Allosteric Modulation of the RNA Polymerase Catalytic Reaction Is an Essential Component of Transcription Control by Rifamycins

SummaryRifamycins, the clinically important antibiotics, target bacterial RNA polymerase (RNAP). A proposed mechanism in which rifamycins sterically block the extension of nascent RNA beyond three nucleotides does not alone explain why certain RNAP mutations confer resistance to some but not other rifamycins. Here we show that unlike rifampicin and rifapentin, and contradictory to the steric model, rifabutin inhibits formation of the first and second phosphodiester bonds. We report 2.5 Å resolution structures of rifabutin and rifapentin complexed with the Thermus thermophilus RNAP holoenzyme. The structures reveal functionally important distinct interactions of antibiotics with the initiation σ factor. Strikingly, both complexes lack the catalytic Mg2+ ion observed in the apo-holoenzyme, whereas an increase in Mg2+ concentration confers resistance to rifamycins. We propose that a rifamycin-induced signal is transmitted over ∼19 Å to the RNAP active site to slow down catalysis. Based on structural predictions, we designed enzyme substitutions that apparently interrupt this allosteric signal

Structure of RNA polymerase bound to ribosomal 30S subunit

In bacteria, mRNA transcription and translation are coupled to coordinate optimal gene expression and maintain genome stability. Coupling is thought to involve direct interactions between RNA polymerase (RNAP) and the translational machinery. We present cryo-EM structures of E. coli RNAP core bound to the small ribosomal 30S subunit. The complex is stable under cell-like ionic conditions, consistent with functional interaction between RNAP and the 30S subunit. The RNA exit tunnel of RNAP aligns with the Shine-Dalgarno-binding site of the 30S subunit. Ribosomal protein S1 forms a wall of the tunnel between RNAP and the 30S subunit, consistent with its role in directing mRNAs onto the ribosome. The nucleic-acid-binding cleft of RNAP samples distinct conformations, suggesting different functional states during transcription-translation coupling. The architecture of the 30S*RNAP complex provides a structural basis for co-localization of the transcriptional and translational machineries, and inform future mechanistic studies of coupled transcription and translation

Allosteric control of the RNA polymerase by the elongation factor RfaH

Efficient transcription of long polycistronic operons in bacteria frequently relies on accessory proteins but their molecular mechanisms remain obscure. RfaH is a cellular elongation factor that acts as a polarity suppressor by increasing RNA polymerase (RNAP) processivity. In this work, we provide evidence that RfaH acts by reducing transcriptional pausing at certain positions rather than by accelerating RNAP at all sites. We show that ‘fast’ RNAP variants are characterized by pause-free RNA chain elongation and are resistant to RfaH action. Similarly, the wild-type RNAP is insensitive to RfaH in the absence of pauses. In contrast, those enzymes that may be prone to falling into a paused state are hypersensitive to RfaH. RfaH inhibits pyrophosphorolysis of the nascent RNA and reduces the apparent Michaelis–Menten constant for nucleotides, suggesting that it stabilizes the post-translocated, active RNAP state. Given that the RfaH-binding site is located 75 Å away from the RNAP catalytic center, these results strongly indicate that RfaH acts allosterically. We argue that despite the apparent differences in the nucleic acid targets, the time of recruitment and the binding sites on RNAP, unrelated antiterminators (such as RfaH and λQ) utilize common strategies during both recruitment and anti-pausing modification of the transcription complex

Highly Divergent RfaH Orthologs from Pathogenic Proteobacteria Can Substitute for Escherichia coli RfaH both In Vivo and In Vitro

The transcriptional enhancer protein RfaH positively regulates production of virulence factors in Escherichia coli and Salmonella enterica serovar Typhimurium via a cis element, ops. Genes coding for RfaH orthologs were identified in conceptually translated genomes of bacterial pathogens, including Vibrio and Yersinia spp. We cloned the rfaH genes from Vibrio cholerae, Yersinia enterocolitica, S. enterica serovar Typhimurium, and Klebsiella pneumoniae into E. coli expression vectors. Purified RfaH orthologs, including the most divergent one from V. cholerae, were readily recruited to the E. coli transcription elongation complex. Postrecruitment stimulation of transcript elongation appeared to vary with the degree of similarity to E. coli RfaH. V. cholerae RfaH was particularly defective in reducing downstream pausing and termination; this defect was substantially alleviated by an increase in its concentration. When overexpressed episomally, all of the rfaH genes complemented the disruption of the chromosomal copy of the E. coli gene. Thus, despite the apparently accelerated divergent evolution of the RfaH proteins, the mechanism of their action is conserved well enough to make them transcriptionally active in the E. coli system

Usuwanie jonów fluorkowych z wody kopalnianej

Murmansk Region is home to some major mining and mineral sites. One of the most challenging environmental problems in the mining industry is mine water treatment. For example, the rocks of the deposit operated by Lovozero Mining and Mineral Processing Company contain villiomite (NaF). It is highly soluble in water, and the mine waters at the site have a high content of fluoride ions – significantly above the maximum permissible values. Lab-scale experiments were conducted to test various reagents and different initial concentrations of fluoride ions in the treatment of model solutions and mine water. Depending on the initial concentrations, magnesium and calcium-containing sorbents are proposed for the defluorization of water. Using scanning electron microscopy and microprobe analysis, it was found that fluorine can be bound in poorly soluble compounds, such as, for example, fluorite. Pilot trials are planned.Region Murmański jest regionem silnie uprzemysłowionym, jest lokalizacją wielu kopalń. Jednym z najtrudniejszych problemów środowiskowych w górnictwie jest uzdatnianie wód kopalnianych. Przedstawiono przykład kopalni Lovozero Mining and Mineral Processing. Złoże zawiera villiaumit (NaF). Jest to minerał dobrze rozpuszczalny w wodzie. Wody kopalniane w tym miejscu mają wysoką zawartość jonów fluorkowych - znacznie powyżej maksymalnych dopuszczalnych wartości. Przeprowadzono eksperymenty na skalę laboratoryjną w celu przetestowania różnych odczynników i różnych początkowych stężeń jonów fluorkowych w obróbce roztworów modelowych i wody kopalnianej. W zależności od początkowych stężeń do odfluoryzacji wody proponuje się sorbenty zawierające magnez i wapń. Za pomocą skaningowej mikroskopii elektronowej i analizy mikropróbek stwierdzono, że fluor może wiązać się w słabo rozpuszczalnych związkach, takich jak na przykład fluoryt. Planowane są próby pilotażowe

Hydrometallurgical Processing of Low-Grade Sulfide Ore and Mine Waste in the Arctic Regions: Perspectives and Challenges

The authors describe the opportunities of low-grade sulfide ores and mine waste processing with heap and bacterial leaching methods. By the example of gold and silver ores, we analyzed specific issues and processing technologies for heap leaching intensification in severe climatic conditions. The paper presents perspectives for heap leaching of sulfide and mixed ores from the Udokan (Russia) and Talvivaara (Finland) deposits, as well as technogenic waste dumps, namely, the Allarechensky Deposit Dumps (Russia). The paper also shows the laboratory results of non-ferrous metals leaching from low-grade copper-nickel ores of the Monchepluton area, and from tailings of JSC Kola Mining and Metallurgical Company