DNA topology: dynamic DNA looping.
The DNA in repressive loops is often tightly bent. DNA flexibility imposes significant constraints on their topology suggesting that they may exist as perturbations in plectonemic DNA.
Binding Sites↗
Biomedical subjects
Andrew Travers
Publications and source records attributed to Andrew Travers.
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↗
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↗
The evolution of the genetic code revisited.
The evolution of the genetic code in terms of the adoption of new codons has previously been related to the relative thermostability of codon-anticodon interactions such that the most stable interactions have been hypothesised to represent the most ancient coding capacity. This derivation is critically dependent on the accuracy of the experimentally determined stability parameters. A new set of parameters recently determined for B-DNA reveals that the codon-anticodon pairs for the codes in non-plant mitochondria on the one hand and prokaryotic and eukaryotic organisms on the other can be unequivocally divided into two classes - the most stable base steps define a common code specified by the first two bases in a codon while the less stable base steps correlate with divergent usage and the adoption of a 3-letter code. This pattern suggests that the fixation of codons for A, G, P, V, S, T, D/E, R may have preceded the divergence of the non-plant mitochondrial line from other organisms. Other variations in the code correlate with the least stable codon-anticodon pairs.
Anticodon↗
Feasibility and applicability of paramedic-based prehospital fibrinolysis in a large North American center.
BACKGROUND: Although considered the highest level of evidence and critical-to-test novel therapies, clinical trials are unrepresentative of the "real world" as they lack a true patient denominator, which limits general applicability of results. Accordingly, in conjunction with the Assessment of the Safety and Efficacy of a New Thrombolytic Regimen 3+ trial, we evaluated a comprehensive contemporary cohort of patients with ST segment elevation myocardial infarction (STEMI) to investigate: feasibility, applicability, safety, and efficacy of de novo paramedic-based prehospital fibrinolysis (PHF) program. METHODS: Prospective observational comparative cohort of all patients with STEMI encountered during the Assessment of the Safety and Efficacy of a New Thrombolytic Regimen 3+ enrollment period. Time-to-treatment, systematic electrocardiographic (ECG) analysis, peak creatine kinase, inhospital clinical events, and mortality were assessed. RESULTS: During the 22-month study period, 1095 patients with STEMI were admitted to hospital; 46% (119/258) of eligible patients received PHF (< or = 6 hours of symptom onset by ambulance). Paramedics contacted the study physician 3.6 times per week: 33% (119/357) of patients enrolled, and ECG transmission failure is 6%. Time-to-treatment was reduced with PHF versus inhospital (1 hour 43 minutes vs 2 hours 38 minutes; P < .001). Despite higher baseline Thrombolysis in Myocardial Infarction Scores and greater ECG territory at risk (ST), prehospital patients achieved more favorable outcomes: peak creatine kinase (1413 vs 1549 U/L; P = .122), Q wave at discharge (56.3% vs 70.7%; P = .003), and intracranial hemorrhage (0% vs 0.8%; P < 1.0), respectively. Inhospital mortality for PHF versus inhospital patients was 3.4% versus 4.8% (P = .627), with an adjusted odds ratio of 0.60 (confidence interval, 0.19-1.87). CONCLUSION: Feasibility and applicability of PHF was demonstrated with a substantial reduction in treatment delay and favorable clinical outcomes. Extending the unrealized potential of paramedic-based PHF programs in North America is feasible and desirable.
Aged↗
Relative affinities of DNA sequences for the histone octamer depend strongly upon both the temperature and octamer concentration.
Using a novel competition assay to determine the relative strength of different histone octamer-binding sites, we have compared three natural and two synthetic sites. We show that the relative affinities of these sites for the histone octamer depend upon both the temperature and octamer concentration. In particular, under certain conditions, a natural octamer-binding site from a yeast promoter outcompetes a synthetic sequence of comparable affinity to the strongest previously described positioning sequence. Under other conditions, this synthetic sequence is the preferred octamer ligand. We infer that sequence selection by the histone octamer depends strongly upon both the sequence-dependent anisotropy of DNA bending and on DNA deformability and that these parameters may contribute differently to nucleosome formation. These findings indicate that previous studies designed to identify strong octamer-binding sites may fail to select some natural strong binding sites.
Anisotropy↗
DNA dynamics: bubble 'n' flip for DNA cyclisation?
A recent demonstration of the facile in vitro formation of DNA microcircles of fewer than 100 base pairs throws new light on the basis of DNA flexibility.
