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Our Groups' Previous Work in Studying the Pressure of DNA Viruses - Packing DNA in Bacteriophage

Bacteriophage lambda, shown in the electron micrograph, consists of a protein capsid 30 nm in radius that has a long cylindrical tail. Its genome, double stranded DNA (dsDNA), is protected by the capsid from attack by nuclease enzymes that would break it down into its nucleotides and therefore lose the genetic information needed to replicate the phage. The DNA contains 48.6 kilo-base pairs; if it were fully extended it would be 17 micrometers long. When the phage is replicated in the host cell, an early form of the capsid, the procapsid, is formed and the DNA is driven into it by a molecular motor at one of the procapsid vertices. This is quite feat! Imagine packing a length of string into an object that is only 1/400th its size. To make the job harder, add negative charges to the string and make it stiff. The stiffness of ds DNA is very high; a measure of this stiffness is its persistence length. It is difficult to bend objects on a scale smaller than the persistence length. The persistence length of dsDNA is 50 nm, and to bend it so that it can fit into the capsid is therefore highly costly in energy.

Why does nature go to all this trouble? The answer lies in the way that phage infect bacteria. The tip of the phage tail recognizes a receptor on the surface of the bacterium E. coli and binds to it. (The receptor, a membrane protein called LamB, is there because it has the function of transporting maltose into the bacterium.) The binding to LamB causes a conformational change in the tail that opens it, allowing the DNA to come out. Because it has been stored in the capsid in such a highly condensed state, the DNA exits the tail with a high force and is injected into the bacterium, leaving the capsid behind. The injection process is passive; all that is happening is the liberation of the energy that was put into the DNA when it was packaged by the motor (which, of course, required an expenditure of energy).We have been carrying out theoretical and experimental research to understand the energetics of DNA packaging and its biological implications. Theory [Kindt et al., 2001, and Tzlil et al., 2003] predicts how the packaging force depends on the fraction of the genome packaged (or conversely how the ejection force depends on the fraction of genome ejected) and the charge on the DNA. We are examining these questions in in vitro experiments. It has been known that with the addition of detergent LamB can be dissolved in buffer solution. Moreover, the receptor remains completely functional so that when λ phage is added to the solution it will bind to the receptor and open, thereby ejecting its DNA. In our experiments, we counterbalance the ejection force by an external osmotic force provided by adding the polymer poly(ethylene glycol) (PEG) to the solution. Although the capsid wall has pores that allow small molecules to enter, the polymer is so big that it remains outside and creates a large osmotic pressure difference.

The experiments consist of measuring the extent of ejection as a function of the counterbalancing osmotic force, which can be controlled by changing the PEG concentration. We use a biochemical trick to measure the fraction of DNA ejected. The enzyme DNase, which like PEG is too large to enter the capsid, is added to the solution. It cuts the DNA up into its nucleotides. The capsids and any DNA that they may remain in them are driven to the bottom of the sample tube by centrifugation and the concentration of nucleotides left in the supernatant solution is determined from the optical absorption at 260 nm. The results of our first measurements [Evilevitch, et al. 2003] are shown on the right, where the extent (percentage) of genome ejected is plotted vs external osmotic pressure. These data are consistent with the fact that the ejection force falls sharply in the early stages of ejection (or conversely, the force required to package DNA increases steeply only towards the end of the packaging process [see Kindt et al., 2001, and Tzlil et al., 2003]). An analysis of the experiments allows the pressure exerted by the DNA on the inside of the capsid to be estimated [Evilevitch et al., 2004]. It is remarkably high - of the order of 50 atm! Phage capsids are very strong despite the fact that they are held together by relatively weak forces, not covalent bonds. In continuing work, we are studying the effect of electrostatic interactions on the ejection force by adding positive ions to the solution. These enter the capsid and interact with the DNA and reduce its charge. In collaboration with the group of Professor [story:phillips Rob Phillips] at Caltech, we have also been studying the effects of genome length on the packaging force. Work has also begun on the kinetics of ejection in which fluorescence microscopy will be employed to visualize the DNA as it passes through a membrane containing LamB or enters a live bacterium.