Biomedical subjects

Georgi Muskhelishvili

Publications and source records attributed to Georgi Muskhelishvili.

11 recordsLinked to original sources

PubMed2006

The algT gene of Pseudomonas syringae pv. glycinea and new insights into the transcriptional organization of the algT-muc gene cluster.

The phytopathogenic bacterium Pseudomonas syringae pv. glycinea infects soybean plants and causes bacterial blight. In addition to P. syringae, the human pathogen Pseudomonas aeruginosa and the soil bacterium Azotobacter vinelandii produce the exopolysaccharide alginate, a copolymer of d-mannuronic and l-guluronic acids. Alginate production in P. syringae has been associated with increased fitness and virulence in planta. Alginate biosynthesis is tightly controlled by proteins encoded by the algT-muc regulatory gene cluster in P. aeruginosa and A. vinelandii. These genes encode the alternative sigma factor AlgT (sigma(22)), its anti-sigma factors MucA and MucB, MucC, a protein with a controversial function that is absent in P. syringae, and MucD, a periplasmic serine protease and homolog of HtrA in Escherichia coli. We compared an alginate-deficient algT mutant of P. syringae pv. glycinea with an alginate-producing derivative in which algT is intact. The alginate-producing derivative grew significantly slower in vitro growth but showed increased epiphytic fitness and better symptom development in planta. Evaluation of expression levels for algT, mucA, mucB, mucD, and algD, which encodes an alginate biosynthesis gene, showed that mucD transcription is not dependent on AlgT in P. syringae in vitro. Promoter mapping using primer extension experiments confirmed this finding. Results of reverse transcription-PCR demonstrated that algT, mucA, and mucB are cotranscribed as an operon in P. syringae. Northern blot analysis revealed that mucD was expressed as a 1.75-kb monocistronic mRNA in P. syringae.

Alginates↗

PubMed2006

RNA polymerase and an activator form discrete subcomplexes in a transcription initiation complex.

Using high-resolution atomic force microscopy (AFM) we show that in a ternary complex of an activator protein, FIS, and RNA polymerase containing the sigma(70) specificity factor at the Escherichia coli tyrT promoter the polymerase and the activator form discrete, but connected, subcomplexes in close proximity. This is the first time that a ternary complex between an activator, a sigma(70) polymerase holoenzyme and promoter DNA has been visualised. Individually FIS and RNA polymerase wrap approximately 80 and 150 bp of promoter DNA, respectively. We suggest that the architecture of the ternary complex provides a general paradigm for the facilitation of direct, but weak, interactions between polymerase and an activator.

DNA↗

PubMed2006

Homeostatic regulation of supercoiling sensitivity coordinates transcription of the bacterial genome.

Regulation of cellular growth implies spatiotemporally coordinated programmes of gene transcription. A central question, therefore, is how global transcription is coordinated in the genome. The growth of the unicellular organism Escherichia coli is associated with changes in both the global superhelicity modulated by cellular topoisomerase activity and the relative proportions of the abundant DNA-architectural chromatin proteins. Using a DNA-microarray-based approach that combines mutations in the genes of two important chromatin proteins with induced changes of DNA superhelicity, we demonstrate that genomic transcription is tightly associated with the spatial distribution of supercoiling sensitivity, which in turn depends on chromatin proteins. We further demonstrate that essential metabolic pathways involved in the maintenance of growth respond distinctly to changes of superhelicity. We infer that a homeostatic mechanism organizing the supercoiling sensitivity is coordinating the growth-phase-dependent transcription of the genome.

Bacterial Proteins↗

PubMed2005

Bacterial chromatin.

Recent studies have revealed that the bacterial nucleoid is a dynamic entity that alters its overall structure in response to changes in both growth rate and growth phase. These structural changes are correlated with, and might be driven by, changes in the distribution and utilization of DNA supercoiling. In turn, these parameters in addition to the delimitation of topological domains are dependent both on the relative proportions of the abundant nucleoid-associated proteins and on transcriptional activity. The domain structure itself is dynamic.

Bacteria↗

PubMed2005

DNA supercoiling - a global transcriptional regulator for enterobacterial growth?

A fundamental principle of exponential bacterial growth is that no more ribosomes are produced than are necessary to support the balance between nutrient availability and protein synthesis. Although this conclusion was first expressed more than 40 years ago, a full understanding of the molecular mechanisms involved remains elusive and the issue is still controversial. There is currently agreement that, although many different systems are undoubtedly involved in fine-tuning this balance, an important control, and in our opinion perhaps the main control, is regulation of the rate of transcription initiation of the stable (ribosomal and transfer) RNA transcriptons. In this review, we argue that regulation of DNA supercoiling provides a coherent explanation for the main modes of transcriptional control - stringent control, growth-rate control and growth-phase control - during the normal growth of Escherichia coli.

DNA, Bacterial↗

PubMed2004

Buffering of stable RNA promoter activity against DNA relaxation requires a far upstream sequence.

The stable RNA promoters of Escherichia coli are exquisitely sensitive to variations in the superhelical density of DNA. Previously, we have shown that binding of the DNA architectural protein FIS at the upstream activating sequences (UASs) of stable RNA promoters prevents the transcription complexes from inactivation induced by changes in the supercoiling level of DNA. Here, we identify a strong FIS binding site 89 bp upstream of the previously described cluster of FIS binding sites located between positions -64 and -150 in the rrnA P1 UAS. Binding of FIS to this 'far upstream sequence' allows the recruitment of additional FIS molecules to the region. We demonstrate that, upon DNA relaxation, the maintenance of promoter activity requires, in addition to UAS, the presence of the far upstream sequence. The far upstream sequence shows no effect in the absence of an intact cluster. This requirement for the integrity of the region encompassing the far upstream sequence and the UAS cluster is correlated with the in vitro modulation of binding of FIS to UAS and interaction of RNA polymerase with the UP element and the region around the transcriptional start point. Our results suggest that, at the rrnA P1 promoter, the entire region comprising the UAS and the far upstream sequence is involved in the assembly of the transcription initiation complex. We propose that the extensive engagement of upstream DNA in this nucleoprotein complex locally compensates for the lack of torsional strain in relaxed DNA, thus increasing the resistance of the promoter to global DNA relaxation.

