I need to summarize this section of a research paper on materials and mthods. I
ID: 87122 • Letter: I
Question
I need to summarize this section of a research paper on materials and mthods. I have no idea wat it is even saying. please explain and summarize the idea of the method if you can.:
DNA curvature prediction and electrophoretic mobility shift assays.TheupaCpromoter region was analyzedin silicousing bend.it, aprogram that enables the prediction of a curvature propensity plot calcu-lated with DNase I-based parameters (http://hydra.icgeb.trieste.it/dna/)(73). The curvature is calculated as a vector sum of dinucleotide geome-tries (roll, tilt, and twist angles) and expressed as degrees per helical turn(10.5°/helical turn1°/bp). Experimentally tested curved motifs producecurvature values of 5 to 25°/helical turn, whereas straight motifs give val-ues below 5°/helical turn. TheupaC250-bp promoter region was ampli-fied using primers upaC.pro.ext-5+250 and upaC.pro.ext-3-1ATG, and its intrinsic curvature was assessed by comparing its electrophoretic mo-bility with that of an unbent marker fragment (Promega; 100-bp DNAladder) on a 0.5% Tris-borate-EDTA (TBE), 7.5% PAGE gel at 4°C forretarded gel electrophoretic mobility.Gel shift assays were performed essentially as previously described (3).A DNA mixture comprising an equimolar ratio of the PCR-amplified upaC promoter region and TaqI-SspI-digested pBR322 was incubated at room temperature for 15 min with increasing amounts of native purified H-NS protein (a gift from S. Rimsky) in 30l of reaction mixture containing 40 mM HEPES (pH 8), 60 mM potassium glutamate, 8 mM mag-nesium aspartate, 5 mM dithiothreitol, 10% glycerol, 0.1% octylphenoxy-polyethoxyethanol, 0.1 mg/ml BSA (H-NS binding buffer). DNAfragments and DNA-protein complexes were resolved by gel electropho-resis (0.5% TBE, 3% MS agarose gel run at 50 V at 4°C) and visualized after staining with ethidium bromide.
Explanation / Answer
This server predicts DNA curvature from DNA sequences. The curvature is calculated as a vector sum of dinucleotide geometries (roll, tilt and twist angles) using the BEND algorithm of Godsell and Dickerson, and expressed as degrees per helical turn (10.5 °/helical turn = 1 °/basepair).
This calculation - like all predictions - is approximate. Experimentaly tested curved motifs produce curvature values of 5 to 25 °/helical turn, while straight motifs give values below 5 °/helical turn.
The angle can be calculated as the angle of normal vectors of the first and last basepair plains. In order to get values that are independent of the DNA length, one can present curvature as degree/basepair or degree/helical turn. From this quantity it is easy to calculate the number of nucleotides or helical turns necessary to form a full circle of 360°. For example a value18 °/helical turn defines a circle of 20 helical turns or 210 nucleotides (20x18=360, and 1 helical turn =10.5 residues).
The prediction of curvature is based on statistically derived roll, tilt and twist angles of dinucleotide steps. We suppose that a dinucleotide step (i.e. two successive basepairs) will curve to the same extent, no matter where it is in a DNA structure. This big and not necessarily correct simplification allows one to derive statistical average values from 3D structures, or from other measurements, such as electrophoretic mobility analysis, nucleosome binding or DNaseI cutting assays.
The calculation is then rather simple: we divide the DNA sequence into overlapping dinucleotides, and assign the corresponding roll, tilt and twist angles to each step. We suppose (another simplification) that the molecule is an ideal B-DNA with successive basepairs 3.4 Angstroms apart. Then we can calculate the DNA path as a result of one vertical translation (3.4 A) and three rotations (roll, tilt, twist). In principle, this calculation is quite complicated, but i) for small angles, such as occur in DNA, and ii) for short DNA segments, the problem is simplified into a straightforward vectorial addition of the geometric parameters. This procedure is complemented with averaging [Goodsell and Dickerson, 1994], and the curvature is determined for a standard segment length, in our case 31 basepairs or approximately 3 helical turns. This value is assigned to the center of the segment. Plotting this value along the sequence one gets a curvature vs. sequence plot, that shows peaks at highly curved positions.