3D models from "Human TFIID binds to core promoter DNA in a reorganized structural state"

<p><em>3D models from the publication: </em></p> <p><strong>Human TFIID binds to core promoter DNA in a reorganized structural state</strong></p> <p>Michael A. Cianfrocco, George A. Kassavetis, Patricia Grob, Jie Fang, Tamar Juven-Gershon, James T. Kadonaga, and Eva Nogales</p> <p><em>Cell. Jan 17, 2013; 152(1-2): 120–131.</em></p> <p>________________________________</p> <p>To create specific figures from the publication, first download the appropriate files:</p> <p>1) Download the .zip archived file <em>Cianfrocco_et_al_2013_3D_Models.zip</em>.</p> <p>2) Download and install the latest version of <em>UCSF Chimera.</em></p> <p>3) Within Chimera, go to<em> File > Restore Session</em>, selecting the file <em>Figures_Chimera_Session.py</em> from the unzipped folder from step #1.</p> <p>4) Once opened, Chimera will display an interactive session showing <em>Example_Snapshot.png.</em></p> <p>________________________________</p> <p>After Chimera is installed and the .zip file is uncompressed, following these steps for generating specific parts of the figures. Each set of instructions assume that you are starting from provided Chimera session file: </p> <p><strong>Figure 3C: </strong></p> <p><strong>1)</strong><em> Tools > Volume Data > Volume Viewer</em></p> <p><strong>2) </strong>On the new window, click on <em>Data > IID-IIA-SCP rearranged</em></p> <p><strong>3) </strong>Now select 'surface' from the style choices. </p> <p><strong>4)</strong> Go to the model panel:<em> Favorites > Model Panel</em></p> <p><strong>5) </strong>Uncheck the boxes next to <em>SCP30_promoter</em> and <em>Surface of SCP30_promoter</em></p> <p><strong>Figure 3D:</strong></p> <p><strong>1)</strong> Go to the model panel: <em>Favorites > Model Panel</em></p> <p><strong>2) </strong>Uncheck box for<em> IID-IIA-SCP rearranged</em></p> <p><strong>3)</strong> Check box for <em>TFIID rearranged</em></p> <p><strong>4)</strong> Uncheck the boxes next to <em>SCP30_promoter</em> and <em>Surface of SCP30_promoter</em></p> <p><strong>Figure 3E:</strong></p> <p><strong>1)</strong> Go to the model panel: <em>Favorites > Model Panel</em></p> <p><strong>2)</strong> Uncheck box for <em>IID-IIA-SCP rearranged</em></p> <p><strong>3)</strong> Check box for <em>IID-IIA-SCP canonical</em></p> <p><strong>4)</strong> Uncheck the boxes next to <em>SCP30_promoter</em> and <em>Surface of SCP30_promoter</em></p> <p><strong>Figure 3F:</strong></p> <p><strong>1) </strong>Go to the model panel: <em>Favorites > Model Panel</em></p> <p><strong>2)</strong> Uncheck box for <em>IID-IIA-SCP rearranged</em></p> <p><strong>3)</strong> Check box for <em>TFIID canonical</em></p> <p><strong>4)</strong> Uncheck the boxes next to <em>SCP30_promoter</em> and <em>Surface of SCP30_promoter</em></p> <p><strong>Figure 4A:</strong></p> <p><strong>1)</strong> Go to the model panel: <em>Favorites > Model Panel</em></p> <p><strong>2)</strong> Uncheck box for <em>IID-IIA-SCP rearranged</em></p> <p><strong>3)</strong> Check box for <em>IID-IIA-SCP(+30) rearranged</em></p> <p><strong>4)</strong> Uncheck the boxes next to <em>SCP30_promoter</em> and <em>Surface of SCP30_promoter</em></p> <p><strong>Figure 6A & B: Core promoter elements</strong></p> <p><strong>1) </strong>Open Chimera and load session.