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

 

Rifampicin is a naturally made, non-peptide antibiotic.  It is bactericidal, killing by disabling the protein expression system universally conserved by all bacteria.  Specifically, rif inhibits the RNA polymerase protein, which is responsible for binding to a strand of DNA as a template and using it to construct a strand of mRNA

 

Rifampicin inhibits RNA polymerase by bonding tightly in the RNA exit channel.  Therefore, after transcription begins, the RNA transcript, trying to exit the RNAP through the exit channel, runs into the rifampicin sitting in the middle of the channel.  This effectively halts transcription when the RNA transcript is merely two or three nucleotides in length.

 

Despite this highly efficient method for killing bacteria, rifampicin is by no means a perfect antibiotic.  The biggest problem arises from the fact that bacteria can easily acquire strong resistance or even immunity to rifampicin through a variety of mutations, most of them a single amino acid substitution.  The problem is an interesting paradox, since the reason rifampicin works so well is that it is a rigid molecule, and sits tightly in the pocket where it binds, allowing the bonds to be very strong. However, this also means that if an amino acid on the edge of the channel with a small sidechain is replaced with an amino acid with a large sidechain, rifampicin may not be able to bind, simply because it cannot fit in the space.  This occurs in three primary positions, one of which occurs 9% of the time, another of which occurs 36% of the time, and the last of which occurs an alarming 41% of the time.

 

Because bacteria gains immunity to rif in a relatively short amount of time, it used only in very special circumstances in an effort to keep bacteria vulnerable to rif, hopefully making it effective when it does need to be used.  The most common usage of rif is as part of a tuberculosis therapy program.  Tuberculosis (TB) is an extremely contagious, extremely lethal bacterial disease, which often lurks in the lungs until the host has a weakened immune system, at which time it manifests itself, resulting in symptoms which are debilitating at best.  In fact, TB remains the leading cause of preventable death by a pathogen in the world.  However, unless the bacteria is immune already, rifampicin is very effective, cutting the therapy time from two years down to six months.  The problem is that with cases in the US declining to the point of nonexistence, pharmaceutical companies no longer see it as a source of profit, and all but three have ceased making it, even though TB is still common among people in developing countries, who are unable to pay for the treatment.  In fact, the three that are still making it are only doing so because they are being forced to do so by the World Health Organization.

Reset View

Highlight rifampicin in RNA exit channel (viewed through channel).  β (beta) flap covering channel colored dark blue.

This script shows RNAP from the perspective of looking strait into the RNA exit channel.  Rifampicin is flashing in red, and as you can see, it completely blocks the exit channel.  The blue region is the β flap, which partially covers the RNA exit channel.

Toggle backbone and spacefill view for RNAP

 

 

View interaction of rifampicin with RNAP.  Part of β (beta) subunit removed to highlight interactions.

This script is designed to show exactly how rifampicin bonds to RNAP and which amino acids are directly involved in the process. Those amino acids which bond with rifampicin are in CPK coloring, with their sidechains displayed. Rifampicin is in red. It should be noted that part of the β subunit of RNAP has been melted away to provide a clearer view of this interaction.

Zoom back to view RNA transcript exit channel

Inhibition of transcript elongation due to interference by Rifampicin

This button adds DNA and RNA in order to show exactly how rifampicin blocks the RNA transcript from exiting through the RNA exit channel. The DNA is in green, with the template strand in dark green. The red strand is the RNA transcript. As you can see, 2-3 nucleotides after transcription begins, the RNA runs into rifampicin, and is halted. Note that the β subunit of RNAP has been removed to provide a clearer view.

 

JMol scripts and text by Trevor Topf

PDB file: 1I6V (modeled and modified by Seth Darst)

Campbell, E.A., Korzheva, N., Mustaev, A., Murakami, K., Nair, S., Goldfarb, A., Darst, S.A. Structural mechanism for rifampicin inhibition of bacterial RNA polymerase. Cell v104 pp.901-912 , 2001