The Headful Packaging Nuclease of Bacteriophage T4
Bacteriophage T4 is a double stranded DNA virus that infects the bacterium, Escherichia coli. The virus spends the last 10 minutes of its life packaging 171 kb double stranded genome into a pre-assembled capsid. A powerful molecular motor is required for the virus DNA packaging process. The T4 packaging motor is composed of a portal complex and a terminase complex. The terminase complex is comprised of the small terminase gp16 and the large terminase gp17. The large terminase gp17 plays two roles during translocation: 1) ATP hydrolysis that provides energy 2) DNA cleavage to make genome length pieces that fit into the capsid. Most bacteriophages replicate their genome as a concatemer, a branched head to tail polymer of the viral genome. During packaging the concatemeric DNA is cut twice, once to initiate and again to terminate the packaging after the 171 kb genome is packaged into the capsid. The termination cut is dependent on the headful length of genome packaging. The mechanism and regulation of headful nuclease are poorly understood. Previously the catalytic center residues of the gp17 nuclease have been determined and the crystal structure of the nuclease domain has been resolved. The structure showed that the catalytic groove is surrounded by basic residues which presumably hold the DNA during catalysis. Mutational approach showed that these residues are dedicated for DNA binding during cleavage. Biochemical studies demonstrate that: 1) gp17 is a weak endonuclease; 2) sequence non specific gp17 nuclease prefers longer DNA substrates and makes blunt ends; 3) the activity is highly sensitive to NaCl and Mg2+ concentrations, nucleotide-state of the N-domain and the presence of the gp16. These results collectively suggest a novel regulatory mechanism of the phage headful nuclease. In physiological environment (e.g. in presence of high salt, ATP and gp16) the nuclease efficiently makes the first cut and remains attached to the ends of the DNA. This prevents further cleavage and facilitates the assembly of the packaging complex. After completion of headful packaging motor detaches from the portal and makes the termination cut. A series of such events leads to packaging of one viral genome into one viral capsid.
Stats
Viewed 265 timesDownloaded 36 times