Seventeen base pairs of region I encode a novel tripartite binding signal for SV40 T antigen.

Three sequence components direct high affinity binding of dimeric SV40 T antigen to SV40 origin region I. Two signals are encoded by two directly repeated 5'-GAGGC-3' pentanucleotides. Approximately equal contributions to binding stability are made by each pentanucleotide, and both spacing and orientation of the pentanucleotides are important for binding affinity. The third vital component is contained in a 5'-TTTTTTG-3' spacer sequence that separates the pentanucleotides. Sequence-specific features of the spacer stabilize binding to the adjacent pentanucleotides. The asymmetry of the spacer suggests that a novel binding mechanism is involved. Because the alignment of T antigen on mutant and wild-type DNAs is similar, we propose that any two of the three sequence signals are sufficient to determine the unique arrangement of a bound protein dimer.