Biomedical subjects

Padraig Deighan

Publications and source records attributed to Padraig Deighan.

5 recordsLinked to original sources

PubMed2006

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.

Bacterial Proteins↗

PubMed2005

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. Here we describe an artificial activator that functions by contacting a normally occluded surface of promoter-bound RNAP holoenzyme. This artificial activator consists of a DNA-binding protein fused to the bacteriophage T4-encoded transcription regulator AsiA. On its own, AsiA inhibits transcription by Escherichia coli RNAP because it remodels the holoenzyme, disrupting an intersubunit interaction that is required for recognition of the major class of bacterial promoters. However, when tethered to the DNA via a DNA-binding protein, AsiA can exert a strong stimulatory effect on transcription by disrupting the same intersubunit interaction, contacting an otherwise occluded surface of the holoenzyme. We show that mutations that affect the intersubunit interaction targeted by AsiA modulate the stimulatory effect of this artificial activator. Our results thus demonstrate that changes in the accessibility of a normally occluded surface of the RNAP holoenzyme can modulate the activity of a gene-specific regulator of transcription.

Base Sequence↗

PubMed2005

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. Two cis-acting elements within the central portion of the 314-bp invertible DNA switch were identified as contributors to Rho-dependent termination and dissected. These fim sequence elements show similarities to well-characterized Rho utilization (rut) sites and consist of a boxA motif and a C-rich and G-poor region of approximately 40 bp. Deletion of the boxA motif alone had only a subtle negative effect on Rho function. However, when this element was deleted in combination with the C-rich, G-poor region, Rho function was considerably decreased. Altering the C-to-G ratio in favor of G in this portion of the switch also strongly attenuated transcription termination. The implications of the existence of a fimE-specific Rho-dependent terminator within the invertible switch are discussed in the context of the fim regulatory circuit.

Base Composition↗

PubMed2003

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. Especially helpful have been studies of the contributions of these proteins to the regulation of specific genes such as the gal operon, and genes coding for stable RNA species, topoisomerases, and the histone-like proteins themselves. Recent advances have also been assisted by insights into the effects the histone-like proteins exert on DNA structure not only at specific promoters but throughout the genome.

Bacterial Proteins↗

PubMed2003

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. flexneri among these proteins and between the proteins and each other's genes. Such three-way interactions among H-NS-like proteins have not been described previously. The expression of the sfh, stpA and hns genes was studied at the transcriptional and post-transcriptional levels. The Sfh protein displays growth phase-dependent regulation that distinguishes it from both H-NS and StpA. Like H-NS and StpA, Sfh can bind to its own promoter region, it negatively autoregulates transcription of its own gene, and when overexpressed all three proteins cross-repress transcription of each other's genes. The presence of highly conserved oligomerization domains within these molecules suggested the possibility of protein-protein interactions. Like H-NS and StpA, the purified Sfh protein forms homodimers in solution. Using the yeast two-hybrid assay we show that each of the three proteins also forms homodimers in vivo and, additionally, each protein can form heterodimers with either of its homologues. This raises the possibility that Sfh may modulate the activities of H-NS and StpA, and vice versa.

Amino Acid Sequence↗