Mechanism of Bacteriophage Lambda Genome Ejection

Recent in vitro experiments with lambda have shown that ejection of its genome is driven by high pressure in the viral capsid, and that only half the genome is ejected before the capsid pressure equals the host osmotic pressure.  These facts raise the question: what physical mechanism drives the delivery in vivo of the remainder of the genome? An in vitro experiment was designed to test if transcription of the injected half of the genome leads to extraction of the rest. Using polyethylene glycol (PEG) to mimic cytoplasm pressure, transcription yields of short DNA templates were found to be weakly dependent on osmotic pressure. Prior to conducting the in vitro ejection experiment, we purified lambda phage, E. coli membrane receptor LamB, and lambda-encoded antiterminator protein N. N is the only lambda gene that is transcribed by E. coli RNA polymerase in the absence of viral gene products, and whose transcription is towards the phage capsid. Lambda phage, LamB and PEG will be flowed into the space between two cover slips under a fluorescence microscope to trigger partial ejection of lambda genome. The length of genome ejected will be measured before and after transcription with and without protein N. This project will help elucidate the genome ejection mechanism of lambda as well as bacteriophage in general.