JMol Tutorial created with support from Richard Ebright (Waksman Institute, Rutgers University) and Tim Herman (Center for Biomolecular Modeling).

Please email tshata@mybiology.com if you have any questions or to report problems with this tutorial.  Thank you.

Using this tutorial:
This website runs the Jmol molecule viewer. You will need a Java-enabled browser to view this website. Refresh your browser to resize applet to your screen size.

You can follow the tutorial below while watching short animated scripts by clicking the appropriate buttons.  At anytime, if you want to change the view of the structure, do the following:
Rotate: Click on mouse and drag
Zoom: Scroll wheel on your mouse or +Shift and left click
Move: +Ctrl and right click

If you are familiar with RasMol commands, you can also use them through the JMol console.

Introduction
RNA Polymerase is the enzyme responsible for the transcription of DNA. This enzyme is made up of five polypeptides; beta prime (β’), beta (β), two alpha units (αI and αII), and omega (ω) which collectively make up the "core enzyme". Together with the sigma factor (σ), the RNA polymerase "holoenzyme" initiates transcription by recognizing different promoter sequences.
Transcription is a key step in gene regulation. In the familiar example of the lac operon, a repressor is expressed from the regulatory gene and binds to the operator to inhibit transcription of lactose-utilization genes in absence of lactose. On the other hand, when lactose is present, the repressor becomes inactivated to make transcription of the lac genes possible.
Yet, presence of lactose alone is not sufficient to turn on the lac genes. A second condition is necessary: absence of glucose and production of cAMP. Cyclic AMP (cAMP), produced during glucose starvation, activates catabolite activator protein (CAP), also known as cAMP binding protein (CBP). This activation mechanism not only makes transcription at the lac genes possible, but also significantly increases expression levels.

Written by Ariana Lichtenstein

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CAP activation by DNA binding

Elevated levels of cyclic AMP (cAMP) (colored red) produced during glucose starvation activates catabolite activator protein (CAP), a two-fold structurally symmetrical dimer. This binds to a 22 bp long two-fold structurally symmetrical sequence of DNA, located 61 base pairs upstream from the promoter. This interaction (CAP sidechains shown in blue) is mediated by the a helix-turn-helix motif found in the CAP.

script prepared by Rachel VanWert

RNAP recruitment by CAP (CAP-αCTD-σ⁷⁰ interaction)

Once CAP is bound to DNA, the activating region 1 (AR1) of CAP (colored blue) is recognized by the 287 determinant of αCTD (colored yellow). The 573-604 determinant of σ⁷⁰ (colored pink) recognizes and binds to the 261 determinant of αCTD (colored white). This binding of RNA polymerase to CAP can increase transcription 10 to 100-fold. The interaction between αCTD and σ⁷⁰ aids in the stabilization of RNA polymerase. The interaction is the means by which the upstream promoter can be communicated to the core RNA polymerase enzyme.

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Activation without CAP

When CAP is not present, αCTD also functions as a DNA binding region that recognizes specific DNA sequences called the UP-element. This interaction occurs between the 265 determinant of αCTD (colored red) and DNA.

script prepared by Caitlin Jennings

σ⁷⁰ - promoter interaction

Sigma factors (σ) aid RNA polymerase in initiating transcription at promoters.  The predominant sigma factor, σ⁷⁰, directs the RNAP to specific promoter DNA sites to initiate transcription. σ⁷⁰ contains regions 1, 2, 3 and 4. Region 2 (orange), 3 (blue), and 4 (dark green) recognize the -10, extended -10, and -35 regions of the promoter, respectively. At least two of these three interactions are required for transcription.  Promoter sequences can deviate from the ideal "consensus sequence."  This affects the relative strength of a given promoter.  

