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 antibiotocs 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.

Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial RNA polymerase (RNAP). Here we report a cryo-EM structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5-Å resolution. The structure shows that Lpm binds at the base of the RNAP "clamp." The structure exhibits an open conformation of the RNAP clamp, suggesting that Lpm traps an open-clamp state. Single-molecule fluorescence resonance energy transfer experiments confirm that Lpm traps an open-clamp state and define effects of Lpm on clamp dynamics. We suggest that Lpm inhibits transcription by trapping an open-clamp state, preventing simultaneous interaction with promoter -10 and -35 elements. The results account for the absence of cross-resistance between Lpm and other RNAP inhibitors, account for structure-activity relationships of Lpm derivatives, and enable structure-based design of improved Lpm derivatives.

SUMMARY Capistruin, a ribosomally synthesized post-translationally modified peptide produced by Burkholderia thailandensis E264, efficiently inhibits growth of Burkholderia and closely related Pseudomonas strains. The functional target of capistruin is unknown. Capistruin is a threaded-lasso peptide (lariat peptide), comprising an N-terminal 9-amino-acid ring followed by a 10-amino-acid C-terminal tail that is threaded through the ring. The structure of capistruin is similar to that of microcin J25 (MccJ25), a threaded-lasso antibacterial peptide that is produced by some strains of Escherichia coli and targets DNA-dependent RNA polymerase (RNAP). Here, we show that capustruin, like MccJ25, inhibits wild-type E. coli RNAP but not mutant, MccJ25-resistant, E. coli RNAP. We show further that an E. coli strain resistant to MccJ25 due to a mutation in an RNAP subunit gene exhibits resistance to capistruin. The results indicate that the structural similarity of capistruin and MccJ25 reflects functional similarity and suggest that the functional target of capistruin, and possibly other threaded-lasso peptides, is bacterial RNAP.

The replication of the genome requires the removal of RNA primers from the Okazaki fragments and their replacement by DNA. In prokaryotes, this process is completed by DNA polymerase I by means of strand displacement DNA synthesis and 5-nuclease activity. Here, we demonstrate that the strand displacement DNA synthesis is facilitated by the collective participation of Ser 769 , Phe 771 , and Arg 841 present in the fingers subdomain of DNA polymerase I. The steady and presteady state kinetic analysis of the properties of appropriate mutant enzymes suggest that: (a) Ser 769 and Phe 771 together are involved in the strand separation via the formation of a flap structure, and (b) Arg 841 interacts with the template strand to achieve the optimal strand separation and DNA synthesis. The amino acid residues Ser 769 and Phe 771 are constituents of the O1-helix, which together with O and O2 helices form a 3-helix bundle structure. We note that this 3-helix bundle motif also exists in prokaryotic RNA polymerase. Thus in both DNA and RNA polymerases, this motif may have been adopted to achieve the strand separation function.Strand displacement synthesis is an essential process in the removal and replacement of RNA primer moieties of Okazaki fragments. In prokaryotes, DNA polymerase I (pol I) 3 carries out this function by its 5Ј-nuclease and 5Ј-3Ј polymerase activities. Whereas early studies indicate a coordination of 5Ј-nuclease with the polymerase activity (1), the precise mechanism underlying this process is not clear. It appears that the participation of 5Ј-nuclease activity is not necessary for the strand displacement synthesis because the Klenow fragment of Escherichia coli DNA polymerase I has been known to catalyze strand displacement DNA synthesis (2). Thus, the strand separation activity resides in the polymerase domain of pol I.Despite the availability of numerous DNA-bound structures of polymerases (3-8), no significant information pertaining to strand displacement could be discerned because none of these crystal structures contained sufficiently long single-stranded template overhang or a downstream doublestranded DNA. One exception to this is the DNA-bound crystal structure of mammalian DNA polymerase ␤, where the enzyme-DNA complex contains gapped DNA (9). However, this enzyme has no strand displacement activity. In the DNA-bound crystal structures of pol I family DNA polymerases, the immediate unpaired template nucleotide assumes a flipped conformation (6) such that it cannot pair with the incoming dNTP substrate. The base moiety of the template (n ϩ 1) 4 nucleotide is positioned out of the DNA helical axis by more than 90°(4, 10, 11). In the enzyme-DNA-dNTP bound ternary complex, this nucleotide rearranges its conformation and pairs with the incoming dNTP substrate (6). In the crystal structures of polymerases, because of the short length of the single-stranded template overhang, only few interactions of downstream DNA with the enzyme protein could be discerned (3, 4, 6 -8, 11-13 4 The numbering scheme for...

