Physiological Levels of Salt and Polyamines Favor Writhe and Limit Twist in DNA

Quantitative analysis of single molecule experiments show that adding either of two natural polyamines, spermine or spermidine, produced more compact plectonemes in DNA in physiological concentrations of monovalent salt. They also promoted plectoneme formation at lower values of torsion in measurements of extension versus twist. Quantifying changes in the plectonemic DNA using some results from simple rod models suggested that exposure to polyamines reduced the radii and increased the densities of plectonemes. Thus, polyamines may limit the twist density by favoring writhe which maintains the B-form. Although polymerases may significantly stretch the double helix, denature DNA, and produce twist instead of writhe, natural polyamines stabilize base-pairing, limit twist to maintain the B-form, and promote supercoiling, which is conducive to replication and transcription and essential for DNA packaging

Direct Characterization of Transcription Elongation by RNA Polymerase I

<div><p>RNA polymerase I (Pol I) transcribes ribosomal DNA and is responsible for more than 60% of transcription in a growing cell. Despite this fundamental role that directly impacts cell growth and proliferation, the kinetics of transcription by Pol I are poorly understood. This study provides direct characterization of <i>S</i>. <i>Cerevisiae</i> Pol I transcription elongation using tethered particle microscopy (TPM). Pol I was shown to elongate at an average rate of approximately 20 nt/s. However, the maximum speed observed was, in average, about 60 nt/s, comparable to the rate calculated based on the <i>in vivo</i> number of active genes, the cell division rate and the number of engaged polymerases observed in EM images. Addition of RNA endonucleases to the TPM elongation assays enhanced processivity. Together, these data suggest that additional transcription factors contribute to efficient and processive transcription elongation by RNA polymerase I <i>in vivo</i>.</p></div

Pol I elongation rates.

<p>The average rate (a) was calculated from the beginning to the end of each elongation run. The maximum rate (b) in each of the 65 traces was found as described in the Materials and Methods, “Particle Tracking and Transcription Rate Analysis.” The distributions of (a) and (b) were then fitted with exponential functions with mean values of 20.7 (average) and 58.4 (maximum) nt/s rates of elongation (R-values of 0.95 and 0.98 respectively). These mean values are significantly different with respective 95% confidence intervals of (14.1–27.3) and (48.6–68.4) relative to the fitted mean values. Note that for an exponential distribution, the standard deviation is equal to the mean.</p

DNA and protein components.

<p>A diagram (not to scale) of the rDNA elements and proteins used in the transcription assays reported here (top). A diagram of the chromosomal context of the rDNA fragment used in the TPM measurements of RNA Pol I elongation (bottom). Note that since Pol I only transcribes rDNA, the DNA tether selected for our experiments was a portion of the natural Pol I template.</p

Pol I processivity measured by TPM.

<p>Elongation complexes arrested after transcribing different distances along the DNA template, with only seven out of sixty-five complexes reaching the expected run-off site and releasing DNA. The percentage of elongation complexes that reached a given position along the template is plotted for single molecule experiments with (purple) or without (gold) added RNAse. Note that position 556 corresponds to the halt site and 2444 nucleotides can be transcribed before runoff at position 2500.</p

Specifically bound lambda repressor dimers promote adjacent non-specific binding

<div><p>Genetic switches frequently include DNA loops secured by proteins. Recent studies of the lambda bacteriophage repressor (CI), showed that this arrangement in which the protein links two sets of three operators separated by approximately 2.3 kbp, optimizes both the stability and dynamics of DNA loops, compared to an arrangement with just two sets of two operators. Because adjacent dimers interact pairwise, we hypothesized that the odd number of operators in each set of the lambda regulatory system might have evolved to allow for semi-specific, pair-wise interactions that add stability to the loop while maintaining it dynamic. More generally, additional CI dimers may bind non-specifically to flanking DNA sequences making the genetic switch more sensitive to CI concentration. Here, we tested this hypothesis using spectroscopic and imaging approaches to study the binding of the lambda repressor (CI) dimer protein to DNA fragments. For fragments with only one operator and a short flanking sequence, fluorescence correlation spectroscopy measurements clearly indicated the presence of two distinct DNA-CI complexes; one is thought to have a non-specifically bound CI dimer on the flanking sequence. Scanning force micrographs of CI bound to DNA with all six operators revealed wild-type or mutant proteins bound at operator positions. The number of bound, wild-type proteins increased with CI concentration and was larger than expected for strictly specific binding to operators. In contrast, a mutant that fails to oligomerize beyond a dimer, D197G, only bound to operators. These data are evidence that CI cooperativity promotes oligomerization that extends from operator sites to influence the thermodynamics and kinetics of CI-mediated looping.</p></div

Specifically bound lambda repressor dimers promote adjacent non-specific binding - Fig 3

<p><b>Variations in the numbers of particles (<i>a</i>) and counts per particle (<i>b</i>) as a function of CI protein concentration.</b> Squares (filled) and circles (open) correspond to WT DNA constructs containing the OL3 or OR3 sites, respectively.</p

Variation in the amplitude of the mutant CI protein-DNA complex.

<p>The amplitudes of <i>a1</i> (filled squares) and <i>a2</i> (open circles) used to fit the auto-correlation data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194930#pone.0194930.e002" target="_blank">Eq 2</a>) are plotted as a function of mutant CI protein (D197G) concentration for (<i>a</i>) WT OL1, (<i>b</i>) WT OL3, or (<i>c</i>) WT OR3 DNA. For each DNA, <i>a</i>_<i>1</i> dropped from an initial maximum to a final minimum with a reciprocal rise in <i>a</i>_<i>2</i> as [CI] increased from 0 to 500 nM. In contrast to the wild-type protein, no third species developed at higher concentrations.</p

Specifically bound lambda repressor dimers promote adjacent non-specific binding - Fig 5

<p><b>Titrations of unlabeled DNA to assay competitive CI-binding to (<i>a</i>) wild-type OL3 or (<i>b</i>) wild-type OR3 DNA.</b> Increasing the concentration of unlabeled DNA (filled squares) from 0 to 0.3 μM in the solution used for FCS measurements reduced the fraction of CI bound. This decrease was characterized by a plateau for both OL3 and OR3. Clearly, competition by the unlabeled DNA progressively reduced the CI available for binding to the fluorescently labeled DNA. This is evidence against the presence of higher molecular weight complexes in which a head-to-head interaction of CI dimers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194930#pone.0194930.g001" target="_blank">Fig 1</a>) might stabilize FCS particles containing a labeled and an unlabeled DNA segment.</p

Measurement of positions and volumes for CI multimers which appeared as amorphous particles along DNA fragments containing OL and OR binding sites.

<p>(<i>a</i>) The positions of these protein particles were measured with respect to the nearest end at different concentrations. Even at the highest protein concentration almost all particles were found near 120 and 240 nanometers into the contour, where high affinity binding sites are located. (<i>b</i>) The sizes of CI particles increased with concentration as can be seen in a cumulative histogram of the volumes measured in scanning force nanographs recorded for DNA exposed to 200 (circles), 400 (crosses), or 800 (triangles) nM CI. (<i>c</i>) The D197G mutant CI protein also formed specifically positioned particles on the OL and OR bindings sites with little binding elsewhere. (<i>d</i>) However the particles of mutant protein, that does not form multimers larger than dimers, did not increase in size as a function of CI concentration.</p