A simple, robust, broadly applicable insertion mutagenesis method to create random fluorescent protein: target protein fusions.

A simple, broadly applicable method was developed using an in vitro transposition reaction followed by transformation into Escherichia coli and screening plates for fluorescent colonies. The transposition reaction catalyzes the random insertion of a fluorescent protein open reading frame into a target gene on a plasmid. The transposition reaction is employed directly in an E.

A Bacterial Cell-Based Assay To Study SARS-CoV-2 Protein-Protein Interactions.

Methods for detecting and dissecting the interactions of virally encoded proteins are essential for probing basic viral biology and providing a foundation for therapeutic advances. The dearth of targeted therapeutics for the treatment of coronavirus disease 2019 (COVID-19), an ongoing global health crisis, underscores the importance of gaining a deeper understanding of the interactions of proteins encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we describe the use of a convenient bacterial cell-based two-hybrid (B2H) system to analyze the SARS-CoV-2 proteome.

Control of a programmed cell death pathway in Pseudomonas aeruginosa by an antiterminator.

In Pseudomonas aeruginosa the alp system encodes a programmed cell death pathway that is switched on in a subset of cells in response to DNA damage and is linked to the virulence of the organism. Here we show that the central regulator of this pathway, AlpA, exerts its effects by acting as an antiterminator rather than a transcription activator. In particular, we present evidence that AlpA positively regulates the alpBCDE cell lysis genes, as well as genes in a second newly identified target locus, by recognizing specific DNA sites within the promoter, then binding RNA polymerase directly and allowing it to bypass intrinsic terminators positioned downstream.

Diatoms: A novel cause of granulomatous inflammation of the head and neck.

Objective: We report the first 4 cases of intraoral nonnecrotizing granulomatous foreign body reactions to diatoms, plausibly as a result of exogenous material introduced following iatrogenic or traumatic injury.

Study Design: Clinical and histopathologic findings of 4 intraoral cases of nonnecrotizing granulomatous foreign body reaction to diatoms, single-celled algae belonging to the taxonomic phylum Bacillariophyta, are reported.

Results: The lesions presented either in the jaws or in the soft tissue overlying the alveolar bone, in some instances mimicking an inflammatory lesion of odontogenic etiology.

p19 Captures RNase III-Cleaved Double-Stranded RNAs Formed by Overlapping Sense and Antisense Transcripts in Escherichia coli.

Antisense transcription is widespread in bacteria. By base pairing with overlapping sense RNAs, antisense RNAs (asRNA) can form double-stranded RNAs (dsRNA), which are cleaved by RNase III, a dsRNA endoribonuclease. The ectopic expression of plant p19 in stabilizes ∼21-nucleotide (nt) dsRNA RNase III decay intermediates, which enabled us to characterize otherwise highly unstable asRNA by deep sequencing of p19-captured dsRNA.

A novel RNA polymerase-binding protein that interacts with a sigma-factor docking site.

Sporulation in Bacillus subtilis is governed by a cascade of alternative RNA polymerase sigma factors. We previously identified a small protein Fin that is produced under the control of the sporulation sigma factor σ to create a negative feedback loop that inhibits σ -directed gene transcription. Cells deleted for fin are defective for spore formation and exhibit increased levels of σ -directed gene transcription.

An Amino Acid Substitution in RNA Polymerase That Inhibits the Utilization of an Alternative Sigma Factor.

Sigma (σ) factors direct gene transcription by binding to and determining the promoter recognition specificity of RNA polymerase (RNAP) in bacteria. Genes transcribed under the control of alternative sigma factors allow cells to respond to stress and undergo developmental processes, such as sporulation in , in which gene expression is controlled by a cascade of alternative sigma factors. Binding of sigma factors to RNA polymerase depends on the coiled-coil (or clamp helices) motif of the β' subunit.

Nascent RNA length dictates opposing effects of NusA on antitermination.

The NusA protein is a universally conserved bacterial transcription elongation factor that binds RNA polymerase (RNAP). When functioning independently, NusA enhances intrinsic termination. Paradoxically, NusA stimulates the function of the N and Q antiterminator proteins of bacteriophage λ.

Mycobacterial mistranslation is necessary and sufficient for rifampicin phenotypic resistance.

Errors are inherent in all biological systems. Errors in protein translation are particularly frequent giving rise to a collection of protein quasi-species, the diversity of which will vary according to the error rate. As mistranslation rates rise, these new proteins could produce new phenotypes, although none have been identified to date.

Phage-encoded inhibitor of Staphylococcus aureus transcription exerts context-dependent effects on promoter function in a modified Escherichia coli-based transcription system.

Promoter recognition in bacteria is mediated primarily by the σ subunit of RNA polymerase (RNAP), which makes sequence-specific contacts with the promoter -10 and -35 elements in the context of the RNAP holoenzyme. However, the RNAP α subunit can also contribute to promoter recognition by making sequence-specific contacts with upstream (UP) elements that are associated with a subset of promoters, including the rRNA promoters. In Escherichia coli, these interactions between the RNAP α subunit (its C-terminal domain [CTD], in particular) and UP element DNA result in significant stimulation of rRNA transcription.

Efficient and specific gene knockdown by small interfering RNAs produced in bacteria.

