An IncY plasmid-encoded single-stranded DNA-binding protein from Escherichia coli shows the identical pattern of stacked tryptophan residues as the chromosomal ssb gene product.
In an extension of earlier studies on the Escherichia coli plasmid-encoded single-stranded DNA-binding proteins pIP71a SSB, F SSB and R64 SSB [Khamis, M. I., Casas-Finet, J. R., Maki, A. H., Ruvolo, P. P. & Chase, J. W. (1987) Biochemistry 26, 3347-3354; Casas-Finet, J. R., Khamis, M. I., Maki, A. H., Ruvolo, P. P. & Chase, J. W. (1987) J. Biol. Chem. 262, 8574-8593], we have investigated the binding of pIP231a SSB to natural and heavy-atom-derivatized single-stranded homopolynucleotides. Fluorimetric equilibrium binding isotherms indicate that pIP231a SSB has a greater solubility at low ionic strength than any other plasmid SSB protein investigated. Furthermore, its complex with mercurated poly(uridylic acid) [poly(Hg5U)] shows a greater resistance to disruption by salt than the other plasmid SSB complexes. Essentially complete binding of pIP231a SSB to poly(Hg5U) could be achieved, and time-resolved optically detected triplet-state magnetic resonance (ODMR) techniques could be applied to the complex. These methods allowed complete resolution of the three Trp chromophores of pIP231a SSB. Comparison of wavelength-selected ODMR results with those obtained for the poly(Hg5U) complex of a point-mutated chromosomal ssb gene product (Eco SSB) carrying substitutions of Phe for Trp [Khamis, M. I., Casas-Finet, J. R., Maki, A. H., Murphy, J. B. & Chase, J. W. (1987) J. Biol. Chem. 262, 10938-10945] confirm that Trp40 and Trp54 of pIP231a SSB are stacked in the complex, while Trp88 is not. This is the same distribution of stacked Trp residues found in Eco SSB. These results are confirmed further by specific effects observed on the ODMR signals of pIP231a SSB upon binding to poly(Br5U) and poly(dT), which are known to be caused by the stacking of Trp54 with nucleic acid bases.
Casas-Finet
J R
JR
Department of Chemistry, University of California, Davis 95616.
Jhon
N I
NI
Khamis
M I
MI
Maki
A H
AH
Ruvolo
P P
PP
Chase
J W
JW
D001426:Bacterial Proteins / Q000235:genetics*; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000032:analysis / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics*; D004926:Escherichia coli / Q000032:analysis / Q000235:genetics*; D005798:Genes, Bacterial; D009682:Magnetic Resonance Spectroscopy; D010957:Plasmids; D011119:Polynucleotides / Q000494:pharmacology; D012995:Solubility; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis / Q000235:genetics*
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011119:Polynucleotides; D014364:Tryptophan