INTERRELATIONS OF SECONDARY STRUCTURE STABILITY AND DNA-BINDING AFFINITY IN THE BACTERIOPHAGE SPO1-ENCODED TYPE-II DNA-BINDING PROTEIN TF1

TF1, a, homodimeric DNA-binding and -bending protein with a preference for hydroxymethyluracil-containing DNA is the Bacillus subtilis-encoded homolog of the bacterial HU proteins and of the E. coli integration host factor. A temperature-sensitive mutation at amino acid 25 of TF1 (L25 → A) and two intragenic second site revertants at amino acids 15 (E15 → G) and 32 (L32 → I) were previously identified and their effects on virus development were examined. The DNA-binding properties of these proteins and the thermal stability of their secondary structures have now been analyzed. Amino acids 15 and 32 are far removed from the putative DNA-binding domains of TF1 but changes there exert striking effects on DNA affinity that correlate with effects on structure. The double mutant protein TF1-G15I32 binds to a preferred site in hydroxymethyluracil-containing DNA 40 times more tightly, denatures at higher temperature (Δt(m) = 21°C), and also exchanges subunits much more slowly than does the wild-type protein. The L25 → A mutation makes TF1 secondary structure and DNA-binding highly salt concentration-dependent. The E15 → G mutation partly suppresses this effect: secondary structure of TF1-A25G15 is restored at 21°C by 1 M NaCl or, at low NaCl concentration, by binding to DNA. © 1994 Academic Press Limited.