Structural basis for rifamycin resistance of bacterial RNA polymerase by the three most clinically important RpoB mutations found in Mycobacterium tuberculosis

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136512/1/mmi13606.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136512/2/mmi13606_am.pd

Structural and mechanistic basis of σ-dependent transcriptional pausing

In σ-dependent transcriptional pausing, the transcription initiation factor σ, translocating with RNA polymerase (RNAP), makes sequence-specific protein-DNA interactions with a promoter-like sequence element in the transcribed region, inducing pausing. It has been proposed that, in σ-dependent pausing, the RNAP active center can access off-pathway “backtracked” states that are substrates for the transcript-cleavage factors of the Gre family, and on-pathway “scrunched” states that mediate pause escape. Here, using site-specific protein-DNA photocrosslinking to define positions of the RNAP trailing and leading edges and of σ relative to DNA at the λPR’ promoter, we show directly that σ-dependent pausing in the absence of GreB in vitro predominantly involves a state backtracked by 2-4 bp, and that σ-dependent pausing in the presence of GreB in vitro and in vivo predominantly involves a state scrunched by 2-3 bp. Analogous experiments with a library of 47 (∼16,000) transcribed-region sequences show that the state scrunched by 2-3 bp--and only that state--is associated with the consensus sequence, T-3N-2Y-1G+1, (where -1 corresponds to the position of the RNA 3’ end), which is identical to the consensus for pausing in initial transcription, and which is related to the consensus for pausing in transcription elongation. Experiments with heteroduplex templates show that sequence information at position T-3 resides in the DNA nontemplate strand. A cryo-EM structure of a complex engaged in σ-dependent pausing reveals positions of DNA scrunching on the DNA nontemplate and template strands and suggests that position T-3 of the consensus sequence exerts its effects by facilitating scrunching

The simulation model for a flood management by flood control facilities

The rapid spread of storm floods over large areas requires flood management throughout the river basin by the creation an innovative system of flood control facilities of various functional purposes distributed in the area. The central part of the system is the hydro system with hydro power plant. In addition, the flood control facilities on the side tributaries with self-regulating reservoir are included in the system. To assess the effect of controlling extreme water discharges by flood control facilities, it is necessary to develop special mathematical models reflecting the specifics of their operation. Unified mathematical models of the operation modes of a hydro complex with hydroelectric power station and flood control facility are created. They are implemented in a computer program that provides the ability to determine the main parameters and operating characteristics of hydro systems when performing multivariate calculations in a wide range of initial data. This makes possible specifying the parameters and operation modes of each hydro system with the current economic and environmental requirements, to assess the energy-economic and environmental consequences in the operation of the system of flood control facilities distributed in the area. The article analyses the results of the extreme water discharge’s regulation by the hydro system on the main river and flood control facilities on the side tributaries, considering environmental requirements

The simulation model for a flood management by flood control facilities

The rapid spread of storm floods over large areas requires flood management throughout the river basin by the creation an innovative system of flood control facilities of various functional purposes distributed in the area. The central part of the system is the hydro system with hydro power plant. In addition, the flood control facilities on the side tributaries with self-regulating reservoir are included in the system. To assess the effect of controlling extreme water discharges by flood control facilities, it is necessary to develop special mathematical models reflecting the specifics of their operation. Unified mathematical models of the operation modes of a hydro complex with hydroelectric power station and flood control facility are created. They are implemented in a computer program that provides the ability to determine the main parameters and operating characteristics of hydro systems when performing multivariate calculations in a wide range of initial data. This makes possible specifying the parameters and operation modes of each hydro system with the current economic and environmental requirements, to assess the energy-economic and environmental consequences in the operation of the system of flood control facilities distributed in the area. The article analyses the results of the extreme water discharge’s regulation by the hydro system on the main river and flood control facilities on the side tributaries, considering environmental requirements

Production of Biohydrogen from Organ-Containing Waste for Use in Fuel Cells

The production of low-carbon hydrogen based on renewable energy sources is considered a promising direction in the development of the modern world economy. The purpose of the presented research is to develop technologies and study the processes of converting biogases into hydrogen, as well as its use in low-temperature fuel cells. The methodology for organizing a multi-stage laboratory experiment for obtaining biogas, its purification from impurities and, in the future, the production of biohydrogen was developed based on field studies in Peter the Great St. Petersburg Polytechnic University. The results of modeling studies have shown that during biogas reforming, it is possible to obtain a hydrogen mixture with a hydrogen content of 98% vol and methane 2% vol. Based on the results of the research, the possibility of using the significant potential of “weak” biogas containing methane 30–45% vol to produce biohydrogen (more than 93% vol) was proved. A technique for using biohydrogen in low-temperature fuel cells for energy generation has been substantiated and tested

Production of Biohydrogen from Organ-Containing Waste for Use in Fuel Cells

The production of low-carbon hydrogen based on renewable energy sources is considered a promising direction in the development of the modern world economy. The purpose of the presented research is to develop technologies and study the processes of converting biogases into hydrogen, as well as its use in low-temperature fuel cells. The methodology for organizing a multi-stage laboratory experiment for obtaining biogas, its purification from impurities and, in the future, the production of biohydrogen was developed based on field studies in Peter the Great St. Petersburg Polytechnic University. The results of modeling studies have shown that during biogas reforming, it is possible to obtain a hydrogen mixture with a hydrogen content of 98% vol and methane 2% vol. Based on the results of the research, the possibility of using the significant potential of “weak” biogas containing methane 30–45% vol to produce biohydrogen (more than 93% vol) was proved. A technique for using biohydrogen in low-temperature fuel cells for energy generation has been substantiated and tested

Structural basis of transcription initiation by bacterial RNA polymerase holoenzyme

12 p.-5 fig.-1 tab.The bacterial RNA polymerase (RNAP) holoenzyme containing σ factor initiates transcription at specific promoter sites by de novo RNA priming, the first step of RNA synthesis where RNAP accepts two initiating ribonucleoside triphosphates (iNTPs) and performs the first phosphodiester bond formation. We present the structure of de novo transcription initiation complex that reveals unique contacts of the iNTPs bound at the transcription start site with the template DNA and also with RNAP and demonstrate the importance of these contacts for transcription initiation. To get further insight into the mechanism of RNA priming, we determined the structure of initially transcribing complex of RNAP holoenzyme with 6-mer RNA, obtained by in crystallo transcription approach. The structure highlights RNAP-RNA contacts that stabilize the short RNA transcript in the active site and demonstrates that the RNA 5'-end displaces σ region 3.2 from its position near the active site, which likely plays a key role in σ ejection during the initiation-to-elongation transition. Given the structural conservation of the RNAP active site, the mechanism of de novo RNA priming appears to be conserved in all cellular RNAPs.This work was supported, in whole or in part, by National Institutes of Health Grant GM087350-A1 (to K. S. M.). This work was also supported in part by the Spanish government through Grant BFU2010-16336 (to C. F.-T.). This work was also supported by the Russian Foundation for Basic Research (Grants 14-04-01696 and 14-04-31994) and the Russian Academy of Sciences Presidium Program in Molecular and Cellular Biology (to A. K.).Peer reviewe