48 research outputs found
IHF-binding sites inhibit DNA loop formation and transcription initiation
No full textTranscriptional activation of enhancer and Sigma(54)-dependent promoters requires efficient interactions between enhancer-binding proteins (EBP) and promoter bound Sigma(54)-RNA polymerase (E Sigma(54)) achieved by DNA looping, which is usually facilitated by the integration host factor (IHF). Since the lengths of the intervening region supporting DNA-loop formation are similar among IHF-dependent and IHF-independent promoters, the precise reason(s) why IHF is selectively important for the frequency of transcription initiation remain unclear. Here, using kinetic cyclization and in vitro transcription assays we show that, in the absence of IHF protein, the DNA fragments containing an IHF-binding site have much less looping-formation ability than those that lack an IHF-binding site. Furthermore, when an IHF consensus-binding site was introduced into the intervening region between promoter and enhancer of the target DNA fragments, loop formation and DNA-loop-dependent transcriptional activation are significantly reduced in a position-independent manner. DNA-looping-independent transcriptional activation was unaffected. The binding of IHF to its consensus site in the target promoters clearly restored efficient DNA looping formation and looping-dependent transcriptional activation. Our data provide evidence that one function for the IHF protein is to release a communication block set by intrinsic properties of the IHF DNA-binding site.http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000268115200005&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701Biochemistry & Molecular BiologySCI(E)PubMed8ARTICLE123878-38863
() Typical IHF–DNA complexes imaged as described in Materials and Methods
No full text<p><b>Copyright information:</b></p><p>Taken from "Analysis of scanning force microscopy images of protein-induced DNA bending using simulations"</p><p>Nucleic Acids Research 2005;33(7):e68-e68.</p><p>Published online 20 Apr 2005</p><p>PMCID:PMC1083423.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The protein-induced bend is indicated by the arrows. Owing to its size, the IHF protein cannot be unambiguously identified in the images, demonstrating the need for an analytical approach using other than visual characteristics. The scale bar is 50 nm. Gray scale represents height ranging from 0 nm (dark) to 2 nm (bright). () Distributions of EED values normalized by contour lengths of IHF–DNA complexes (top) and bare DNA molecules (bottom), demonstrating the effect of DNA bending. () Histogram of bending angles estimated using tangents from IHF–DNA complexes. The bimodal distribution shows that not all DNA molecules have IHF bound; by fitting to a double Gaussian distribution, we estimate that ∼50% of the imaged molecules have IHF bound
Compensatory trunk movements in patients with hip osteoarthritis accuracy and reproducibility of a body-fixed sensor-based assessment
No full textReininga IHF, Stevens M, Wagenmakers R, Boerboom AL, Groothoff JW, Bulstra SK, Zijlstra W: Compensatory trunk movements in patients with hip osteoarthritis; Accuracy and reproducibility of a body-fixed sensor-based assessment. Am J Phys Med Rehabil 2011;90:681-687. This study examined the accuracy and reproducibility of a body-fixed sensor-based assessment for quantifying frontal plane angular movements of the (upper) thorax and pelvis of patients with hip osteoarthritis at different walking speeds. To evaluate accuracy, the angular movements of sensors attached to the thorax and pelvis of three patients were compared with results based on an optical motion analysis system. Accuracy was high, with small and consistent mean differences (<1.0 degree) and corresponding standard deviations (<1.3 degrees) between optical motion analysis system and body-fixed sensor data. To evaluate reproducibility, angular trunk movements were assessed twice in 15 patients. Reproducibility was high (intraclass correlation coefficients ranged from 0.86 to 0.97), and the values of the mean differences between the test and retest were small, with the 95% confidence interval containing zero. This body-fixed sensor-based assessment is an accurate and reproducible method for quantifying frontal plane compensatory trunk movements during gait of patients with hip osteoarthritis at different walking speeds
Electrophoretic mobility-shift assay of IHF binding to oligonucleotide A
No full text<p><b>Copyright information:</b></p><p>Taken from "Pressure dissociation of integration host factor–DNA complexes reveals flexibility-dependent structural variation at the protein–DNA interface"</p><p></p><p>Nucleic Acids Research 2007;35(6):1761-1772.</p><p>Published online 25 Feb 2007</p><p>PMCID:PMC1874591.</p><p>© 2007 The Author(s)</p>2. IHF concentrations in Lanes 1–10 are 0, 20, 40, 60, 81, 99, 120, 165, 201 and 240 nM, respectively. This pseudo-color image was generated by coloring the emission collected through a 520-nm band pass filter green (FAM fluorescence) and coloring the emission collected through a 580-nm band pass filter red (TAMRA fluorescence). With excitation at 488 nm, the unliganded oligonucleotide is green, reflecting only FAM fluorescence. The yellow color of the mobility-shifted band results from a combination of green and red fluorescence, indicating efficient FRET due to the wrapped DNA in the bound complex
