Message (Crammed) in a Bottle

by Kristopher Hite in Tom Paine's Ghost

To sell a bottle of Champagne as "bubbly" a vendor must ensure that the wine has absorbed between five and six atmospheres of carbon dioxide. To anyone who has ever popped the cork on champagne you know there is enough force there to do some serious damage to an innocent bystander in the event of poor aim.



Viruses spend part of their existence as a kind of bottle (capsid) with a message inside (DNA or RNA). The message inside carries the code or blueprint to make more viruses, more messages, more bottles. This message isn't short. If it were written out as As Ts Gs an Cs on regular 8 X 11" printer paper it would take up between 100-200 pages, depending on the specific virus we're talking about. Imagine trying to jam 100 leaves of paper into a wine bottle! The analogy isn't prefect as the viral message looks more like a rope than sheets of paper. Regardless, the message has to be crammed into an extremely tight space. The bottle's volume in this case being 0.065 cubic millimicrons. When the packing is done the DNA or RNA has been condensed from a free floating strand 6,ooo times like a tightly coiled garden hose at immense density. The pressure inside that tiny bottle reaches nearly 60 atmospheres. 10 times the pressure in the champagne bottle!





This raises many questions. How does a virus manage to pull or suck that long piece of rope inside its shell? Especially at the end when the pressure is at its peak? <--- The illustrated capsid cutaway shows the immense density with which the rope is crammed. How does such a tiny bottle not break under such intense pressure? Where's the cork and how does it stay put? And who or what pops the cork?



Dr. Carlos Bustamante of the University of California, Berkeley is answering these questions in incredible detail using single-molecule biophysical techniques. His lab is able to "grab the cat by the tail" and measure individual molecules directly. They specialize in DNA translocases. These are molecular machines or machine-like entities as he calls them.  Tiny "motors" that convert chemical energy into physical energy. When your eyes move to read these words the muscles are using the same chemical reaction's to pull your eyeballs from side to side as a virsus uses to pull that rope inside the bottle. This reaction is the hydrolysis of Adenosine triphosphate, the famed ATP, the energy "currency" of the cell. 


From Morais, M.C., Koti, J.S., Bowman, V.D., Reyes-Aldrete, E., Anderson, D.L., and Rossmann, M.G. 2008. Structure 16:1267–1274. © 2008, from Elsevier.


The viral machine is displayed in panel B. The capsid is pictured in gray while the motor sits at the "gate" and pulls the DNA inside. 



The motor is made of a five piece ring of small RNA molecules (pink) and five ATPases
(lavender).
In this particular single molecule study they are using two polystyrene
beads to tether two ends of a long piece of DNA (the set up is pictured in panel A below). The strand is held
in what is called an optical trap - those red sideways vase looking things. I blogged about how optical tweezers work before here's the link if you want to read about how this works. The goal in this study was to capture the capsid
on one bead (coated with capsid protein antibodies) and the other end
of the DNA  strand is bound to the other bead by a biotin-strepdavidin bridge. Not only did the researchers accomplish this feat but they were able to tug on either side of the system to measure how much force the motor was exerting on the DNA strand as it was sucking it into the capsid.


From Moffitt, J.R., Chemla, Y.R., Aathavan, K., Grimes, S., Jardine, P.J., Anderson, D., and Bustamante, C. 2009. Nature 457:446–1450. © 2009 Nature Publishing Group, a division of Macmillan Publishers Limited.




They discovered that with zero tension the capsid is packed with DNA at
the rate of 100-120 base pairs per
second. These studies also build a model for how they think the motor is
working.  As said before, the motor requires a chemical reaction (hydrolysis of ATP) to work.  In this controlled environment the researches can set the concentration of ATP. When and only when all five site on the motor are loaded with their ATP molecules can the motor pull the strand.  Like a revolver laying with the cylinder open and someone pouring bullets on top of it, the "gun" can only fire after it is fully loaded by the random addition of bullets. When the concentration of ATP is low the rate of firing is less frequent, higher  - more frequent.



The simplicity of this model as understood by observing single molecules, for me, made years of Michaelis-Menten kinetics (the dry math that drives people away from studying biochemistry) come to life in a visual context.  I could see in my mind's eye exactly how concentration affects the rate of a reaction.
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