Bacteroides expand the functional versatility of a conserved transcription factor and transcribed DNA to program capsule diversity.

The genomes of human gut bacteria in the genus Bacteroides include numerous operons for biosynthesis of diverse capsular polysaccharides (CPSs). The first two genes of each CPS operon encode a locus-specific paralog of transcription elongation factor NusG (called UpxY), which enhances transcript elongation, and a UpxZ protein that inhibits noncognate UpxYs. This process, together with promoter inversions, ensures that a single CPS operon is transcribed in most cells.

The yin and yang of the universal transcription factor NusG.

RNA polymerase (RNAP), the central enzyme of transcription, intermittently pauses during the elongation stage of RNA synthesis. Pausing provides an opportunity for regulatory events such as nascent RNA folding or the recruitment of transregulators. NusG (Spt5 in eukaryotes and archaea) regulates RNAP pausing and is the only transcription factor conserved across all cellular life.

In Vitro Transcription Assay to Quantify Effects of H-NS Filaments on RNA Chain Elongation by RNA Polymerase.

Bacterial chromosomal DNA is structured and compacted by proteins known as bacterial chromatin proteins (i.e., nucleoid-associated proteins or NAPs).

expand the functional versatility of a universal transcription factor and transcribed DNA to program capsule diversity.

Human gut species encode numerous (eight or more) tightly regulated capsular polysaccharides (CPS). Specialized paralogs of the universal transcription elongation factor NusG, called UpxY (Y), and an anti-Y UpxZ (Z) are encoded by the first two genes of each CPS operon. The Y-Z regulators combine with promoter inversions to limit CPS transcription to a single operon in most cells.

Bacterial chromatin proteins, transcription, and DNA topology: Inseparable partners in the control of gene expression.

DNA in bacterial chromosomes is organized into higher-order structures by DNA-binding proteins called nucleoid-associated proteins (NAPs) or bacterial chromatin proteins (BCPs). BCPs often bind to or near DNA loci transcribed by RNA polymerase (RNAP) and can either increase or decrease gene expression. To understand the mechanisms by which BCPs alter transcription, one must consider both steric effects and the topological forces that arise when DNA deviates from its fully relaxed double-helical structure.

RNA polymerase SI3 domain modulates global transcriptional pausing and pause-site fluctuations.

Transcriptional pausing aids gene regulation by cellular RNA polymerases (RNAPs). A surface-exposed domain inserted into the catalytic trigger loop (TL) of Escherichia coli RNAP, called SI3, modulates pausing and is essential for growth. Here we describe a viable E.

Compensatory evolution in NusG improves fitness of drug-resistant M. tuberculosis.

Drug-resistant bacteria are emerging as a global threat, despite frequently being less fit than their drug-susceptible ancestors. Here we sought to define the mechanisms that drive or buffer the fitness cost of rifampicin resistance (RifR) in the bacterial pathogen Mycobacterium tuberculosis (Mtb). Rifampicin inhibits RNA polymerase (RNAP) and is a cornerstone of modern short-course tuberculosis therapy.

Evolutionary flexibility and rigidity in the bacterial methylerythritol phosphate (MEP) pathway.

Terpenoids are a diverse class of compounds with wide-ranging uses including as industrial solvents, pharmaceuticals, and fragrances. Efforts to produce terpenoids sustainably by engineering microbes for fermentation are ongoing, but industrial production still largely relies on nonrenewable sources. The methylerythritol phosphate (MEP) pathway generates terpenoid precursor molecules and includes the enzyme Dxs and two iron-sulfur cluster enzymes: IspG and IspH.

Structural and functional basis of the universal transcription factor NusG pro-pausing activity in Mycobacterium tuberculosis.

Transcriptional pauses mediate regulation of RNA biogenesis. DNA-encoded pause signals trigger pausing by stabilizing RNA polymerase (RNAP) swiveling and inhibiting DNA translocation. The N-terminal domain (NGN) of the only universal transcription factor, NusG/Spt5, modulates pausing through contacts to RNAP and DNA.

Structural basis for substrate selection by the SARS-CoV-2 replicase.

The SARS-CoV-2 RNA-dependent RNA polymerase coordinates viral RNA synthesis as part of an assembly known as the replication-transcription complex (RTC). Accordingly, the RTC is a target for clinically approved antiviral nucleoside analogues, including remdesivir. Faithful synthesis of viral RNAs by the RTC requires recognition of the correct nucleotide triphosphate (NTP) for incorporation into the nascent RNA.

Structural basis for intrinsic transcription termination.