Region 3.2 (yellow), also known as the linker region, connects regions 3 and 4. The linker region is believed to play a role in the ability of RNAP to start transcription without a primer by recruiting and stabilizing initiating nucleotides on the template DNA strand (red). Notice the Mg ion (pink) in the active site.

script prepared by Kelly Peeler

σ⁷⁰- promoter recognition

Promoter recognition by σ⁷⁰ is facilitated by interactions between regions 2, 3, and 4 of the σ factor and the -10, extended -10 (x -10), and -35 regions of the promoter, respectively.  Select aromatic amino acids in region 2 interact with the six nucleotide long -10 region of the promoter.  Upstream of this is the extended -10, also a six nucleotide sequence, which interacts with an α-helix in region 3 of the σ factor.  The -35 region of the promoter, 19 base pairs upstream of the -10 region and again six nucleotides long, interacts with a helix-turn-helix motif in region 4. These interactions between specific residues of σ factor and promoter sequences promote the melting of the transcription bubble and beginning of transcription.  Promoter sequences on DNA are shown green and protein regions in blue.

Display σ⁷⁰ as backbone to highlight structural motifs

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script prepared by Zack Cordero

Highlighting the transcription bubble in the core enzyme

By removing part of the β' subunit, the transcription bubble can be exposed. The transcription bubble is a region where double stranded DNA is melted into separate template (colored red) and non-template strands (colored pink).  This melting occurs between the -11 and +3 base pairs, in relation to the transcription start site.³  A single magnesium ion (seen here in bright pink) is found at the active site of the enzyme.

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script prepared by Nick Molé

Free RNA nucleotide entrance channel (Secondary channel)

Zoom into RNA nucleotide entrance channel.  The Mg²⁺ ion in the active site (pink) and the template strand (red) are both visible through the channel.

script prepared by Jonathan Bregman

Thank you to our SMART Team cooperating scientist, Dr. Richard Ebright, for his time to help us understand the structure and function of this complex and for his input for the organization and scripts written for this website.  The original Chime tutorial has been converted to this Jmol based site with the help of Bill Lane.

We also thank Dr. Helen Berman and the PDB for their time and cooperation with this project.  Special thanks to Dr. Shuchismita Dutta, and to Kyle Burkhardt for editing the PDB file here so that it could be used for our project.  Their time helped us better understand the nature of crystallography and the significance of the work done by crystallographers and the scientists at the PDB. Finally, thank you to all others that worked on this structure to make it available to the science community. 

References

¹ Lawson, C., Swigon, D., Murakami, K., Darst, S., Berman, H., and Ebright, R.: (2004) Catabolite activator protein (CAP): DNA binding and transcription activation.  Curr Opin Struct Biol,. 14,10-20 (2004).

² Ptashne, Mark, and Alexander Gann. Genes & Signals. New York: Cold Spring Harbor Laboratory Press, 2001.

³ Watson, James D., T. Baker, S. Bell, A. Gann, M. Levine, and R. Losick. Molecular Biology of the Gene, Fifth Ed. Benjamin Cummings, 2004.

PDB ID: 1LB2

Benoff, B., Yang, H., Lawson, C. L., Parkinson, G., Lui, J., Blatter, E., Ebright, Y. W., Berman, H. M., Ebright, R. H.:
Structural Basis of Transcription Activation: The Structure of CAP-Alphactd-DNA Complex
Science 297 pp. 1562 (2002)

PDB ID: 1KU7
Campbell, E. A., Muzzin, O., Chlenov, M., Sun, J. L., Olson, C. A., Weinman, O., Trester-Zedlitz, M. L., Darst, S. A.: Structure of the Bacterial RNA Polymerase Promoter Specificity Sigma Subunit Mol. Cell 9 pp. 527 (2002)

PDB ID: 1L9Z

Murakami, K. S., Masuda, S., Campbell, E. A., Muzzin, O., Darst, S. A.: Structural Basis of Transcription Initiation: An RNA Polymerase Holoenzyme-DNA Complex Science 296 pp. 1285 (2002)