ABSTRACT Search Engine Driven Author Disambiguation
No full textIn scholarly digital libraries, author disambiguation is an important task that attributes a scholarly work with specific authors. This is critical when individuals share the same name. We present an approach to this task that analyzes the results of automatically-crafted web searches. A key observation is that pages from rare web sites are stronger source of evidence than pages from common web sites, which we model as Inverse Host Frequency (IHF). Our system is able to achieve an average accuracy of 0.836
Patient satisfaction at and after discharge. Effect of a time lag
Get PDFObjective: Patient satisfaction is an important outcome measure for evaluating the quality of medical care. It is remarkable that consistently high satisfaction ratings have been reported over the last 30 years. There are indications that the time point of administration of a patient satisfaction questionnaire has an influence on satisfaction ratings. This study aimed at investigating whether the assessment of patient satisfaction at different time points resulted in different outcomes. Methods: Patient satisfaction was measured twice. The sample consisted of 152 orthopedic patients who filled in the questionnaire at hospital discharge and one to 12 months after discharge. Results: At follow-up, satisfaction ratings decreased significantly. Satisfaction with postoperative information decreased the most after discharge. Conclusion: The results of this study indicate that the time point of administration of a patient satisfaction questionnaire does influence satisfaction ratings. Practice implications: Patient satisfaction outcomes collected during hospitalization and after discharge may not be interpreted similarly. 9 (c) 2005 Elsevier Ireland Ltd. All rights reserved
A study of Escherichia coli integration host factor interaction with DNA
No full textThe sequence elements required for IHF binding to the H\sp\prime and H1 sites in attP of were examined. The H\sp\prime site naturally contains a dA+dT element 5\sp\prime to the core consensus element WATCAANNNNTTR, while the H1 site does not. It was found that both elements are required for IHF to bind to the H\sp\prime site. In contrast, the core consensus determinant alone is sufficient for IHF binding to the H1 site. Placement of a dA+dT element upstream of the H1 core consensus element significantly increased the affinity, suggesting that the presence of a dA+dT element enhances IHF binding.Mutants of IHF were examined for their ability to bind to various IHF binding sites in vitro and in vivo, and to promote recombination of in vitro. The relative affinity of mutant IHF proteins was increased by the presence of the dA+dT region, confirming the enhancer-like properties of the dA+dT element. It was also found that these mutant proteins not only retain their DNA-bending ability but make any protein-protein contacts necessary to form a recombination-proficient intasome.Gel mobility-shift assays using circularly permuted DNA fragments containing IHF sites showed that IHF forms a more compact protein-DNA complex with a site containing a dA+dT element when compared to a site lacking this sequence element. Additionally, artificial placement of a dA+dT element upstream of the H1 site in the genome of weakened recombination in vitro. Therefore, the precise nucleoprotein structure required for recombination is disrupted in this modified substrate. These results support the hypothesis that the DNA at an IHF site is bent in a different manner when the dA+dT element is present. It was demonstrated that IHF can form a specific protein-DNA crosslink with binding sites containing or lacking this dA+dT element. These results confirm the importance of flanking DNA and a dA+dT element in the binding and bending of a site by IHF.Made available in DSpace on 2011-05-07T11:55:55Z (GMT). No. of bitstreams: 2
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Crystal Structures of Native and AdoMet Bound rRNA Methyltransferase from Sinorhizobium meliloti : Structural Insights into rRNA Recognition. Evolutionary, Structural and Functional Studies on Nucleoid-Associated Proteins HU and IHF
No full textDNA- and RNA-binding proteins play a central role in gene regulation, which includes transcriptional control, alternative splicing, post-translational and transcriptional modifications like methylation and acetylation among other roles. In this way, they control most of the working machinery of the cell in direct or indirect manner. Although more than 60 years ago the structure of DNA was proposed by Watson and Crick, our understanding of how RNA- and DNA-binding proteins interact with the genome and transcriptome remains scarce. One of the most important questions in biology is how a large number of DNA- and RNA-binding proteins find their target, interact and later disassociate. These nucleic acid binding proteins either recognizes the unique structural and chemical signatures of the bases (base readout) which give the specificity or it recognizes a sequence-dependent shape (shape readout).
