Elena Severinova

Crystal Structure of a σ70 Subunit Fragment from E. coli RNA Polymerase

Abstract The 2.6 A crystal structure of a fragment of the σ 70 promoter specificity subunit of E. coli RNA polymerase is described. Residues involved in core RNA polymerase binding lie on one face of the structure. On the opposite face, aligned along one helix, are exposed residues that interact with the −10 consensus promoter element (the Pribnow box), including four aromatic residues involved in promoter melting. The structure suggests one way in which DNA interactions may be inhibited in the absence of RNA polymerase and provides a framework for the interpretation of a large number of genetic and biochemical analyses.

Three-dimensional structure of E. coil core RNA polymerase: Promoter binding and elongation conformations of the enzyme

The structure of E. coli core RNA polymerase (RNAP) has been determined to approximately 23 A resolution by three-dimensional reconstruction from electron micrographs of flattened helical crystals. The structure reveals extensive conformational changes when compared with the previously determined E. coli RNAP holoenzyme structure, but resembles the yeast RNAPII structure. While each of these structures contains a thumb-like projection surrounding a channel 25 A in diameter, the E. coli RNAP holoenzyme thumb defines a deep but open groove on the molecule, whereas the thumb of E. coli core and yeast RNAPII form part of a ring that surrounds the channel. This may define promoter-binding and elongation conformations of RNAP, as E. coli holoenzyme recognizes promoter sites on double-stranded DNA, while both E. coli core and yeast RNAPII are elongating forms of the polymerase and are incapable of promoter recognition.

STREPTOLYDIGIN-RESISTANT MUTANTS IN AN EVOLUTIONARILY CONSERVED REGION OF THE BETA ' SUBUNIT OF ESCHERICHIA COLI RNA POLYMERASE

Mutations conferring streptolydigin resistance onto Escherichia coli RNA polymerase have been found exclusively in the β subunit (Heisler, L. M., Suzuki, H., Landick, R., and Gross, C. A.(1993) J. Biol. Chem. 268, 25369-25375). We report here the isolation of a streptolydigin-resistant mutation in the E. coli rpoC gene, encoding the β‘ subunit. The mutation is the Phe793 → Ser substitution, which occurred in an evolutionarily conserved segment of the β‘ subunit. The homologous segment in the eukaryotic RNA polymerase II largest subunit harbors mutations conferring α-amanitin resistance. Both streptolydigin and α-amanitin are inhibitors of transcription elongation. Thus, the two antibiotics may inhibit transcription in their respective systems by a similar mechanism, despite their very different chemical nature.

Disruption of Escherichia coli HepA, an RNA Polymerase-associated Protein, Causes UV Sensitivity*

During the development of purification procedures for Escherichia coli RNA polymerase (RNAP), we noticed the consistent co-purification of a 110-kDa polypeptide. Here, we report the identification of the 110-kDa protein as the product of thehepA gene, a member of the SNF2 family of putative helicases. We have cloned the hepA gene and overexpressed and purified the HepA protein. We show in vitro that RNAP preparations have an ATPase activity only in the presence of HepA and that HepA binds core RNAP competitively with the promoter specificity ς70 subunit with a 1:1 stoichiometry and a dissociation constant (K d ) of 75 nm. An E. coli strain with a disruption in the hepA gene shows sensitivity to ultraviolet light.

Pribnow Box Recognition and Melting by Escherichia coli RNA Polymerase

The core RNA polymerases from bacterial and eukaryotic cells, which are homologous in structure and function (Allison et al. 1985; Biggs et al. 1985; Ahearn et al. 1987; Sweetser et al. 1987; Darst et al. 1989, 1991; Schultz et al. 1993; Polyakov et al. 1995), are catalytically active in RNA chain elongation but are incapable of promoter recognition and specific initiation. Promoter-specific transcription initiation requires additional protein factors. In bacteria, specific initiation by RNA polymerase (RNAP) requires a single polypeptide known as a σ factor, which binds to core RNAP to form the holoenzyme (Burgess et al. 1969; Travers and Burgess 1969). One primary σ factor directs the bulk of transcription during exponential growth. Specialized, alternative σ factors direct transcription of specific regulons during unusual physiological or developmental conditions (reviewed in Helmann and Chamberlin 1988; Gross et al. 1992). The primary and most of the alternative σ factors comprise a highly homologous family of proteins (Stragier et al. 1985; Gribskov and Burgess 1986) with four regions of highly conserved amino acid sequence (Fig. 1; reviewed in Lonetto et al. 1992). Based on the results of genetic and biochemical experiments, specific functions have been assigned to some of the conserved regions(summarized in Fig. 1).