [0002] RNA in all cellular organisms is synthesized by a complex molecular machine, the DNA-dependent RNA polymerase (RNAP). In its simplest bacterial form, the. enzyme comprises at least 4 subunits with a total molecular mass of around 400 kDa. The eukaryotic enzymes comprise upwards of a dozen subunits with a total molecular mass of around 500 kDa. The essential core component of the RNAP (subunit composition α2ββ′) is evolutionarily conserved from bacteria to man [Archambault and Friesen, Microbiological Reviews, 57:703-724 (1993)]. Sequence homologies point to structur...
Claims
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IPC IPC(8): C12Q1/68G06F19/00C12N9/12C12Q1/18G16B15/10G16B20/00
CPCC07K2299/00C12N9/1247G06F19/18G01N2333/9125G06F19/16C12Q1/18G16B15/00G16B20/00G16B15/10
Inventor DARST, SETHZHANG, GONGYICAMPBELL, ELIZABETHMINAKIN, LEONIDSEVERINOV, KONSTANTIN
Owner AFFINIUM PHARMA