No abstract

MicroRNAs (miRNAs) are a group of small noncoding RNA molecules with significant capacity to regulate the gene expression at the post-transcriptional level in a sequence-specific manner either through translation repression or mRNA degradation triggering a fine-tuning biological impact. They have been implicated in several processes, including cell growth and development, signal transduction, cell proliferation and differentiation, metabolism, apoptosis, inflammation, and immune response modulation. However, over the last few years, extensive studies have shown the relevance of miRNAs in human pathophysiology. Common human parasitic diseases, such as Malaria, Leishmaniasis, Amoebiasis, Chagas disease, Schistosomiasis, Toxoplasmosis, Cryptosporidiosis, Clonorchiasis, and Echinococcosis are the leading cause of death worldwide. Thus, identifying and characterizing parasite-specific miRNAs and their host targets, as well as host-related miRNAs, are important for a deeper understanding of the pathophysiology of parasite-specific diseases at the molecular level. In this review, we have demonstrated the impact of human microRNAs during host−parasite interaction as well as their potential to be used for diagnosis and prognosis purposes.

The antibiotic myxopyronin (Myx) functions by inhibiting bacterial RNA polymerase (RNAP). The binding site on RNAP for Myx-the RNAP "switch region SW1/SW2 subregion"-is different from the binding site on RNAP for the RNAP inhibitor currently used in broad-spectrum antibacterial therapy, rifampin (Rif). Here, we report the frequency, spectrum, and fitness costs of Myx resistance in Staphylococcus aureus. The resistance rate for Myx is 4 ؋ 10 ؊8 to 7 ؋ 10 ؊8 per generation, which is equal within error to the resistance rate for Rif (3 ؋ 10 ؊8 to 10 ؋ 10 ؊8 per generation). Substitutions conferring Myx resistance were obtained in the RNAP ␤ subunit [six substitutions: V1080(1275)I, V1080(1275)L, E1084(1279)K, D1101(1296)E, S1127(1322)L, and S1127 (1322) Mf50 (18,20,23,49). Myx exhibits broad-spectrum antibacterial activity, with potent antibacterial activity against most Gram-positive species and some Gram-negative species. Myx is under investigation as a potential lead compound for broad-spectrum antibacterial therapy. M yxopyronin (Myx) is an ␣-pyrone antibiotic produced by Myxococcus fulvusMyx functions by inhibiting bacterial RNA polymerase (RNAP) (3,18,20,32,49). The binding site on RNAP for Myx is located in the RNAP "switch region" and comprises the RNAP switch region structural elements termed "switch 1" and "switch 2," (switch region SW1/SW2 subregion) (3,18,32,49). The binding site on RNAP for Myx is different from the binding site on RNAP for the RNAP inhibitor in current use in broad-spectrum antibacterial therapy, rifampin (Rif) (3,18,32,49). Accordingly, Myx exhibits no cross-resistance with Rif (18,19,32,33,49).Previous studies have provided information about spontaneous resistance frequencies and resistance spectra for Myx (31, 32). However, the fitness costs of resistance have not previously been assessed.In this work, we comprehensively evaluate the resistance properties of Myx in Staphylococcus aureus. We report (i) the spontaneous resistance rate for Myx in S. aureus, (ii) the spontaneous resistance spectrum of Myx in S. aureus, and (iii) the fitness costs of substitutions that confer Myx resistance. Hu et al. (19). Rif was purchased from Sigma, Inc. Bacterial strains were obtained from the American Type Culture Collection. MATERIALS AND METHODS Materials. (Ϯ)-Myx B was synthesized as described inMICs. Minimal inhibitory concentrations (MICs) in column 2 of Table 1 were quantified using broth microdilution assays (8). MICs in column 3 of Table 1, and in Tables 3 and 4, were quantified using spiral gradient endpoint assays, essentially as described previously (38,45,54). Spiral gradient endpoint assays employed 150-mm by 4-mm exponentialgradient plates containing Mueller-Hinton II cation-adjusted agar and 0.5 to 100 g/ml of Myx, 0.0008 to 0.2 g/ml of Rif, or 0.2 to 40 g/ml of Rif. Plates were prepared using an Autoplate 4000 spiral plater (Spiral Biotech, Inc.). Cells were grown to early log phase, adjusted to 1 ϫ 10 8 CFU/ml, and swabbed radially onto plates. Plates were incubated for 16 h at 3...

Aberrant pro-survival signaling is a hallmark of cancer cells, but the response to chemotherapy is poorly understood. In this study, we investigate the initial signaling response to standard induction chemotherapy in a cohort of 32 acute myeloid leukemia (AML) patients, using 36-dimensional mass cytometry. Through supervised and unsupervised machine learning approaches, we find that reduction of extracellular-signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) phosphorylation in the myeloid cell compartment 24 h post-chemotherapy is a significant predictor of patient 5-year overall survival in this cohort. Validation by RNA sequencing shows induction of MAPK target gene expression in patients with high phospho-ERK1/2 24 h post-chemotherapy, while proteomics confirm an increase of the p38 prime target MAPK activated protein kinase 2 (MAPKAPK2). In this study, we demonstrate that mass cytometry can be a valuable tool for early response evaluation in AML and elucidate the potential of functional signaling analyses in precision oncology diagnostics.