</p> <p><strong>Figure 6A & B: DNase I footprint</strong></p> <p><strong>1)</strong> Go to the model panel: <em>Favorites > Model Panel</em></p> <p><strong>2)</strong> Uncheck the boxes next to <em>SCP30_promoter</em> and <em>Surface of SCP30_promoter</em></p> <p><strong>3)</strong> Check boxes next to <em>DNase IID-IIA-SCP</em> and <em>DNase IID-IIA-SCP surface</em></p> <p><strong>Figure 6A & B: MPE-Fe footprint</strong></p> <p><strong>1)</strong> Go to the model panel: <em>Favorites > Model Panel</em></p> <p><em></em><strong>2)</strong> Uncheck the boxes next to <em>SCP30_promoter</em> and <em>Surface of SCP30_promoter</em></p> <p><em></em><strong>3)</strong> Check boxes next to <em>MPE_Fe_IID_IIA_SCP</em> and <em>Surface of MPE_Fe_IID_IIA_SCP</em></p> <p><strong>Figure 6C:</strong></p> <p><strong>1) </strong>Go to the model panel:<em> Favorites > Model Panel</em></p> <p><strong>2)  </strong>Uncheck the box next to <em>Surface of SCP30_promoter</em></p> <p><strong>3)</strong> Check boxes next to<em> PDB: 1NVP </em>and<em> PDB: 1VOL.</em></p> <p><strong>Figure S6B:</strong></p> <p><strong>1)</strong> Go to the model panel:<em> Favorites > Model Panel</em></p> <p><em></em><strong>2)</strong> Uncheck box for <em>IID-IIA-SCP rearranged</em></p> <p><em></em><strong>3)</strong> Check box for <em>IID-IIA-SCP(mTATA) rearranged</em></p> <p><em></em><strong>4)</strong> Uncheck the boxes next to <em>SCP30_promoter</em> and <em>Surface of SCP30_promoter</em></p> <p><strong>Supplemental Movie 2: </strong><strong>Segmented 3D maps of TFIID</strong></p> <p><strong>1)</strong> Go to the model panel: <em>Favorites > Model Panel</em></p> <p><strong>2)</strong> Uncheck box for <em>IID-IIA-SCP rearranged</em></p> <p>Then, check the box for the model that you would like shown:</p> <p><em>TFIID canonical, lobe A only: </em>Lobe A (extracted from canonical IID alone)</p> <p><em>TFIID canonical, BC core only: </em>BC core (extracted from canonical IID alone)</p> <p><em>TFIID rearranged, lobe A only: </em>Lobe A (extracted from rearranged IID alone)</p> <p><em>TFIID rearranged, BC core only: </em>BC core (extracted from rearranged IID alone)</p> <p>________________________________</p> <p><strong>File information:</strong></p> <p><em>EMD-2282.spi -</em> TFIID-IIA-SCP, rearranged</p> <p><em>EMD-2283.spi - </em>TFIID-IIA-SCP, canonical</p> <p><em>EMD-2284.spi - </em>holoTFIID, rearranged</p> <p><em>EMD-2287.spi -</em> holoTFIID, canonical</p> <p><em>Figure.png -</em> Example snapshot of opened session</p> <p><em>Figures_Chimera_Session.py -</em> Chimera session file </p> <p><em>IID_IIA_SCP+30_rearranged.spi -</em> 3D model for TFIID-IIA-SCP(+30) (Figure 4A)</p> <p><em>IID_IIA_SCPmTATA_rearranged.spi - </em>3D model for TFIID-TFIIA-SCP(mTATA) (Figure S6B)</p> <p><em>SCP1_30.pdb -</em> PDB coordinate file for SCP(+30) sequence</p> <p><em>holoTFIID_canonical_BC.mrc -</em> BC core from TFIID alone (canonical)</p> <p><em>holoTFIID_canonical_lobeA.mrc -</em> lobe A from TFIID alone (canonical)</p> <p><em>holoTFIID_rearranged_BC.mrc -</em> BC core from TFIID alone (rearranged)</p> <p><em>holoTFIID_rearranged_lobeA.mrc -</em> lobe A from TFIID alone (rearranged)</p> <p><br></p> <p><em><br></em></p> <p> </p> <p><em><br></em></p> <p> </p

