Characterization of a region in phage Mu transposase that is involved in interaction with the Mu B protein.
Mu A protein, the 75-kDa phage transposase, consists of three domains: a 30-kDa NH2 terminus, a 35-kDa central domain, and a 10-kDa COOH terminus (Nakayama, C., Teplow, D. B., and Harshey, R. M. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1809-1813). Genetic and biochemical experiments have demonstrated that the COOH-terminal domain must be present for functional interaction with Mu B protein. To further investigate the COOH-terminal domain of Mu A, we fused this 89-amino acid region to the glutathione S-transferase gene to facilitate subsequent expression and purification. We show that either the glutathione S-transferase-peptide fusion protein or the COOH-terminal peptide severed from glutathione S-transferase is active in Mu B interaction. Addition of the COOH-terminal domain to the in vitro strand transfer reaction inhibits intermolecular strand transfer by a mechanism previously characterized for intact Mu A protein (Baker, T. A., Mizuuchi, M., and Mizuuchi, K. (1991) Cell 65, 1003-1013), although the COOH-terminal domain is 70 times less effective than intact Mu A. The transient interaction between the COOH-terminal domain and Mu B does not inhibit Mu B stimulation of the strand cleavage and intramolecular strand transfer activity of Mu A. Deletion analysis has shown that the last 36 amino acids are sufficient for interaction with Mu B, but that removal of as few as 4 amino acids from the COOH terminus renders the peptide inactive. The recovery of an active COOH-terminal domain of Mu A will facilitate future structure/function studies of the Mu transposase.
Wu
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Department of Biochemistry, University of Western Ontario, London, Canada.
Chaconas
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D010583:Bacteriophage mu / Q000201:enzymology* / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000378:metabolism*; D019895:Transposases; D014764:Viral Proteins / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; C069215:MuB protein, Enterobacteria phage Mu; D014764:Viral Proteins; D009713:Nucleotidyltransferases; D019895:Transposases