Carrier Proteins↗

PubMed2003

Mechanism of transcriptional activation by FIS: role of core promoter structure and DNA topology.

The Escherichia coli DNA architectural protein FIS activates transcription from stable RNA promoters on entry into exponential growth and also reduces the level of negative supercoiling. Here we show that such a reduction decreases the activity of the tyrT promoter but that activation by FIS rescues tyrT transcription at non-optimal superhelical densities. Additionally we show that three different "up" mutations in the tyrT core promoter either abolish or reduce the dependence of tyrT transcription on both high negative superhelicity and FIS in vivo and infer that the specific sequence organisation of the core promoter couples the control of transcription initiation by negative superhelicity and FIS. In vitro all the mutations potentiate FIS-independent untwisting of the -10 region while at the wild-type promoter FIS facilitates this step. We propose that this untwisting is a crucial limiting step in the initiation of tyrT RNA synthesis. The tyrT core promoter structure is thus optimised to combine high transcriptional activity with acute sensitivity to at least three major independent regulatory inputs: negative superhelicity, FIS and ppGpp.

Base Sequence↗

PubMed2003

Transcription factor as a topological homeostat.

Abundant prokaryotic chromatin architectural proteins often function also as global transcriptional regulators. In addition, some of this class of proteins modulate the activity of cellular topoisomerases and hence, the superhelical density of DNA. The relationships between the global effect of these proteins on DNA topology and their local effects exerted on particular promoter regions remain largely unexplored. One of the best-characterised examples of this class of proteins is the pleiotropic regulator of metabolism FIS, which reduces the activity of DNA gyrase and counteracts the increase of the overall superhelicity of DNA during early exponential growth phase. Binding of FIS to supercoiled DNA molecules in vitro leads to the formation of branched structures and consequent multiplication of apical loops, whereas on bending the upstream regions of stable RNA promoters FIS acts as a topological homeostat maintaining high local levels of supercoiling required for promoter activity. We argue that the coordinated effects of FIS on the global and local DNA architecture optimise gene expression by channelling the free energy of negative supercoiling to specific, biologically relevant sites.

Factor For Inversion Stimulation Protein↗

PubMed2002

FIS modulates the kinetics of successive interactions of RNA polymerase with the core and upstream regions of the tyrT promoter.

We have applied laser UV photo-footprinting to characterise kinetically complexes involving the activator protein FIS, RNA polymerase and the tyrT promoter of Escherichia coli. FIS photo-footprints strongly to three binding sites upstream of the core promoter. The polymerase photo-footprints in the near-consensus -35 hexamer on the non-template strand of DNA in a fashion similar to that of stable complexes involving the lacUV5 promoter. The kinetics of the interactions of polymerase alone with the tyrT promoter differ from those observed previously at the lacUV5 promoter. In the absence of FIS, we observe an upstream polymerase-induced signal at -122 within FIS site III that occurs subsequent to changes in the core promoter region and is strongly dependent on negative supercoiling. These observations support the proposal that the upstream region of the promoter is wrapped around the polymerase. We propose that the wrapped DNA allows the polymerase to overcome, at least in part, the barrier to DNA untwisting imparted by the G+C-rich discriminator. We further suggest that FIS plays a similar role and may facilitate polymerase escape.

Base Sequence↗

PubMed2002

Promoter protection by a transcription factor acting as a local topological homeostat.

Binding of the Escherichia coli global transcription factor FIS to the upstream activating sequence (UAS) of stable RNA promoters activates transcription on the outgrowth of cells from stationary phase. Paradoxically, while these promoters require negative supercoiling of DNA for optimal activity, FIS counteracts the increase of negative superhelical density by DNA gyrase. We demonstrate that binding of FIS at the UAS protects the rrnA P1 promoter from inactivation at suboptimal superhelical densities. This effect is correlated with FIS-dependent constraint of writhe and facilitated untwisting of promoter DNA. We infer that FIS maintains stable RNA transcription by stabilizing local writhe in the UAS. These results suggest a novel mechanism of transcriptional regulation by a transcription factor acting as a local topological homeostat.

Aminocoumarins↗

PubMed2002

Transcriptional regulation of fis operon involves a module of multiple coupled promoters.

The transcription of the Escherichia coli fis gene is strongly activated during the outgrowth of cells from stationary phase. The high activity of the promoter of the fis operon requires the transcription factor IHF. Previously, we identified a divergent promoter, div, located upstream of the fis promoter. In this study we demonstrate that at least two additional promoters, designated fis P2 and fis P3, are located in the control region of the fis operon. The fis P2 and div promoters overlap completely, whereas fis P3 and div P are arranged as face-to-face divergent promoters. We show that the div and the tandem fis promoters counterbalance each other, such that their activity is kept on a lower than potentially attainable level. Furthermore, we demonstrate an unusual activation mechanism by IHF, involving a coordinated shift in the balance of promoter activities. We infer that these coupled promoters represent a regulatory module and propose a novel "dynamic balance" mechanism involved in the transcriptional control of the fis operon.

Base Sequence↗