New target for inhibition of bacterial RNA polymerase: 'switch region'

A new drug target - the 'switch region' - has been identified within bacterial RNA polymerase (RNAP), the enzyme that mediates bacterial RNA synthesis. The new target serves as the binding site for compounds that inhibit bacterial RNA synthesis and kill bacteria. Since the new target is present in most bacterial species, compounds that bind to the new target are active against a broad spectrum of bacterial species. Since the new target is different from targets of other antibacterial agents, compounds that bind to the new target are not cross-resistant with other antibacterial agents. Four antibiotics that function through the new target have been identified: myxopyronin, corallopyronin, ripostatin, and lipiarmycin. This review summarizes the switch region, switch-region inhibitors, and implications for antibacterial drug discovery.     

Effects of deficiency and overdose of group 2 sigma factors in triple inactivation strains of Synechocystis sp. strain PCC 6803

Acclimation of cyanobacteria to environmental changes includes major changes in the gene expression patterns partly orchestrated by the replacement of a particular σ subunit with another in the RNA polymerase holoenzyme. The cyanobacterium Synechocystis sp. strain PCC 6803 encodes nine σ factors, all belonging to the σ(70) family. Cyanobacteria typically encode many group 2 σ factors that closely resemble the principal σ factor. We inactivated three out of the four group 2 σ factors of Synechocystis simultaneously in all possible combinations and found that all triple inactivation strains grow well under standard conditions. Unlike the other strains, the ΔsigBCD strain, which contains SigE as the only functional group 2 σ factor, did not grow faster under mixotrophic than under autotrophic conditions. The SigB and SigD factors were important in low-temperature acclimation, especially under diurnal light rhythm. The ΔsigBCD, ΔsigBCE, and ΔsigBDE strains were sensitive to high-light-induced photoinhibition, indicating a central role of the SigB factor in high-light tolerance. Furthermore, the ΔsigBCE strain (SigD is the only functional group 2 σ factor) appeared to be locked in the high-fluorescence state (state 1) and grew slowly in blue but not in orange or white light. Our results suggest that features of the triple inactivation strains can be categorized as (i) direct consequences of the inactivation of a particular σ factor(s) and (ii) effects resulting from the higher probability that the remaining group 2 σ factors associate with the RNA polymerase core.     

The molar ratio of σ73 to core polymerase in the obligate intracellular bacterium, Rickettsia prowazekii

In the obligate Intracellular parasitic bacterium, Rickettsia prowazekii, the molar ratio of σ⁷³ to core RNA polymerase, that is, the degree of saturation of the core polymerase by the catalytically active sigma factor, was very low. This ratio was determined from the radioactivity in rickettsial RNA polymerase immuno-precipitated from crude extracts of infected L929 cells in which the parasite was exponentially growing. If we assume that, as Is true for the σ subunit, in R. prowazekii and Escherichia coli the β’and β subunits of the RNA polymerase have similar methionine and cysteine contents (the radiolabelled amino acids), the molar ratio of σ⁷³ to core polymerase in R prowazekii would be 0.1. This is in striking contrast to E. Coli where the ratio is typically 0.4. it remains to be established whether this low sigma saturation results In a limitation of active RNA polymerase in R. prowazekii and contributes to its slow growth.     

Biochemical studies on the potentiation of antitumor activity of cisplatin by adriamycin

The RNA synthesis in vitro by E. coli RNA polymerase was found to be highly sensitive to cis-diamminedichloroplatinum (cisplatin) inhibition. The degree of inhibition was in proportion to the length of time of template preincubation with cisplatin, suggesting that cisplatin-template binding was involved in the inhibition of RNA polymerase. The effect of adriamycin on this inhibition was studied and it was found that adriamycin significantly enhanced the inhibitory effect of cisplatin and that the total effect was greater than the sum of the effects of each drug used individually. This synergistic effect was not observed when the effect of the combination of adriamycin and cisplatin on in vitro DNA synthesis was studied.     

DNA-binding properties of the Bacillus subtilis and Aeribacillus pallidus AC6 σ(D) proteins

σ(D) proteins from Aeribacillus pallidus AC6 and Bacillus subtilis bound specifically, albeit weakly, to promoter DNA even in the absence of core RNA polymerase. Binding required a conserved CG motif within the -10 element, and this motif is known to be recognized by σ region 2.4 and critical for promoter activity.     

Subunits of RNA polymerase in function and structure; Maturation in vitro of core enzyme from Escherichia coli

Reconstitution of Escherichia coli RNA polymerase was found to be markedly enhanced by DNA as well as by the σ subunit. Among discrete steps of subunit assembly, formation of the primary intermediate α₂β complex and subsequent association of the complex with the β′ subunit are not affected by the presence of DNA and the σ subunit; the α₂ββ′ complex thus formed, however, is virtually inactive and is subject to temperature-dependent activation by DNA and the σ subunit. The α₂ββ′ complex is, therefore, a secondary intermediate in the sequence of enzyme formation, or a premature form of core enzyme.In the course of activation of the premature core complex, the subunit σ interacts with both the α₂β complex and the β′ subunit; DNA acts in much the same way. The enzyme, reconstituted in the presence of DNA, is recovered attached to the DNA, added as an enhancer, and initiates RNA synthesis without prior release from the DNA. A limited number of unique DNA sites appear to be concerned with the enzyme maturation.     

Limited digestion of RNA polymerase from Escherichia coli by trypsin (effect of rifamycin and DNA on the integrity of sigma subunit)

A modified polyacrylamide gel electrophoresis technique is used to separate the polypeptides after digestion of $\text{E. coli}$ RNA polymerase with various concentration of trypsin. The subunits β and β′ and two large breakdown products of molecular weight of 147,000 and 141,000 are distinctly separated. At a very low level of trypsin σ and α are not cleaved while two major breakdown products of molecular weights of 110,000 and 43,000 appear from the larger subunits. At a still higher level of trypsin σ is converted to a polypeptide of molecular weight of 86,000 and other small fragments. DNA protects, to some extent, the σ and this polypeptide and also β and the two large breakdown products from trypsin digestion. It is also observed that rifamycin, an inhibitor of RNA synthesis, enhances the tryptic digestion of σ, only in the absence of MgCl₂.