Speaker: Santiago Ramón-Maiques
IBV-CSIC

Host

Ignasi Fita, IRB Barcelona & IBMB - CSIC

Friday, 26 September 2008, 12.00h Aula Fèlix Serratosa

Abstract

The bacteriophage Mu is a gigantic mobile genetic element or transposon that uses DNA transposition as the mechanism to integrate and amplify its genetic material. Mu has a genome of 38 kb, which encodes two proteins, MuA and MuB, that are required for the transposition. MuA is the transposase, binds the ends of the viral DNA and catalyzes the cut-and-paste reactions during the DNA-transposition reaction. MuB is an ATP-dependent binding protein that regulates the association and dissociation of MuA and the DNA, stimulates MuA activity and it is critical for the selection of target sites on the host chromosome. In its ATP-bound state MuB forms polymers on the DNA, and upon ATP hydrolysis it dissociates from DNA. MuB ATPase activity is very slow and it is strongly enhanced by the interaction with MuA. The structural characterization of MuB has been hampered by its extremely low solubility, particularly in the ATP-bound state, and it has defied all attempts of crystallization. Using electron microscopy we focused on the MuB precipitates and found that MuB forms filamentous polymers in the presence of ATP. Negatively stained samples show that MuB forms filaments of 150Å in diameter with great tendency to aggregate forming filament bundles. When DNA is present, MuB wraps around the DNA forming a continuous nucleoprotein filament. We recorded cryo-EM images and reconstructed the 3D structures of MuB with and without DNA using the single particle approach in combination with Fourier-Bessel methods. The filament is a 1-start helix with a helical pitch of 46Å and a number of proteins per turn that varies between 5.6 and 6.2. These results clarify the behavior of MuB and lead us to propose a model for the complex transposition mechanism of phage Mu.

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