Methyltransferases are enzymes with diverse folds, which perform methyltransfer to various substrates using mainly S-adenosyl-L-methionine (AdoMet) as a methyl donor. RNA methylation is one of the most crucial post-transcriptional modifications which influences a wide variety of cellular processes like metabolic stabilization of RNA, quality control in protein synthesis, resistance to antibiotics, mRNA reading frame maintenance, splicing, viral nucleoprotein stabilization among others. Specificity in recognition and methylation in ribosomal RNA (rRNA) methyltransferases is very crucial, as rRNA is highly conserved and lack of specificity would influence the stabilization of RNA and thus, will affect the ribosome. In recent years, rRNA modifications which confer resistance to ribosomal antibiotics have also been observed. The mechanism of recognition to their unique rRNA target site with high selectivity and their evolution still remains an enigma. Thus, the evolution of antibiotic resistance-conferring methyltransferases in pathogenic organisms needs to be investigated from the structural and evolutionary perspective.
In the last two decades, many global regulators in both eukaryotes and prokaryotes have been discovered, which promiscuously bind to a large number of DNA sequences. In prokaryotes, they are called as ‘Nucleoid-associated proteins’ (NAPs), which influence the transcriptional process and exhibit multi-specificity or promiscuity. They also take part in the formation of many multi-protein complexes. HU and Integration Host Factor (IHF) are NAPs which belong to prokaryotic DNA-bending protein family (DNABII family). HU and IHF play crucial architectural roles in bacterial DNA condensation and additionally play a regulatory role in many cellular processes. Although sharing structural similarity, the DNA binding and bending features of HU and IHF are strikingly different, allowing them to selectively regulate genes from different genomic locations. HU binds to DNA in a sequence promiscuous manner while IHF is moderately sequence specific. The molecular mechanism of DNA binding multi-specificity (differential specificity with varied binding affinity) of HU/IHF proteins remains unexplored, as little attention has been paid to the determinants at the sequence level.
Now, the fundamental question which the author attempted to understand is the structural and evolutionary determinants of specificity in DNA- and RNA-binding proteins. The candidate has taken nucleoid-associated protein HU and SPOUT superfamily RNA methyltransferase as model systems. As the very limited number of structural folds makes up the DNA- and RNA-binding proteins, it is intriguing to examine closely related nucleic acid binding domains or folds carrying out specific functions. Also, we observed that some proteins having a particular structural fold (or homologous ancestry) bind to DNA or RNA with high specificity, while its other homolog binds promiscuously. These observations tempted us to find the sequence and structural determinants which guide this phenomenon, not just specific to only a single protein family, but, determinants are of more general nature, where results can possibly be applied to other nucleic acid binding proteins too.
The first part of the thesis reports the crystal structures of native and AdoMet bound ribosomal RNA Methyltransferase from Sinorhizobium meliloti (smMtase), by single anomalous dispersion (SAD) phasing on seleno-methionine substituted crystal, which diffracted to 2.28Å and 2.9 Å resolutions respectively in space group P212121. smMtase belong to an rRNA binding SPOUT superfamily protein, which is fused with an RNA binding L30e domain at the N-terminus. We focused our study on these types of proteins among the large superfamily (henceforth termed as SPOUTL30).