Synthetic small interfering RNAs (siRNAs) are an indispensable tool to investigate gene function in eukaryotic cells and may be used for therapeutic purposes to knock down genes implicated in disease. Thus far, most synthetic siRNAs have been produced by chemical synthesis. Here we present a method to produce highly potent siRNAs in Escherichia coli.

Crystal structure of the bacteriophage T4 late-transcription coactivator gp33 with the β-subunit flap domain of Escherichia coli RNA polymerase.

Activated transcription of the bacteriophage T4 late genes, which is coupled to concurrent DNA replication, is accomplished by an initiation complex containing the host RNA polymerase associated with two phage-encoded proteins, gp55 (the basal promoter specificity factor) and gp33 (the coactivator), as well as the DNA-mounted sliding-clamp processivity factor of the phage T4 replisome (gp45, the activator). We have determined the 3.0 Å-resolution X-ray crystal structure of gp33 complexed with its RNA polymerase binding determinant, the β-flap domain.

Initial transcribed region sequences influence the composition and functional properties of the bacterial elongation complex.

The bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytic core enzyme (α(2)ββ'ω) in complex with a σ factor that is essential for promoter recognition and transcription initiation. During early elongation, the stability of interactions between σ and the remainder of the transcription complex decreases. Nevertheless, there is no mechanistic requirement for release of σ upon the transition to elongation.

A regulator from Chlamydia trachomatis modulates the activity of RNA polymerase through direct interaction with the beta subunit and the primary sigma subunit.

The obligate intracellular human pathogen Chlamydia trachomatis undergoes a complex developmental program involving transition between two forms: the infectious elementary body (EB), and the rapidly dividing reticulate body (RB). However, the regulators controlling this development have not been identified. To uncover potential regulators of transcription in C.

The bacteriophage lambda Q antiterminator protein contacts the beta-flap domain of RNA polymerase.

The multisubunit RNA polymerase (RNAP) in bacteria consists of a catalytically active core enzyme (alpha(2)beta beta'omega) complexed with a sigma factor that is required for promoter-specific transcription initiation. During early elongation the stability of interactions between sigma and core decreases, in part because of the nascent RNA-mediated destabilization of an interaction between region 4 of sigma and the flap domain of the beta-subunit (beta-flap). The nascent RNA-mediated destabilization of the sigma region 4/beta-flap interaction is required for the bacteriophage lambda Q antiterminator protein (lambdaQ) to engage the RNAP holoenzyme.

The bacteriophage lambdaQ anti-terminator protein regulates late gene expression as a stable component of the transcription elongation complex.

The Q protein of bacteriophage lambda (lambdaQ) is a transcription anti-terminator required for the expression of the phage's late genes under the control of promoter P(R'). To effect terminator read-through, lambdaQ must gain access to RNA polymerase (RNAP) via a promoter-restricted pathway. In particular, lambdaQ modifies RNAP by binding a specific DNA site embedded in P(R') and interacting with RNAP in the context of a specific paused early elongation complex.

Conformational toggle triggers a modulator of RNA polymerase activity.

Members of a recently discovered class of transcription factor, which includes the Gre factors that stimulate transcript cleavage, function by directly modulating the catalytic properties of RNA polymerase (RNAP). Now, three research groups have determined crystal structures of a Gre homolog, Gfh1, which inhibits all RNAP catalytic activities. Strikingly, these structures reveal a puzzling discrepancy between the Gfh1 and GreA conformations, but the discovery that a pH-dependent conformational toggle alters Gfh1 activity suggests an elegant solution.

Characterization of the detachable Rho-dependent transcription terminator of the fimE gene in Escherichia coli K-12.

The fim genetic switch in the chromosome of Escherichia coli K-12 is an invertible DNA element that harbors the promoter for transcription of the downstream fim structural genes and a transcription terminator that acts on the upstream fimE regulatory gene. Switches oriented appropriately for structural gene transcription also allow fimE mRNA to read through, whereas those in the opposite orientation terminate the fimE message. We show here that termination is Rho dependent and is suppressed in a rho mutant or by bicyclomycin treatment when fimE mRNA is expressed by the fimE gene, either from a multicopy recombinant plasmid or in its native chromosomal location.

An artificial activator that contacts a normally occluded surface of the RNA polymerase holoenzyme.

Many activators of transcription are sequence-specific DNA-binding proteins that stimulate transcription initiation through interaction with RNA polymerase (RNAP). Such activators can be constructed artificially by fusing a DNA-binding protein to a protein domain that can interact with an accessible surface of RNAP. In these cases, the artificial activator is directed to a target promoter bearing a recognition site for the DNA-binding protein.

Three-way interactions among the Sfh, StpA and H-NS nucleoid-structuring proteins of Shigella flexneri 2a strain 2457T.

Shigella flexneri 2a strain 2457T has been found to express Sfh, a new member of the H-NS-like family of nucleoid-structuring proteins. With H-NS and its paralogue, StpA, this brings to three the number of these proteins expressed in this bacterium. This raises the possibility that three-way interactions may occur in S.

Regulation of gene expression by histone-like proteins in bacteria.

Histone-like proteins in bacteria contribute to the control of gene expression, as well as participating in other DNA transactions such as recombination and DNA replication. They have also been described, somewhat vaguely, as contributors to the organization of the bacterial nucleoid. Our view of how these proteins act in the cell is becoming clearer, particularly in the cases of Fis, H-NS and HU, three of the most intensively studied members of the group.