10 research outputs found
Algorithm for discrete TI model.
<p><b>(A)</b> A pair of convergent promoters pX (present on sense/top DNA strand) and pY (present on antisense/bottom DNA strand) separated by overlapping DNA of length L is shown. Promoters pX and pY drive expression of genes <i>X</i> and <i>Y</i> respectively, which produce full-length transcripts <i>x</i> and <i>y</i> (denoted by bold arrows) respectively. For each i<sup>th</sup> and j<sup>th</sup> round of transcription from pX and pY promoters respectively, RNAP (denoted by large grey ovals) form DNA-bound RNAP complexes at the respective promoter region following a binding (Ï„<sub>BX</sub> and Ï„<sub>BY</sub>) and initiation (Ï„<sub>IX</sub> and Ï„<sub>IY</sub>) process. After firing, the center of RNAP moves to first position of the overlapping region to form an elongation complex (EC, denoted by smaller grey ovals). The time taken for each i<sup>th</sup> EC (fired from pX) to reach k<sup>th</sup> position on sense strand (t<sub>X,i,k</sub>) as well as the time taken for each j<sup>th</sup> EC (fired from pY) to reach h<sup>th</sup> position on the antisense (t<sub>Y,j,h</sub>) strand along the overlapping DNA are tracked. The footprint of an EC is denoted by <i>fp</i>. <b>(B)</b> For each i<sup>th</sup> and j<sup>th</sup> rounds of transcription, the model calculates the outcome of TI depending on the region where opposing RNAPs meet. Occlusion and sitting duck interference occur at the promoters pX (left panels) or pY (right panels), and RNAP collisions between ECs occur along the overlapping DNA (middle panel) following the mathematical constraints shown. Upon RNAP collision, one or both ECs on the sense and antisense strand fall off the DNA and result in production of truncated transcripts <i>x</i><sub><i>k</i></sub> and <i>y</i><sub><i>h</i></sub> (denoted by dashed arrows) from pX and pY respectively. In absence of any kind of TI, transcription is successful, producing a full-length transcript (<i>x</i>, <i>y</i>). Once 30,000 rounds of transcription from the stronger promoter have been calculated the net rate of production of full-length (<i>k</i><sub><i>x</i></sub> and <i>k</i><sub><i>y</i></sub>) and truncated RNA (<math><mrow><msub><mi>k</mi><mrow><msub><mi>x</mi><mi>k</mi></msub></mrow></msub></mrow></math> and <math><mrow><msub><mi>k</mi><mrow><msub><mi>y</mi>h</msub></mrow></msub></mrow></math>) are obtained.</p
Algorithm for discrete TI model.
<p><b>(A)</b> A pair of convergent promoters pX (present on sense/top DNA strand) and pY (present on antisense/bottom DNA strand) separated by overlapping DNA of length L is shown. Promoters pX and pY drive expression of genes <i>X</i> and <i>Y</i> respectively, which produce full-length transcripts <i>x</i> and <i>y</i> (denoted by bold arrows) respectively. For each i<sup>th</sup> and j<sup>th</sup> round of transcription from pX and pY promoters respectively, RNAP (denoted by large grey ovals) form DNA-bound RNAP complexes at the respective promoter region following a binding (Ï„<sub>BX</sub> and Ï„<sub>BY</sub>) and initiation (Ï„<sub>IX</sub> and Ï„<sub>IY</sub>) process. After firing, the center of RNAP moves to first position of the overlapping region to form an elongation complex (EC, denoted by smaller grey ovals). The time taken for each i<sup>th</sup> EC (fired from pX) to reach k<sup>th</sup> position on sense strand (t<sub>X,i,k</sub>) as well as the time taken for each j<sup>th</sup> EC (fired from pY) to reach h<sup>th</sup> position on the antisense (t<sub>Y,j,h</sub>) strand along the overlapping DNA are tracked. The footprint of an EC is denoted by <i>fp</i>. <b>(B)</b> For each i<sup>th</sup> and j<sup>th</sup> rounds of transcription, the model calculates the outcome of TI depending on the region where opposing RNAPs meet. Occlusion and sitting duck interference occur at the promoters pX (left panels) or pY (right panels), and RNAP collisions between ECs occur along the overlapping DNA (middle panel) following the mathematical constraints shown. Upon RNAP collision, one or both ECs on the sense and antisense strand fall off the DNA and result in production of truncated transcripts <i>x</i><sub><i>k</i></sub> and <i>y</i><sub><i>h</i></sub> (denoted by dashed arrows) from pX and pY respectively. In absence of any kind of TI, transcription is successful, producing a full-length transcript (<i>x</i>, <i>y</i>). Once 30,000 rounds of transcription from the stronger promoter have been calculated the net rate of production of full-length (<i>k</i><sub><i>x</i></sub> and <i>k</i><sub><i>y</i></sub>) and truncated RNA (<math><mrow><msub><mi>k</mi><mrow><msub><mi>x</mi><mi>k</mi></msub></mrow></msub></mrow></math> and <math><mrow><msub><mi>k</mi><mrow><msub><mi>y</mi>h</msub></mrow></msub></mrow></math>) are obtained.</p
Effect of TI and AR on switch response.
<p><b>(A)</b> TI model predicts the rate of production of truncated (dashed) and full-length RNA (bold). Interactions between different species arise due to presence of secondary structures and complementary sequences. RNA duplexes are targeted for fast degradation. <b>(B, C)</b> Different switch responses of full-length <i>x</i> and <i>y</i> levels depending on presence or absence of TI and AR. Sharpest response is obtained when both mechanisms are coupled, <i>H</i> denotes value of Hill coefficient.</p
Antisense transcription coupled with protein feedback gives rise to bistability.
