Abstract
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)].
Highlights
From the $Department of Molecular Bwbgy, Albert Einstein Collegeof Medicine, Bronx, New York 10461and the $Department of Molecular Biophysicsand Bwchemistry, Yale University, New Haven, Connecticut 06510
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 M M to less than 0.5 MM.The SSB-1 tetramer appears to be stable to elevated temperature (45 "C) but the monomer is not
The abbreviations used are: ssDNA, single-stranded DNA; SSB, the E. coli single-stranded DNA binding protein encoded by the ssb gene, which has been referred to as a DNA helix-destabilizing protein (EcoHD-protein I); FSSB, ssDNA binding protein encoded by E. coli sex factor F with extensive homology to SSB; SDS, sodium dodecyl sulfate; HPLC, high-pressure liquid chromatography
Summary
Bacterial Strains and Plasmids-The E. coli K12strains used were KLC1038 and KLC1039. These are, respectively, ssb-1 derivatives of N99 XcZ+ (N99=IncZ; galK-, thi-, suo)and M5248=N99(Xbio275, N+, cZ857, DH1). Minimum estimates of the apparent association constant for poly(dT) were calculated from the per cent quenching observed at the stoichiometric point in the titration assuming an apparent binding site size of 10 nucleotides per SSB or SSB-1 monomer This lattervalue was determined by extrapolating the initial and final slopes of titrations done a t protein concentrations above 3 p~ and is slightly less than thevalue of about 11that we observedwith single-stranded fd DNA (Chase et al, 1984). Further DNA sequence analysis indicates that the thirdnucleotide in the codon for serine 39 is deoxycytidylate rather than deoxyadenylate as was previously reported This correction does not change the amino acid residue at this position it does introduce a BstNI recognition site
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