The author also has conducted a phylogenetic study, which revealed 11 major clades, out of which we focused our present study in understanding the sequence conservation and variations of 5 (A-E) clades, for which structural, biochemical and functional data is available. These proteins share homology to antibiotic resistance conferring methyltransferases. The availability of experimentally determined structures of native and AdoMet bound smMtase along with an analysis of other homologous crystal structures has enabled a critical examination of factors influencing RNA binding specificity. Also, the thesis reports for the first time an evolutionary and structural inter-connectivity of the three conserved motifs (I-III) in SPOUT superfamily, which is responsible for AdoMet binding and catalysis. The results highlight that both the location of conserved positive and negatively charged residues influence the RNA binding, specificity, and affinity. The conservation of these residues could be at superfamily, family or at clade level, and the position of these charged residues at specific sites, alters their salt-bridge geometry, which ultimately fixes the conformation of RNA-binding residues, thus defining a particular binding site specific to its cognate RNA. The study conducted by the author reveals that the dynamics of salt-bridge and other directional interactions like hydrogen bonding and aromatic interactions essentially determines the specificity of SPOUTL30.
The second part of the thesis reports evolutionary, structural and functional studies on nucleoid-associated proteins HU and IHF. To understand the sequence determinants, which influence the degree of DNA binding specificity, we undertook a phylogenetic study in conjunction with analysis of three-dimensional structures. The phylogenetic analysis revealed three major clades, belonging to HU, IHFα, and IHFβ like proteins with reference to E. coli. The author observed statistically significant amino acid compositional bias in the DNA binding sites of HU and IHF clade proteins. The author proposes that the molecular mechanisms giving rise to specificity or multi-specificity depend on a combination effect of the amino acid composition of the binding site, its flexibility, ionic and steric constraints. In continuation of this part of the thesis, the candidate examined the role of protein interacting interface of HU-IHF family proteins, understanding its evolutionary history and utilizing it in designing inhibitors for Mycobacterium tuberculosis HU (MtbHU). The present results give a model example of an evolutionary study of a protein interface of nucleoid-associated protein, which is used to understand the interface and computationally design inhibitors targeting it.
The author was a part of the study (Bhowmick et al. 2014, Nature communications) which has determined the crystal structure of Mycobacterium tuberculosis HU, inhibited it using stilbene derivatives (SD1 and SD4) which curtailed the Mtb cell growth. In the present thesis, the candidate observed from microarray analysis that the SD1 stimulon consists of genes involved majorly in lipid biosynthesis pathway, ribosomal genes which affect the overall translation, aerobic respiration pathways, antigenic membrane proteins involved in pathogenicity. Nearly half of the genes in affected by SD1 are essential in nature, thus could explain the curtailing of cellular growth. The whole study provides a system inspired view of probing as well, inhibiting global regulator HU using novel chemical molecules
Panel A shows the pressure FRET ratio baseline data (open circle) and polynomial smoothing curve (solid line for oligonucleotide A
No full text<p><b>Copyright information:</b></p><p>Taken from "Pressure dissociation of integration host factor–DNA complexes reveals flexibility-dependent structural variation at the protein–DNA interface"</p><p></p><p>Nucleic Acids Research 2007;35(6):1761-1772.</p><p>Published online 25 Feb 2007</p><p>PMCID:PMC1874591.</p><p>© 2007 The Author(s)</p>6 in the absence of IHF compared with unprocessed data for 10 nM DNA and 25 nM IHF (filled square) (10 mM Tris pH 8.0, 100 mM NaCl and 1 mM EDTA). Panel B compares fraction bound for oligonucleotides A.2 (filled diamond) and A.6 (filled square) at 10 nM DNA, 25 nM IHF, i.e. same A.6 data as panel A and same reaction conditions. Solid and dashed curves are the fits and 95% confidence intervals to these individual experiments, using equations () as described in the text
