Structural basis of archaeal FttA-dependent transcription termination.

The ribonuclease FttA (also known as aCPSF and aCPSF1) mediates factor-dependent transcription termination in archaea. Here we report the structure of a Thermococcus kodakarensis transcription pre-termination complex comprising FttA, Spt4, Spt5 and a transcription elongation complex (TEC). The structure shows that FttA interacts with the TEC in a manner that enables RNA to proceed directly from the TEC RNA-exit channel to the FttA catalytic centre and that enables endonucleolytic cleavage of RNA by FttA, followed by 5'→3' exonucleolytic cleavage of RNA by FttA and concomitant 5'→3' translocation of FttA on RNA, to apply mechanical force to the TEC and trigger termination.

Structural basis of RfaH-mediated transcription-translation coupling.

The NusG paralog RfaH mediates bacterial transcription-translation coupling in genes that contain a DNA sequence element, termed an ops site, required for pausing RNA polymerase (RNAP) and for loading RfaH onto the paused RNAP. Here, we report cryo-electron microscopy structures of transcription-translation complexes (TTCs) containing Escherichia coli RfaH. The results show that RfaH bridges RNAP and the ribosome, with the RfaH N-terminal domain interacting with RNAP and the RfaH C-terminal domain interacting with the ribosome.

Structural basis of long-range transcription-translation coupling.

Structures recently have been reported of molecular assemblies that mediate transcription-translation coupling in . In these molecular assemblies, termed "coupled transcription-translation complexes" or "TTC-B", RNA polymerase (RNAP) interacts directly with the ribosome, the transcription elongation factor NusG or its paralog RfaH forms a bridge between RNAP and ribosome, and the transcription elongation factor NusA optionally forms a second bridge between RNAP and ribosome. Here, we have determined structures of coupled transcription-translation complexes having mRNA spacers between RNAP and ribosome longer than the maximum-length mRNA spacer compatible with formation of TTC-B.

Structural basis of RfaH-mediated transcription-translation coupling.

Unlabelled: The NusG paralog RfaH mediates bacterial transcription-translation coupling on genes that contain a DNA sequence element, termed an site, required for pausing RNA polymerase (RNAP) and for loading RfaH onto the paused RNAP. Here we report cryo-EM structures of transcription-translation complexes (TTCs) containing RfaH. The results show that RfaH bridges RNAP and the ribosome, with the RfaH N-terminal domain interacting with RNAP, and with the RfaH C-terminal domain interacting with the ribosome.

The Mysterious Amurian Grig Storozhenko, 1980 (Orthoptera: Prophalangopsidae): New Data on Its Distribution, Ecology and Biology.

New data on distribution, ecology and biology of the rare extant species of the almost completely ancient family Prophalangopsidae (Orthoptera) are given. This montane species prefers humid areas with relatively low summer temperatures. Habits, mating behaviour and life history of are extremely similar to those of the North American representatives of the genus .

Structural basis of archaeal FttA-dependent transcription termination.

Unlabelled: The ribonuclease FttA mediates factor-dependent transcription termination in archaea . Here, we report the structure of a transcription pre-termination complex comprising FttA, Spt4, Spt5, and a transcription elongation complex (TEC). The structure shows that FttA interacts with the TEC in a manner that enables RNA to proceed directly from the TEC RNA-exit channel to the FttA catalytic center and that enables endonucleolytic cleavage of RNA by FttA, followed by 5'→3' exonucleolytic cleavage of RNA by FttA and concomitant 5'→3' translocation of FttA on RNA, to apply mechanical force to the TEC and trigger termination.

Structural basis of Rho-dependent transcription termination.

Rho is a ring-shaped hexameric ATP-dependent molecular motor. Together with the transcription elongation factor NusG, Rho mediates factor-dependent transcription termination and transcription-translation-coupling quality control in Escherichia coli. Here we report the preparation of complexes that are functional in factor-dependent transcription termination from Rho, NusG, RNA polymerase (RNAP), and synthetic nucleic acid scaffolds, and we report cryogenic electron microscopy structures of the complexes.

Redesign of Rifamycin Antibiotics to Overcome ADP-Ribosylation-Mediated Resistance.

Rifamycin antibiotics are a valuable class of antimicrobials for treating infections by mycobacteria and other persistent bacteria owing to their potent bactericidal activity against replicating and non-replicating pathogens. However, the clinical utility of rifamycins against Mycobacterium abscessus is seriously compromised by a novel resistance mechanism, namely, rifamycin inactivation by ADP-ribosylation. Using a structure-based approach, we rationally redesign rifamycins through strategic modification of the ansa-chain to block ADP-ribosylation while preserving on-target activity.

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 σ-dependent pausing in the presence of GreB in vitro and in vivo predominantly involves a state scrunched by 2–3 bp.

