A regulator that inhibits transcription by targeting an intersubunit interaction of the RNA polymerase holoenzyme.

The structures of the bacterial RNA polymerase holoenzyme have provided detailed information about the intersubunit interactions within the holoenzyme. Functional analysis indicates that one of these is critical in enabling the holoenzyme to recognize the major class of bacterial promoters. It has been suggested that this interaction, involving the flap domain of the beta subunit and conserved region 4 of the sigma subunit, is a potential target for regulation. Here we provide genetic and biochemical evidence that the sigma region 4/beta-flap interaction is targeted by the transcription factor AsiA. Specifically, we show that AsiA competes directly with the beta-flap for binding to sigma region 4, thereby inhibiting transcription initiation by disrupting the sigma region 4/beta-flap interaction.

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 the hepA 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 sigma70 subunit with a 1:1 stoichiometry and a dissociation constant (Kd) of 75 nM. An E. coli strain with a disruption in the hepA gene shows sensitivity to ultraviolet light.

Inhibition of Escherichia coli RNA polymerase by bacteriophage T4 AsiA.

The 10 kDa bacteriophage T4 antisigma protein AsiA binds the Escherichia coli RNA polymerase promoter specificity subunit, sigma 70, with high affinity and inhibits its transcription activity. AsiA binds to sigma 70 primarily through an interaction with sigma 70 conserved region 4.2, which has also been implicated in sequence-specific recognition of the -35 consensus promoter element. Here we show that AsiA forms a stable ternary complex with core RNA polymerase (RNAP) and sigma 70 and thus does not inhibit sigma 70 activity by preventing its binding to core RNAP. We investigated the effect of AsiA on open promoter complex formation and abortive initiation at two -10/-35 type promoters and two "extended -10" promoters. Our results indicate that the binding of AsiA to sigma 70 and the interaction of sigma 70 region 4.2 with the -35 consensus promoter element of -10/-35 promoters is mutually exclusive. In contrast, AsiA has much less effect on open promoter complex formation and abortive initiation from extended -10 promoters, which lack a -35 consensus element and do not require sigma 70 conserved region 4.2. From these results we conclude that T4 AsiA inhibits E. coli RNAP sigma 70 holoenzyme transcription at -10/-35 promoters by interfering with the required interaction between sigma 70 conserved region 4.2 and the -35 consensus promoter element.

Domain organization of the Escherichia coli RNA polymerase sigma 70 subunit.

We used limited trypsin digestion to determine the domain organization of the Escherichia coli RNA polymerase sigma 70 subunit. Trypsin-resistant fragments containing sigma 70 conserved region 2 (sigma 70(2)), and carboxy-terminal fragments containing conserved regions 3 and 4 (sigma 70(3-4)) were identified by a combination of amino acid sequencing and mass spectrometry. The domains were studied for partial biochemical functions of sigma 70.sigma 70(2) bound core RNA polymerase competitively with intact sigma 70. In contrast to sigma 70(2) alone, the RNA polymerase holoenzyme formed with sigma 70(2) specifically bound a single-stranded DNA oligomer with a sequence corresponding to the non-template strand of the -10 promoter element (the Pribnow box). Sigma 70(2) also forms crystals that are suitable for X-ray analysis. Sigma 70(3-4) bound the T4 AsiA protein with high affinity. The epitope for T4 AsiA on sigma 70 was further localized to within sigma 70[551-608], comprising sigma conserved region 4.2.

Crystal structure of a sigma 70 subunit fragment from E. coli RNA polymerase.

The 2.6 A crystal structure of a fragment of the sigma 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.

The beta subunit Rif-cluster I is only angstroms away from the active center of Escherichia coli RNA polymerase.

Ribonucleotide analogs bound in the initiating site of Escherichia coli RNA polymerase-promoter complex were cross-linked to the beta subunit. Using limited proteolysis and chemical degradation, the cross-link was mapped to a segment of beta between amino acids Val516 and Arg540. This region (Rif-cluster I) is known to harbor many rifampicin-resistant (RifR) mutations. The results demonstrate that Rif-culster I is part of the "5'-face" of the active center and provide structural basis for the long-known effects of RifR mutations on transcription initiation, elongation, and termination.

Three-dimensional structure of E. coli 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 beta 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 beta' subunit. The mutation is the Phe793-->Ser substitution, which occurred in an evolutionarily conserved segment of the beta' subunit. The homologous segment in the eukaryotic RNA polymerase II largest subunit harbors mutations conferring alpha-amanitin resistance. Both streptolydigin and alpha-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.

Assembly of functional Escherichia coli RNA polymerase containing beta subunit fragments.

The Escherichia coli rpoB gene, which codes for the 1342-residue beta 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 beta subunit, fragments encoded by segments of rpoB flanking the dispensable regions were individually overexpressed and purified. We show that these beta-subunit polypeptide fragments, when added with purified recombinant beta', sigma, and alpha subunits of RNAP, reconstitute a functional enzyme in vitro. These results demonstrate that the beta subunit is composed of at least three distinct domains and open another avenue for in vitro studies of RNAP assembly and structure.

The sigma subunit conserved region 3 is part of "5'-face" of active center of Escherichia coli RNA polymerase.

Ribonucleotide analogs bound in the initiating site of Escherichia coli RNA polymerase holoenzyme in open promoter complexes were cross-linked to the beta and sigma 70 subunits. Using limited proteolysis and chemical degradation, the cross-link site in sigma 70 was mapped to a segment between amino acids Glu508 and Met561 containing the C-terminal part of conserved region 3. This result, when reconciled with genetic data on the interaction of sigma 70 conserved regions 2 and 4 with the -10 and -35 promoter regions, respectively, allows us to model the orientation of the sigma 70 subunit domains within the open promoter complex.

A non-essential domain of Escherichia coli RNA polymerase required for the action of the termination factor Alc.

An evolutionarily nonconserved region of approximately 250 amino acids can be deleted from the amino-terminal part of the beta subunit of Escherichia coli RNA polymerase without effect on the enzyme's basic function. The non-essential segment is located between two highly conserved motifs and is flanked by sequences participating in the rifampicin-binding site. The results define the second non-essential domain in the beta subunit, in addition to the more distal dispensable segment identified previously. The Alc protein of bacteriophage T4 participates in the host transcription shutoff after infection by causing premature termination of transcription on E. coli DNA. Point mutations which prevent Alc action in vivo change amino acids in the non-essential NH2-terminal domain of the beta subunit. These point mutations as well as deletions which remove the non-essential region also prevent Alc action. Thus, in the RNA polymerase molecule, the proximal non-essential domain of beta may function as an acceptor of Alc or other regulatory factors.