Chromatin↗
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↗
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↗
A 'one-pot' assay for the accessibility of DNA in a nucleosome core particle.
The accessibility of nucleosomal DNA to transcription factors and other sequence-specific DNA binding proteins is of importance in the consideration of mechanisms of transcriptional control. Here, we report a simple novel assay which determines this accessibility at eight different rotationally equivalent sites on nucleosomal DNA and shows that linker histones and the chromosomal HMGB proteins, HMG-D and HMG-Z, have opposite effects on the accessibility of nucleosomal DNA. We compare this assay to previously described methods.
Binding Sites↗
A conserved role but different partners for the transcriptional corepressor CoREST in fly and mammalian nervous system formation.
Identification of conserved proteins that act to establish the neuronal phenotype has relied predominantly on structural homologies of the underlying genes. In the case of the repressor element 1 silencing transcription factor (REST), a central player in blocking the neuronal phenotype in vertebrate non-neural tissue, the invertebrate homolog is absent, raising the possibility that distinct strategies are used to establish the CNS of invertebrates. Using a yeast two-hybrid screen designed specifically to identify functional analogs of REST, we show that Drosophila melanogaster uses a strategy that is functionally similar to, but appears to have evolved independently of, REST. The gene at the center of the strategy in flies encodes the repressor Tramtrack88 (Ttk88), a protein with no discernable homology to REST but that nonetheless is able to interact with the same transcriptional partners. Ttk88 uses the REST corepressor Drosophila CoREST to coordinately regulate a set of genes encoding the same neuronal hallmarks that are regulated by REST in vertebrates. Our findings indicate that repression is an important mechanism for regulating neuronal phenotype across phyla and suggest that co-option of a similar corepressor complex occurred to restrict expression of genes critical for neuronal function to a compartmentalized nervous system.
Amino Acid Sequence↗
Conducting research using the emergency exception from informed consent: the Public Access Defibrillation (PAD) Trial experience.
BACKGROUND: The Public Access Defibrillation (PAD) Trial, a prospective, multicenter, randomized clinical trial comparing two prehospital resuscitation strategies, was conducted under the regulations for exception from informed consent (21CFR50.24) in 24 communities in North America. These regulations place additional requirements for human subject protection on investigators and Institutional Review Boards (IRBs), including conducting community consultation (CC) and public disclosure (PD). OBJECTIVE: To describe the IRB approval process at study sites and the number and types of community consultation and public disclosure activities conducted. METHODS: The 24 study sites in the United States and Canada submitted IRB applications, CC and PD plans, and a structured report on IRB process and investigator perceptions to the Clinical Trial Center at the University of Washington. RESULTS: The primary IRBs for all 24 trial sites and a total of 101 IRBs approved the study. The median interval from submission to approval was 108 days (IQR 43-196), and the mean number of revisions was two (range 0-7). Investigators conducted nearly 12,000 activities to achieve CC and PD; activities varied greatly from site to site in both type and quantity. CONCLUSION: The length of time to obtain IRB approval and the extent of community consultation and public disclosure varied greatly among trial sites in meeting the current regulations for conducting emergency research with exception from informed consent. This suggests that more specific guidance may be useful and that determination of effective strategies for community consultation and public disclosure is needed.
Cardiopulmonary Resuscitation↗
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↗
HMG-D and histone H1 alter the local accessibility of nucleosomal DNA.
There is evidence that HMGB proteins facilitate, while linker histones inhibit chromatin remodelling, respectively. We have examined the effects of HMG-D and histone H1/H5 on accessibility of nucleosomal DNA. Using the 601.2 nucleosome positioning sequence designed by Widom and colleagues we assembled nucleosomes in vitro and probed DNA accessibility with restriction enzymes in the presence or absence of HMG-D and histone H1/H5. For HMG-D our results show increased digestion at two spatially adjacent sites, the dyad and one terminus of nucleosomal DNA. Elsewhere varying degrees of protection from digestion were observed. The C-terminal acidic tail of HMG-D is essential for this pattern of accessibility. Neither the HMG domain by itself nor in combination with the adjacent basic region is sufficient. Histone H1/H5 binding produces two sites of increased digestion on opposite faces of the nucleosome and decreased digestion at all other sites. Our results provide the first evidence of local changes in the accessibility of nucleosomal DNA upon separate interaction with two linker binding proteins.
Animals↗
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↗
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↗