<p><b>(A)</b> Successful full-length <i>x</i> and <i>y</i> transcripts that do not undergo antisense interaction are free to be translated into proteins X and Y respectively. Production of inducer molecule, Z, is indirectly activated by protein Y. Protein X implements a negative feed-back loop by binding to operator site O<sub>Y</sub> and repressing promoter pY. Z binds to X and relieves the repression of pY promoter. <b>(B-D)</b> These multiple regulatory layers enable cells to demonstrate a higher-order a bistable switch response to different rates of production of W (denoted by k<sub>WZ</sub>). ON state is characterized by production of proteins Y and Z while OFF state is characterized by production of protein X. Threshold k<sub>WZ</sub> value to switch between OFF to ON states is 11.5 nM/s whereas the threshold for the inverse switch between ON to OFF states occurs at k<sub>WZ</sub> = 8.8 nM/s.</p
TI in the presence of RNAP pause sites.
<p><b>(A)</b> Mechanistic representation of RNAP collision in presence of two pause sites in the sense strand at positions 160 bp (x<sub>k</sub>/L = 0.4) and 320 bp (x<sub>k</sub>/L = 0.8). Pause times are 2 s. Increased production of truncated RNA at the pause sites due to enhanced probability of collision is indicated by arrows. <b>(B)</b> Switch response in <i>x</i> levels maintains sharpness as pause time increases but the dynamic range of <i>x</i> is widened more than one order of magnitude. <i>H</i> denotes the value of Hill coefficient (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133873#sec002" target="_blank">Methods</a>). <b>(C)</b> Expression maps for truncated and full-length RNA from pX at different pause times. Truncated RNA production increases as pause time increases. <b>(D)</b> Expression maps for truncated and full-length RNA from pY at different pause times.</p
Parameters in GN model that impact bistable switch behavior.
<p>Examples of some of the parameters that had a greater effect when fine-tuned. Protein X and Y levels are shown for each modified parameter. Arrows indicate the way the curve shifts as the following parameters are individually tuned relative to the values shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133873#pone.0133873.s001" target="_blank">S1 Table</a>. <b>(A, B)</b><i>f</i><sub>X</sub>, firing rate at pX. <b>(C, D)</b><i>f</i><sub>Y,max</sub>, firing rate at pX at de-repressed state. <b>(E, F)</b><i>f</i><sub>Y,min</sub>, firing rate at pX at repressed state. <b>(G, H)</b> k<sub>bxy</sub>, binding rate of full-length transcripts <i>x</i> and <i>y</i>. <b>(I, J)</b> k<sub>XZ</sub>, binding rate of protein complex X:Z. <b>(K, L)</b> K<sub>OY,</sub> equilibrium binding constant of protein X at O<sub>Y</sub> site.</p
Antisense transcription mechanisms in a set of convergent genes.
<p><b>(A)</b> General set of convergent promoters driving expression of genes <i>X</i> and <i>Y</i> synthesizing transcripts <i>x</i> and <i>y</i> (bold arrows), respectively. Such a system is susceptible to TI and produces overlapping transcripts that may participate in AR. <b>(B)</b> AR can cause translational inhibition, mRNA degradation and transcriptional attenuation due to the interactions that may exist between full-length sense and antisense transcripts as well as truncated RNA produced as a result of RNAP collisions, one of the reported TI mechanisms. <b>(C)</b> Mechanisms of TI: Occlusion caused by passage of an opposing elongating RNAP on the antisense promoter which hinders binding of RNAP to the sense promoter; Sitting duck interference, dislodgement of an initiation complex due to collision with an opposing elongating RNAP; and Collision between opposing elongating RNAP molecules that produces truncated RNA of different sizes susceptible to participate in AR. Both TI and AR mechanisms are likely to be coupled during antisense transcription.</p
List of species and corresponding models.
<p>List of species and corresponding models.</p
<i>Cis</i>-Antisense Transcription Gives Rise to Tunable Genetic Switch Behavior: A Mathematical Modeling Approach
<div><p>Antisense transcription has been extensively recognized as a regulatory mechanism for gene expression across all kingdoms of life. Despite the broad importance and extensive experimental determination of <i>cis</i>-antisense transcription, relatively little is known about its role in controlling cellular switching responses. Growing evidence suggests the presence of non-coding <i>cis</i>-antisense RNAs that regulate gene expression via antisense interaction. Recent studies also indicate the role of transcriptional interference in regulating expression of neighboring genes due to traffic of RNA polymerases from adjacent promoter regions. Previous models investigate these mechanisms independently, however, little is understood about how cells utilize coupling of these mechanisms in advantageous ways that could also be used to design novel synthetic genetic devices. Here, we present a mathematical modeling framework for antisense transcription that combines the effects of both transcriptional interference and <i>cis</i>-antisense regulation. We demonstrate the tunability of transcriptional interference through various parameters, and that coupling of transcriptional interference with <i>cis</i>-antisense RNA interaction gives rise to hypersensitive switches in expression of both antisense genes. When implementing additional positive and negative feed-back loops from proteins encoded by these genes, the system response acquires a bistable behavior. Our model shows that combining these multiple-levels of regulation allows fine-tuning of system parameters to give rise to a highly tunable output, ranging from a simple-first order response to biologically complex higher-order response such as tunable bistable switch. We identify important parameters affecting the cellular switch response in order to provide the design principles for tunable gene expression using antisense transcription. This presents an important insight into functional role of antisense transcription and its importance towards design of synthetic biological switches.</p></div