Schematic representation of the engineered core promoters.

<p>The pRc/CMV vector (Life Technologies) contains the CMV enhancer and TATA box, but lacks any CMV sequences that are downstream of -16 relative to the +1 transcription start site (including the Inr element). Three variants of pRc/CMV were constructed in which the core promoter region (from -36 to +45) was replaced with either the natural CMV core promoter, which contains the CMV TATA and Inr elements, or with SCP2 or SCP3, which contains the CMV TATA and Inr, the <i>Tollo</i> MTE, and the <i>Calm2</i> DPE. Single nucleotide changes in SCP3 (relative to SCP2) are marked by red rectangles. Each of these pRc/CMV-based constructs contains the <i>EGFP</i> reporter gene.</p

Live cell <i>EGFP</i> imaging of short-term expression of pRc/CMV-based constructs, in HeLa S3 and SH-SY5Y cells.

<p>HeLa S3 and SH-SY5Y cells were transiently transfected with either the pRc/CMV, natural CMV, SCP2 or SCP3 vector expressing <i>EGFP</i>. The cells were imaged once a day during 1–4 days post-transfection (P.T.). Each circle displays the whole well image constructed by stitching individual microscopic fields. (A) HeLa S3 cells. (B) SH-SY5Y cells. Data shown are representative of 3 independent experiments for each cell type.</p

Flow cytometric analysis of short-term fluorescence intensity and number of fluorescent HeLa S3 and SH-SY5Y cells.

<p>HeLa S3 and SH-SY5Y cells were transiently transfected with pRc/CMV, natural CMV, SCP2 or SCP3 vector expressing <i>EGFP</i>. The cells were collected 1–4 days post-transfection (P.T.) for flow cytometric analysis. (A) Flow cytometric analysis of fluorescence intensity of all HeLa S3 fluorescent cells and SH-SY5Y fluorescent cells. (B) Flow cytometric analysis of fluorescence intensity of high intensity HeLa S3 fluorescent cells and SH-SY5Y fluorescent cells. (C) Flow cytometric analysis of the number of all HeLa S3 fluorescent cells and SH-SY5Y fluorescent cells. (D) Flow cytometric analysis of the number of high intensity HeLa S3 fluorescent cells and fluorescent SH-SY5Y cells. Data shown are representative of 5 independent experiments using HeLa S3 cells and 6 independent experiments using SH-SY5Y cells. Statistical comparisons between the promoters were done using the Kruskal—Wallis test with pairwise comparisons. Significant p-values (p ≤0.05) are indicated in the results section.</p

Flow cytometric analysis of long-term fluorescence intensity and number of fluorescent HeLa S3 and SH-SY5Y cells.

<p>HeLa S3 and SH-SY5Y cells were transiently transfected with pRc/CMV, natural CMV, SCP2 or SCP3 vector expressing <i>EGFP</i>. The cells were collected 4–8 days post-transfection (P.T.) for flow cytometric analysis. (A) Flow cytometric analysis of fluorescence intensity of all HeLa S3 fluorescent cells and SH-SY5Y fluorescent cells. (B) Flow cytometric analysis of fluorescence intensity of high intensity HeLa S3 fluorescent cells and SH-SY5Y fluorescent cells. (C) Flow cytometric analysis of the number of all HeLa S3 fluorescent cells and SH-SY5Y fluorescent cells. (D) Flow cytometric analysis of the number of high intensity HeLa S3 fluorescent cells and SH-SY5Y fluorescent cells. Data shown are representative of 6 independent experiments using HeLa S3 cells and 5 independent experiments using SH-SY5Y cells. Statistical comparisons between the promoters were done using the Kruskal—Wallis test with pairwise comparisons. Significant p-values (p ≤0.05) are indicated in the results section.</p

A comparison of the four core promoters DNA sequences.

<p>A comparison of the four core promoters DNA sequences.</p

Real-Time quantitative PCR of purified transiently transfected plasmid DNA in HeLa S3 and SH-SY5Y cells.

<p>HeLa S3 and SH-SY5Y cells were transiently transfected with pRc/CMV, natural CMV, SCP2 or SCP3 vector expressing <i>EGFP</i>, and harvested every other day during 8 days post-transfection (P.T.). Plasmid DNA was purified from cells and subjected to qPCR analysis with primers for the GAPDH, <i>EGFP</i> and Neomycin genes. Data shown are the averaged Ct values of 3 independent experiments (each performed in triplicates). (A) 2 days post-transfection. (B) 4 days post-transfection. (C) 6 days post-transfection. (D) 8 days post-transfection. Error bars represent SEM.</p