Efficient and accurate termination is required for gene transcription in all living organisms. Cellular RNA polymerases in both bacteria and eukaryotes can terminate their transcription through a factor-independent termination pathway-called intrinsic termination transcription in bacteria-in which RNA polymerase recognizes terminator sequences, stops nucleotide addition and releases nascent RNA spontaneously. Here we report a set of single-particle cryo-electron microscopy structures of Escherichia coli transcription intrinsic termination complexes representing key intermediate states of the event.

RfaH Counter-Silences Inhibition of Transcript Elongation by H-NS-StpA Nucleoprotein Filaments in Pathogenic Escherichia coli.

Expression of virulence genes in pathogenic Escherichia coli is controlled in part by the transcription silencer H-NS and its paralogs (e.g., StpA), which sequester DNA in multi-kb nucleoprotein filaments to inhibit transcription initiation, elongation, or both.

Transcriptomic Data Sets for Zymomonas mobilis 2032 during Fermentation of Ammonia Fiber Expansion (AFEX)-Pretreated Corn Stover and Switchgrass Hydrolysates.

The transcriptomes of Zymomonas mobilis 2032 were captured during the fermentation of ammonia fiber expansion (AFEX)-pretreated corn stover and switchgrass hydrolysates containing different concentrations of glucose and xylose. RNA samples were collected when Z. mobilis was fermenting glucose or xylose.

A roadmap for designing narrow-spectrum antibiotics targeting bacterial pathogens.

() infection (CDI) continues to be the leading threat of nosocomial deaths worldwide and a major burden on health-care systems. Broad-spectrum antibiotics eradicate the normal gut microbiome, killing protective commensal bacteria and increasing CDI recurrence. In contrast, Fidaxomicin (Fdx) is a narrow-spectrum antibiotic that inhibits growth without affecting crucial gut microbes.

Bacterial H-NS contacts DNA at the same irregularly spaced sites in both bridged and hemi-sequestered linear filaments.

Gene silencing in bacteria is mediated by chromatin proteins, of which H-NS is a paradigmatic example. H-NS forms nucleoprotein filaments with either one or two DNA duplexes. However, the structures, arrangements of DNA-binding domains (DBDs), and positions of DBD-DNA contacts in linear and bridged filaments are uncertain.

Basis of narrow-spectrum activity of fidaxomicin on Clostridioides difficile.

Fidaxomicin (Fdx) is widely used to treat Clostridioides difficile (Cdiff) infections, but the molecular basis of its narrow-spectrum activity in the human gut microbiome remains unknown. Cdiff infections are a leading cause of nosocomial deaths. Fidaxomicin, which inhibits RNA polymerase, targets Cdiff with minimal effects on gut commensals, reducing recurrence of Cdiff infection.

Seeing gene expression in cells: the future of structural biology.

Although much is known about the machinery that executes fundamental processes of gene expression in cells, much also remains to be learned about how that machinery works. A recent paper by O'Reilly reports a major step forward in the direct visualization of central dogma processes at submolecular resolution inside bacterial cells frozen in a native state. The essential methodologies involved are cross-linking mass spectrometry (CLMS) and cryo-electron tomography (cryo-ET).

Conserved Trigger Loop Histidine of RNA Polymerase II Functions as a Positional Catalyst Primarily through Steric Effects.

In all domains of life, multisubunit RNA polymerases (RNAPs) catalyze both the extension of mRNA transcripts by nucleotide addition and the hydrolysis of RNA, which enables proofreading by removal of misincorporated nucleotides. A highly conserved catalytic module within RNAPs called the trigger loop (TL) functions as the key controller of these activities. The TL is proposed to act as a positional catalyst of phosphoryl transfer and transcript cleavage via electrostatic and steric contacts with substrates in its folded helical form.

Crabtree/Warburg-like aerobic xylose fermentation by engineered Saccharomyces cerevisiae.

Bottlenecks in the efficient conversion of xylose into cost-effective biofuels have limited the widespread use of plant lignocellulose as a renewable feedstock. The yeast Saccharomyces cerevisiae ferments glucose into ethanol with such high metabolic flux that it ferments high concentrations of glucose aerobically, a trait called the Crabtree/Warburg Effect. In contrast to glucose, most engineered S.

Obligate movements of an active site-linked surface domain control RNA polymerase elongation and pausing via a Phe pocket anchor.

The catalytic trigger loop (TL) in RNA polymerase (RNAP) alternates between unstructured and helical hairpin conformations to admit and then contact the NTP substrate during transcription. In many bacterial lineages, the TL is interrupted by insertions of two to five surface-exposed, sandwich-barrel hybrid motifs (SBHMs) of poorly understood function. The 188-amino acid, two-SBHM insertion in RNAP, called SI3, occupies different locations in elongating, NTP-bound, and paused transcription complexes, but its dynamics during active transcription and pausing are undefined.