Design, Synthesis, and Characterization of TNP-2198, a Dual-Targeted Rifamycin-Nitroimidazole Conjugate with Potent Activity against Microaerophilic and Anaerobic Bacterial Pathogens.

TNP-2198, a stable conjugate of a rifamycin pharmacophore and a nitroimidazole pharmacophore, has been designed, synthesized, and evaluated as a novel dual-targeted antibacterial agent for the treatment of microaerophilic and anaerobic bacterial infections. TNP-2198 exhibits greater activity than a 1:1 molar mixture of the parent drugs and exhibits activity against strains resistant to both rifamycins and nitroimidazoles. A crystal structure of TNP-2198 bound to a RNA polymerase transcription initiation complex reveals that the rifamycin portion of TNP-2198 binds to the rifamycin binding site on RNAP and the nitroimidazole portion of TNP-2198 interacts directly with the DNA template-strand in the RNAP active-center cleft, forming a hydrogen bond with a base of the DNA template strand.

The Handsome Cross Grasshopper (Germar, 1825) (Orthoptera: Acrididae) as a Neglected Pest in the South-Eastern Part of West Siberian Plain.

is a widely distributed acridid over the Eurasian semi-arid territories, from the Atlantic coast to the Pacific coast. In many semi-arid territories, was and is the most important pest, but in the south-eastern part of West Siberian Plain, it was not considered a pest until the 1960s. We compared two sets of data on the acridid distribution in the region: before 1960 and from 1961 until 2021.

Structural basis of RNA polymerase recycling by the Swi2/Snf2 family of ATPase RapA in Escherichia coli.

After transcription termination, cellular RNA polymerases (RNAPs) are occasionally trapped on DNA, impounded in an undefined post-termination complex (PTC), limiting the free RNAP pool and subsequently leading to inefficient transcription. In Escherichia coli, a Swi2/Snf2 family of ATPase called RapA is known to be involved in countering such inefficiency through RNAP recycling; however, the precise mechanism of this recycling is unclear. To better understand its mechanism, here we determined the structures of two sets of E.

Evaluation of Bacterial RNA Polymerase Inhibitors in a -Based Wound Infection Model in SKH1 Mice.

Chronic wounds infected with pathogens such as represent a worldwide health concern, especially in patients with a compromised immune system. As antimicrobial resistance has become an immense global problem, novel antibiotics are urgently needed. One strategy to overcome this threatening situation is the search for drugs targeting novel binding sites on essential and validated enzymes such as the bacterial RNA polymerase (RNAP).

Structural basis of transcription-translation coupling.

In bacteria, transcription and translation are coupled processes in which the movement of RNA polymerase (RNAP)-synthesizing messenger RNA (mRNA) is coordinated with the movement of the first ribosome-translating mRNA. Coupling is modulated by the transcription factors NusG (which is thought to bridge RNAP and the ribosome) and NusA. Here, we report cryo-electron microscopy structures of transcription-translation complexes (TTCs) containing different-length mRNA spacers between RNAP and the ribosome active-center P site.

RNA extension drives a stepwise displacement of an initiation-factor structural module in initial transcription.

All organisms-bacteria, archaea, and eukaryotes-have a transcription initiation factor that contains a structural module that binds within the RNA polymerase (RNAP) active-center cleft and interacts with template-strand single-stranded DNA (ssDNA) in the immediate vicinity of the RNAP active center. This transcription initiation-factor structural module preorganizes template-strand ssDNA to engage the RNAP active center, thereby facilitating binding of initiating nucleotides and enabling transcription initiation from initiating mononucleotides. However, this transcription initiation-factor structural module occupies the path of nascent RNA and thus presumably must be displaced before or during initial transcription.

Mode of Action of Kanglemycin A, an Ansamycin Natural Product that Is Active against Rifampicin-Resistant Mycobacterium tuberculosis.

Antibiotic-resistant bacterial pathogens pose an urgent healthcare threat, prompting a demand for new medicines. We report the mode of action of the natural ansamycin antibiotic kanglemycin A (KglA). KglA binds bacterial RNA polymerase at the rifampicin-binding pocket but maintains potency against RNA polymerases containing rifampicin-resistant mutations.

Structure-function comparisons of (p)ppApp vs (p)ppGpp for Escherichia coli RNA polymerase binding sites and for rrnB P1 promoter regulatory responses in vitro.

Precise regulation of gene expression is crucial for bacteria to respond to changing environmental conditions. In addition to protein factors affecting RNA polymerase (RNAP) activity, second messengers play an important role in transcription regulation, such as well-known effectors of the stringent response: guanosine 5'triphosphate-3'diphosphate and guanosine 3', 5'-bis(diphosphate) [(p)ppGpp]. Although much is known about importance of the 5' and 3' moieties of (p)ppGpp, the role of the guanine base remains somewhat cryptic.

