Characterization of the structural and functional defect in the Escherichia coli single-stranded DNA binding protein encoded by the ssb-1 mutant gene. Expression of the ssb-1 gene under lambda pL regulation.
The ssb-1 gene encoding a mutant single-stranded DNA binding protein (SSB-1) has been cloned into a vector placing its expression under lambda pL regulation. This construction results in more than 100-fold increased expression of the mutant protein following temperature induction. Tryptic peptide analysis of the mutant protein by high-pressure liquid chromatography and solid-phase protein sequencing has shown that the ssb-1 mutation results in these substitution of tyrosine for histidine at residue 55 of SSB. This change could only occur in one step by a C----T transition in the DNA sequence which has been confirmed. Physicochemical studies of the homogeneous mutant protein have shown that in contrast to that of the wild-type SSB, the tetrameric structure of SSB-1 is unstable and gradually dissociates to monomer as the protein concentration is decreased from about 10 microM to less than 0.5 microM. The SSB-1 tetramer appears to be stable to elevated temperature (45 degrees C) but the monomer is not. We estimate the normal cellular concentration of SSB-1 (single chromosomal gene) to be 0.5-1 microM. Thus, there is a plausible physical explanation for our previous finding that increased expression of ssb-1 reverses the effects of a single gene (chromosomal) copy amount of SSB-1 (Chase, J.W., Murphy, J.B., Whittier, R.F., Lorensen, E., and Sninsky, J.J. (1983) J. Mol. Biol. 164, 193-211). However, even though the in vivo effects of ssb-1 and most of the in vitro defects of SSB-1 protein are reversed simply by increasing SSB-1 protein concentration, the mutant protein is not as effective a helix-destabilizing protein as wild-type SSB as measured by its ability to lower the thermal melting transition of poly[d-(A-T)].
D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D005453:Fluorescence; D005786:Gene Expression Regulation; D006358:Hot Temperature; D007526:Isoelectric Point; D008970:Molecular Weight; D009154:Mutation; D011067:Poly dA-dT / Q000032:analysis
D004268:DNA-Binding Proteins; D011067:Poly dA-dT