JMol Tutorial created with support from Seth Darst (Darst Lab, Rockefeller University) and Tim Herman (Center for Biomolecular Modeling).

Please email tshata@gmail.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 (RNAP) is the enzymatic machinery responsible for transcription, a key regulatory step in gene expression.  The prokaryotic RNAP is a highly conserved, "crab claw" shaped enzyme with a molecular mass of ~400kD.  In order to recognize a promoter to begin transcription, the 5-subunit core enzyme (α,α,β,β’,ω) must bind to one of various sigma (σ) factors; this form of the enzyme is called the holoenzyme.  Each of the different σ factors recognize different promoter elements upstream of genes allowing the cell to respond to various environmental cues.
Once holoenzyme binds the promoter, DNA downstream of this interaction is brought into the enzyme and melted to expose single-stranded DNA.  This stage of transcription initiation is described here using a model of RNA polymerase-open-promoter-complex (RPo).

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Structural characteristics and functions of RNA Polymerase (RNAP)

Clamp

Region on the β and β’ subunits.  These residues are mobile domains that are part of the "crab claw" structure that swing open and closed, effectively changing the size of the active site channel. 

β (beta) flap

The β-flap covers the RNA exit channel through which the newly formed RNA strand is released from the enzyme. This flap binds to domain four of the σ subunit (domain four binds to the -35 promoter element).  The main part of the β flap (the part that binds σ) is called the flap-tip-helix.

β' (beta prime) zipper

The β’ zipper removes the RNA transcript from the RNA DNA hybrid.  The β’ zipper interacts with domain three of the σ subunit (domain three binds to the -10 promoter element).

β' (beta prime) lid

The β’ lid, which covers the RNA exit channel, also contributes to the removal of the RNA transcript from the RNA DNA hybrid.

β' (beta prime) coiled-coil

The β’ coiled-coil binds to domain two of the σ subunit at the most important sigma binding site.  The rudder is located within this coiled-coil. 

β' (beta prime) bridge

The β’ bridge helix functions as a separator of the main active site channel from the secondary channel.  It is very highly conserved and, through inference, it is known to contribute in some way to the processes of catalysis and translocation.

Highlight all characteristics

JMol script and text by Caroline Pinke

α-CTD (alpha subunit carboxyl terminal domain) interaction with DNA and σ (sigma) factor

The interaction of CTD with sigma and the DNA is highlighted here. The σ (sigma) factor, when bound to RNAP, aids in promoter recognition. Alpha CTD, the carboxyl terminal domain of α (alpha) subunit, is connected to the rest of the α subunit by a flexible linker (modeled with spheres in the model). α-CTD also facilitates RNAP binding DNA by recognizing the UP-element, located upstream of the promoter. This additional interaction with the UP-element further stabilizes RNAP’s interaction with the DNA. Alpha CTD also interacts with σ factor, which binds the -10 and -35 promoter regions on the DNA strand.

Highlighted in red is the interaction of alpha CTD with the UP-element. In white and pink are CTD’s interactions with sigma and sigma with CTD, respectively.

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JMol script and text by Jessica Westerman

RNAP-Promoter interactions: promoter recognition and binding by σ (sigma) factor

Core enzyme requires the addition of the σ factor (forming holoenzyme) to be able to recognize the promoter elements. The six base pairs on the -10 region, two on the extended -10 region, and six base pairs on the -35 region of is recognized by domains 2, 3, and 4 of σ factor, respectively.

Promoter sequence can vary in "strength" depending on its deviation from its consensus sequence.  Similarly, each σ factor has an affinity for different promoter sequences.  These interactions contribute to gene regulation in prokaryotes by putting various genes under control of separate σ factors.  RNAP-promoter interactions are, therefore, key to the control of gene expression.

Interaction with the -10 and extended -10 (x-10) promoter elements

The five σ residues important for binding to -10 element and the two residues important for the binding of the x-10 element are highlighted.

Interaction with the -35 promoter elements

The four σ residues important for binding to -35 element are shown.

Highlight of all residues interacting with promoter elements

Promoter elements on DNA are highlighted in blue.

JMol script and text by Angela Ramirez

Closer look at β' (beta prime) bridging helix and secondary channel

Between the β and β’ regions of RNAP there is a single alpha helix called the bridging helix.  The β and β’ regions make up a “crab claw” shape that is characteristic of this RNAP.  The bridging helix connects these two regions, creating two channels. Down-stream DNA enters the larger of these channels where it is split to create the transcription bubble spanning 12-14 nucleotides.  The smaller of the two is the secondary channel, where RNA nucleotides enter the complex towards the single stranded template stand near the RNAP active site.  With its 12 diameter, the secondary channel is only large enough to allow these nucleotides to pass through (the larger diameter of a double-stranded DNA (20 ) is much too large to pass through the secondary channel). 
The bridging helix also seems to be supporting the β and β’ regions apart from each other and creating the space in which the RNA nucleotides can reach the active site Mg2+, which is located directly opposite from the helix itself.  Notice, when looking directly into the secondary channel, that both Mg2+ ion and single stranded template DNA is visible.

 

View into secondary channel

JMol scirpt and text by Taylor Sankovich

Abortive Initiation: a closer look at σ (sigma) factor domain 3.2 (σ3.2) and the beta (β) flap region

Highlighting the σ3.2 region and β flap

Sigma factor 3.2 loop (σ3.2) is located between sigma domains 3 and 4 (σ3 and σ4) and extends into the active-site channel beneath the beta (β) flap of RNAP. During abortive initiation, RNA transcripts only a few nucleotides in length are displaced by the σ3.2 loop and are released from the RNAP complex. After abortive RNA transcripts elongate and are released several times, a transcript grows to approximately 12 nucleotides in length. At this size, the transcript is long enough to dislocating the σ3.2 loop, completing abortive initiation and successfully commencing the synthesis of the complete RNA transcript. Because of the significance of the σ3.2 loop’s location near the RNAP active site, it is also thought that it plays a role in the stabilization of the initiation RNA nucleotide onto the DNA template strand.

The β flap (colored dark blue), which sits above the σ3.2 loop, is a part of core RNAP. In the holoenzyme, the β flap-tip-helix (a section of the β flap colored light blue) is the main part of core enzyme that interacts with σ4.

 JMol script and text by Olivia Delia

The active site Mg2+ ion; conserved residues around RNAP active site.

A magnesium ion is found at the center of the active site of bacterial RNA polymerase, where RNA nucleotides are attached to the growing transcript. A conserved set of seven amino acids found on the β’ (beta prime) subunit surrounds and holds the magnesium ion in place.  Ribonucleotides reach the active site by entering through the secondary channel. As the nucleotides bond, the newly forming strand of RNA exits the enzyme through the RNA-exit channel. Double stranded downstream DNA enters the enzyme through the active site channel and splits, bringing the template strand in close proximity to the active side as the non-template strand travels along the outside of the enzyme. The template strand exits the enzyme through the template-strand channel and reforms a double helix with the non-template strand.

 

Zoom back to active site

JMol script and text by Danika Paulo

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PDB file:

Modeled and provided by Seth Darst

References:

Darst, Seth A. Bacterial RNA polymerase.  Current Opinion in Structural Biology 11:155-162, 2001