The Primary Structure of Escherichia coli RNA Polymerase. Nucleotide Sequence of the rpoB Gene and Amino-Acid Sequence of the beta-Subunit (original) (raw)
Related papers
The primary structure of the E.coli rpoC gene (5321 base pairs) coding the /8'-subunit of BNA polymerase as well as its adjacent segment have been determined. The structure analysis of the peptides obtained by cleavage of the protein with cyanogen bromide and trypsin has confirmed the amino acid sequence of the #'-subunit deduced from the nucleotide sequence aziaJlysis. The 16'-subunit of E.coli RNA polymerase contains 1407 amino acid residues. Its translation is initiated by codon GUG and terminated by codon TAA. It has been detected that the sequence following the terminating codon is strikingly homologous to known sequences of°-independent terminators.
European Journal of Biochemistry, 1989
A method is proposed for localization of the sites of affinity labelling of the p subunit of Escherichiu coli RNA polymerase. The principle of this method is similar to that of the methods of rapid sequencing of nucleic acids. The polypeptide bearing a radioactive affinity label at one of the amino acid residues is subjected to shortterm treatment with cyanogen bromide. The conditions of this reaction are selected in such a way that less than one cleavage occurs on average per polypeptide chain. Two series of radioactive peptides are formed, one involving all the possible N-terminal peptides and the other the C-terminal peptides. The distribution of the lengths of these peptides is studied by means of gel electrophoresis and compared with the theoretical ones based on the known amino acid sequence of the p subunit. Obviously, the affinity label resides between the C-terminus of the shortest N-tcrminal radioactive peptide and the N-terminus of the shortest C-terminal radioactive peptide. In order to increase reliability and resolution of the method, partial trypsinolysis may be employed. The evidence obtained suggests that lysine residues over the regions 1036-1066, 1234-1242, and histidine-1237 are situated in the nearest neighbourhood to, or directly involved in the formation of the active center of initiating substrate binding of the p subunit of E. coli RNA polymerase.
Proceedings of the National Academy of Sciences, 1995
We present a simple, rapid procedure for reconstitution ofEscherichia coli RNA polymerase holoenzyme (RNAP) from individual recombinant a, ,B, (', and a7O subunits. Hexahistidine-tagged recombinant a subunit purified by batch-mode metal-ion-affinity chromatography is incubated with crude recombinant 13, 13', and cr70 subunits from inclusion bodies, and the resulting reconstituted recombinant RNAP is purified by batch-mode metal-ion-affinity chromatography. RNAP prepared by this procedure is indistinguishable from RNAP prepared by conventional methods with respect to subunit stoichiometry, a-DNA interaction, catabolite gene activator protein (CAP)-independent transcription, and CAP-dependent transcription. Experiments with a(1-235), an a subunit C-terminal deletion mutant, establish that the procedure is suitable for biochemical screening of subunit lethal mutants.
Journal of Biological Chemistry, 1996
The  and  subunits of Escherichia coli DNA-dependent RNA polymerase are highly conserved throughout eubacterial and eukaryotic kingdoms. However, in some archaebacteria and chloroplasts, the corresponding sequences are "split" into smaller polypeptides that are encoded by separate genes. To test if such split sites can be accommodated into E. coli RNA polymerase, subunit fragments encoded by the segments of E. coli rpoB and rpoC genes corresponding to archaebacterial and chloroplast split subunits were individually overexpressed. The purified fragments, when mixed in vitro with complementing intact RNA polymerase subunits, yielded an active enzyme capable of catalyzing the phosphodiester bond formation. Thus, the large subunits of eubacteria and eukaryotes are composed of independent structural modules corresponding to the smaller subunits of archaebacteria and chloroplasts.
Crystal Structure of Thermus aquaticus Core RNA Polymerase at 3.3 Å Resolution
Cell, 1999
Since the initial indications of DNA-dependent RNAP activity from a number of systems (Weiss and Gladstone, 1959; Huang et al., 1960; Hurwitz et al., 1960; Stevens, 1960) and the isolation of the RNAP enzyme from bacte-Summary rial sources (Chamberlin and Berg, 1962), a wealth of biochemical, biophysical, and genetic information has The X-ray crystal structure of Thermus aquaticus core accumulated on RNAP and its complexes with nucleic RNA polymerase reveals a "crab claw"-shaped moleacids and accessory factors (von Hippel et al., 1984; cule with a 27 Å wide internal channel. Located on the Erie et al., 1992; Sentenac et al., 1992; Gross et al., back wall of the channel is a Mg 2؉ ion required for 1996). Nevertheless, the enzyme itself, in terms of its catalytic activity, which is chelated by an absolutely structure-function relationship, remains a black box. An conserved motif from all bacterial and eukaryotic celessential step toward understanding the mechanism of lular RNA polymerases. The structure places key functranscription and its regulation is to determine threetional sites, defined by mutational and cross-linking dimensional structures of RNAP and its complexes with analysis, on the inner walls of the channel in close DNA, RNA, and regulatory factors. proximity to the active center Mg 2؉ . Further out from Low-resolution structures of bacterial and eukaryotic the catalytic center, structural features are found that RNAPs, provided by electron crystallography, reveal a may be involved in maintaining the melted transcripmolecule shaped like a crab claw with a groove or chantion bubble, clamping onto the RNA product and/or nel that is an appropriate size for accomodating double-DNA template to assure processivity, and delivering helical DNA (Darst et al., , 1998a(Darst et al., , 1998b; nucleotide substrates to the active center.
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.
Assembly of functional Escherichia coli RNA polymerase containing beta subunit fragments
Proceedings of the National Academy of Sciences, 1995
The Escherichia coli rpoB gene, which codes for the 1342-residue j3 subunit of RNA polymerase (RNAP), contains two dispensable regions centered around codons 300 and 1000. To test whether these regions demarcate domains of the RNAP 1 subunit, fragments encoded by segments of rpoB flanking the dispensable regions were individually overexpressed and purified. We show that these p-subunit polypeptide fragments, when added with purified recombinant P', or, and a subunits of RNAP, reconstitute a functional enzyme in vitro. These results demonstrate that the 1f subunit is composed of at least three distinct domains and open another avenue for in vitro studies of RNAP assembly and structure.