dna footprinting
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eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Himasha M Perera ◽  
Michael A Trakselis

DNA replication requires that the duplex genomic DNA strands be separated; a function that is implemented by ring-shaped hexameric helicases in all Domains. Helicases are composed of two domains, an N- terminal DNA binding domain (NTD) and a C- terminal motor domain (CTD). Replication is controlled by loading of helicases at origins of replication, activation to preferentially encircle one strand, and then translocation to begin separation of the two strands. Using a combination of site-specific DNA footprinting, single-turnover unwinding assays, and unique fluorescence translocation monitoring, we have been able to quantify the binding distribution and the translocation orientation of Saccharolobus (formally Sulfolobus) solfataricus MCM on DNA. Our results show that both the DNA substrate and the C-terminal winged-helix (WH) domain influence the orientation but that translocation on DNA proceeds N-first.


2018 ◽  
Vol 293 (43) ◽  
pp. 16741-16750 ◽  
Author(s):  
Chen Zhu ◽  
Yu Liu ◽  
Lihua Hu ◽  
Min Yang ◽  
Zheng-Guo He

Isoniazid (INH) and ethambutol (EMB) are two major first-line drugs for managing tuberculosis (TB), caused by the microbe Mycobacterium tuberculosis. Although co-use of these two drugs is common in clinical practice, the mechanism for the potential synergistic interplay between them remains unclear. Here, we present first evidence that INH and EMB act synergistically through a transcriptional repressor of the inhA gene, the target gene of INH encoding an enoyl–acyl carrier protein reductase of the fatty acid synthase type II system required for bacterial cell wall integrity. We report that EMB binds a hypothetical transcription factor encoded by the Rv0273c gene, designated here as EtbR. Using DNA footprinting, we found that EtbR specifically recognizes a motif sequence in the upstream region of the inhA gene. Using isothermal titration calorimetry and surface plasmon resonance assays, we observed that EMB binds EtbR in a 1:1 ratio and thereby stimulates its DNA-binding activity. When a nonlethal dose of EMB was delivered in combination with INH, EMB increased the INH susceptibility of cultured M. tuberculosis cells. In summary, EMB induces EtbR-mediated repression of inhA and thereby enhances the mycobactericidal effect of INH. Our findings uncover a molecular mechanism for the synergistic activity of two important anti-TB drugs.


mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Ngoc-Phuong-Thao Nguyen ◽  
Sonja Linder ◽  
Stefanie K. Flitsch ◽  
Bettina Schiel-Bengelsdorf ◽  
Peter Dürre ◽  
...  

ABSTRACT An operon comprising two genes, CA_P0037 and CA_P0036 , that encode proteins of unknown function that were previously shown to be highly expressed in acidogenic cells and repressed in solventogenic and alcohologenic cells is located on the pSOL1 megaplasmid of Clostridium acetobutylicum upstream of adhE2 . A CA_P0037 :: int ( 189/190s ) mutant in which an intron was inserted at position 189/190 in the sense strand of CA_P0037 was successfully generated by the Targetron technique. The resultant mutant showed significantly different metabolic flux patterns in acidogenic (producing mainly lactate, butyrate, and butanol) and alcohologenic (producing mainly butyrate, acetate, and lactate) chemostat cultures but not in solventogenic or batch cultures. Transcriptomic investigation of the CA_P0037 :: int ( 189/190s ) mutant showed that inactivation of CA_P0037 significantly affected the expression of more than 258 genes under acidogenic conditions. Surprisingly, genes belonging to the Fur regulon, involved in iron transport ( CA_C1029-CA_C1032 ), or coding for the main flavodoxin ( CA_C0587 ) were the most significantly expressed genes under all conditions, whereas fur (coding for the ferric uptake regulator) gene expression remained unchanged. Furthermore, most of the genes of the Rex regulon, such as the adhE2 and ldhA genes, and of the PerR regulon, such as rbr3A-rbr3B and dfx , were overexpressed in the mutant. In addition, the whole CA_P0037-CA_P0036 operon was highly expressed under all conditions in the CA_P0037 :: int ( 189/190s ) mutant, suggesting a self-regulated expression mechanism. Cap0037 was shown to bind to the CA_P0037-CA_P0036 operon, sol operon, and adc promoters, and the binding sites were determined by DNA footprinting. Finally, a putative Cap0037 regulon was generated using a bioinformatic approach . IMPORTANCE Clostridium acetobutylicum is well-known for its ability to produce solvents, especially n -butanol. Understanding the regulatory network of C. acetobutylicum will be crucial for further engineering to obtain a strain capable of producing n -butanol at high yield and selectivity. This study has discovered that the Cap0037 protein is a novel regulator of C. acetobutylicum that drastically affects metabolism under both acidogenic and alcohologenic fermentation conditions. This is pioneering work for further determining the regulatory mechanism of Cap0037 in C. acetobutylicum and studying the role of proteins homologous to Cap0037 in other members of the phylum Firmicutes .


Microbiology ◽  
2014 ◽  
Vol 160 (8) ◽  
pp. 1637-1647 ◽  
Author(s):  
Satya Deo Pandey ◽  
Mitali Choudhury ◽  
Manjula Sritharan

The influence of iron levels on the transcription of the hupB gene in Mycobacterium tuberculosis is the focus of this study. Studies in our laboratory showed HupB to be co-expressed with the two siderophores in low-iron organisms. Mycobactin biosynthesis is repressed by the IdeR–Fe2+ complex that binds the IdeR box in the mbtB promoter. Recently, we demonstrated the positive regulatory effect of HupB on mycobactin biosynthesis by demonstrating its binding to a 10 bp HupB box in the mbtB promoter. Earlier, we observed that HupB, expressed maximally in low-iron media (0.02 µg Fe ml−1; 0.36 µM Fe) was still detectable at 8 µg Fe ml−1 (144 µM Fe) when the siderophores were absent and complete repression was seen only at 12 µg Fe ml−1 (216 µM Fe). In this study, we observed elevated levels of hupB transcripts in iron-limited organisms. IdeR, and not FurA, functioned as the iron regulator, by binding to two IdeR boxes in the hupB promoter. Interestingly, the 10 bp HupB box, first reported in the mbtB promoter, was identified in the hupB promoter. Using DNA footprinting and electrophoretic mobility shift assays, we demonstrated the functionality of the HupB box and the two IdeR boxes. The high hupB transcript levels expressed by the organism and the in vitro protein–DNA interaction studies led us to hypothesize the sequence of events occurring in response to changes in the intracellular iron concentration, emphasizing the roles played by IdeR and HupB in iron homeostasis.


2013 ◽  
Vol 1830 (10) ◽  
pp. 4660-4668 ◽  
Author(s):  
Matteo Nadai ◽  
Giovanna Sattin ◽  
Giorgio Palù ◽  
Manlio Palumbo ◽  
Sara N. Richter

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