Minimalism and functionality: Structural lessons from the heterodimeric N4 bacteriophage RNA polymerase II.

Genomes of phages, mitochondria, and chloroplasts are transcribed by a diverse group of transcriptional machineries with structurally related single-subunit RNA polymerases (RNAPs). Our understanding of transcription mechanisms of these enzymes is predominantly based on biochemical and structural studies of three most-studied members, transcription factor-independent phage T7 RNAP, transcription factor-dependent phage N4 virion-encapsidated RNAP, and transcription factor-dependent mitochondrial RNAPs (mtRNAP). Although these RNAPs employ completely different mechanisms for promoter recognition and transcription termination, these enzymes are relatively large and formed by single polypeptides.

Allosteric Effector ppGpp Potentiates the Inhibition of Transcript Initiation by DksA.

DksA and ppGpp are the central players in the stringent response and mediate a complete reprogramming of the transcriptome. A major component of the response is a reduction in ribosome synthesis, which is accomplished by the synergistic action of DksA and ppGpp bound to RNA polymerase (RNAP) inhibiting transcription of rRNAs. Here, we report the X-ray crystal structures of Escherichia coli RNAP in complex with DksA alone and with ppGpp.

X-ray crystal structure of a reiterative transcription complex reveals an atypical RNA extension pathway.

Reiterative transcription is a noncanonical form of RNA synthesis in which a nucleotide specified by a single base in the DNA template is repetitively added to the nascent transcript. Here we determined the crystal structure of an RNA polymerase, the bacterial enzyme from , engaged in reiterative transcription during transcription initiation at a promoter resembling the promoter of The structure reveals that the reiterative transcript detours from the dedicated RNA exit channel and extends toward the main channel of the enzyme, thereby allowing RNA extension without displacement of the promoter recognition σ-factor. Nascent transcripts containing reiteratively added G residues are eventually extended by nonreiterative transcription, revealing an atypical pathway for the formation of a transcription elongation complex.

Gene expression profiling of tumor-initiating stem cells from mouse Krebs-2 carcinoma using a novel marker of poorly differentiated cells.

Using the ability of poorly differentiated cells to natively internalize fragments of extracellular double-stranded DNA as a marker, we isolated a tumorigenic subpopulation present in Krebs-2 ascites that demonstrated the features of tumor-inducing cancer stem cells. Having combined TAMRA-labeled DNA probe and the power of RNA-seq technology, we identified a set of 168 genes specifically expressed in TAMRA-positive cells (tumor-initiating stem cells), these genes remaining silent in TAMRA-negative cancer cells. TAMRA+ cells displayed gene expression signatures characteristic of both stem cells and cancer cells.

Novel Chemical Scaffolds for Inhibition of Rifamycin-Resistant RNA Polymerase Discovered from High-Throughput Screening.

Rifampin has been a cornerstone of tuberculosis (TB) treatment since its introduction. The rise of multidrug-resistant and extensively drug-resistant TB makes the development of novel therapeutics effective against these strains an urgent need. Site-specific mutations in the target enzyme of rifampin, RNA polymerase (RNAP) comprises the majority (~97%) of rifamycin-resistant (Rif) strains of Mycobacterium tuberculosis (MTB).

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

Since 1967, Rifampin (RMP, a Rifamycin) has been used as a first line antibiotic treatment for tuberculosis (TB), and it remains the cornerstone of current short-term TB treatment. Increased occurrence of Rifamycin-resistant (RIF ) TB, ∼41% of which results from the RpoB S531L mutation in RNA polymerase (RNAP), has become a growing problem worldwide. In this study, we determined the X-ray crystal structures of the Escherichia coli RNAPs containing the most clinically important S531L mutation and two other frequently observed RIF mutants, RpoB D516V and RpoB H526Y.

X-ray crystal structures of Escherichia coli RNA polymerase with switch region binding inhibitors enable rational design of squaramides with an improved fraction unbound to human plasma protein.

Squaramides constitute a novel class of RNA polymerase inhibitors of which genetic evidence and computational modeling previously have suggested an inhibitory mechanism mediated by binding to the RNA polymerase switch region. An iterative chemistry program increased the fraction unbound to human plasma protein from below minimum detection levels, i.e.

Alignathon: a competitive assessment of whole-genome alignment methods.

Multiple sequence alignments (MSAs) are a prerequisite for a wide variety of evolutionary analyses. Published assessments and benchmark data sets for protein and, to a lesser extent, global nucleotide MSAs are available, but less effort has been made to establish benchmarks in the more general problem of whole-genome alignment (WGA). Using the same model as the successful Assemblathon competitions, we organized a competitive evaluation in which teams submitted their alignments and then assessments were performed collectively after all the submissions were received.