SUMMARY The global regulatory nucleotide ppGpp (“magic spot”) regulates transcription from a large subset of Escherichia coli promoters, illustrating how small molecules can control gene expression promoter-specifically by interacting with RNA polymerase (RNAP) without binding to DNA. However, ppGpp’s target site on RNAP, and therefore its mechanism of action, have remained unclear. We report here a binding site for ppGpp on E. coli RNAP, identified by crosslinking, protease mapping, and analysis of mutant RNAPs that fail to respond to ppGpp. A strain with a mutant ppGpp binding site displays properties characteristic of cells defective for ppGpp synthesis. The binding site is at an interface of two RNAP subunits, ω and β′, and its position suggests an allosteric mechanism of action involving restriction of motion between two mobile RNAP modules. Identification of the binding site allows prediction of bacterial species in which ppGpp exerts its effects by targeting RNAP.

Anthrax is caused by the spore-forming, gram-positive bacterium Bacillus anthracis. The bacterium's major virulence factors are (a) the anthrax toxins and (b) an antiphagocytic polyglutamic capsule. These are encoded by two large plasmids, the former by pXO1 and the latter by pXO2. The expression of both is controlled by the bicarbonate-responsive transcriptional regulator, AtxA. The anthrax toxins are three polypeptides-protective antigen (PA), lethal factor (LF), and edema factor (EF)-that come together in binary combinations to form lethal toxin and edema toxin. PA binds to cellular receptors to translocate LF (a protease) and EF (an adenylate cyclase) into cells. The toxins alter cell signaling pathways in the host to interfere with innate immune responses in early stages of infection and to induce vascular collapse at late stages. This review focuses on the role of anthrax toxins in pathogenesis. Other virulence determinants, as well as vaccines and therapeutics, are briefly discussed.

SummaryEscherichia coli DksA, GreA, and GreB have similar structures and bind to the same location on RNA polymerase (RNAP), the secondary channel. We show that GreB can fulfill some roles of DksA in vitro, including shifting the promoter-open complex equilibrium in the dissociation direction, thus allowing rRNA promoters to respond to changes in the concentrations of ppGpp and NTPs. However, unlike deletion of the dksA gene, deletion of greB had no effect on rRNA promoters in vivo. We show that the apparent affinities of DksA and GreB for RNAP are similar, but the cellular concentration of GreB is much lower than that of DksA. When overexpressed and in the absence of competing GreA, GreB almost completely complemented the loss of dksA in control of rRNA expression, indicating its inability to regulate rRNA transcription in vivo results primarily from its low concentration. In contrast to GreB, the apparent affinity of GreA for RNAP was weaker than that of DksA, GreA only modestly affected rRNA promoters in vitro, and even when overexpressed GreA did not affect rRNA transcription in vivo. Thus, binding in the secondary channel is necessary but insufficient to explain the effect of DksA on rRNA transcription. Neither Gre factor was capable of fulfilling two other functions of DksA in transcription initiation: co-activation of amino acid biosynthetic gene promoters with ppGpp and compensation for the loss of the ω subunit of RNAP in the response of rRNA promoters to ppGpp. Our results provide important clues to the mechanisms of both negative and positive control of transcription initiation by DksA.

Previous studies have come to conflicting conclusions about the requirement for the subunit of RNA polymerase in bacterial transcription regulation. We demonstrate here that purified RNAP lacking does not respond in vitro to the effector of the stringent response, ppGpp. DksA, a transcription factor that works in concert with ppGpp to regulate rRNA expression in vivo and in vitro, fully rescues the ppGpp-unresponsiveness of RNAP lacking , likely explaining why strains lacking display a stringent response in vivo. These results demonstrate that plays a role in RNAP function (in addition to its previously reported role in RNAP assembly) and highlight the importance of inclusion of in RNAP purification protocols. Furthermore, these results suggest that either one or both of two short segments in the ␤ subunit that physically link to the ppGpp-binding region of the enzyme may play crucial roles in ppGpp and DksA function. In Escherichia coli, transcription is carried out by a multi-subunit RNA polymerase (RNAP) composed of six subunits, including two copies of ␣ and one copy each of ␤, ␤Ј, , and (for a recent review, see Geszvain and Landick 2004). ␣ 2 , ␤, ␤Ј, and comprise core RNAP, which is catalytically active but unable to recognize promoters. The ␣ 2 dimer serves as the scaffold on which ␤ and ␤Ј assemble. ␤ and ␤Ј make up the vast majority of RNAP by mass and create the enzyme's active center. To initiate transcription, one of several types of subunits, most commonly 70 , binds to core to form RNAP holoenzyme. and ␣ are site-specific DNA-binding proteins that account for specific promoter recognition. Although a high-resolution structure of E. coli RNAP has not yet been determined, X-ray structures of the Thermus aquaticus and Thermus thermophilus holoenzymes (Murakami et al. 2002b;Vassylyev et al. 2002), as well as of a T. aquaticus RNAP holoenzyme-DNA complex (Murakami et al. 2002a), elucidate how the RNAP subunits interact with each other and with template DNA., encoded by the E. coli rpoZ gene, is the smallest RNAP subunit at only 10 kDa. has homologs in all three kingdoms of life. It is present in all sequenced freeliving bacteria (although some intracellular parasitic bacteria, such as Chlamydia sp., appear to lack an homolog), in archaea (RpoK), and in eukaryotes (RPB6) (Minakhin et al. 2001). The RNAP structures indicate that there is one copy of per RNAP, and that it interacts with ␤Ј conserved regions D and G and wraps over and around the ␤Ј C-terminal tail, latching ␤Ј to the ␣ 2 ␤ subassembly (Minakhin et al. 2001). The RNAP structures therefore are consistent with the model that functions as a chaperone in enzyme assembly by facilitating the binding of ␤Ј to ␣ 2 ␤ (Gentry and Burgess 1993; Mukherjee et al. 1999;Ghosh et al. 2001Ghosh et al. , 2003. In support of this view, reconstitution of RNAP from its individual subunits is less efficient in the absence of (Mukherjee and Chatterji 1997).In contrast to the insights that the structures of RNAP provide about a role for in enzyme assembly, the str...