A single residue substitution causes a switch from the dual DNA binding specificity of plant transcription factor MYB.Ph3 to the animal c-MYB specificity

Abstract

Transcription factor MYB.Ph3 from Petunia binds to two types of sequences, MBSI and MBSII, whereas murine c-MYB only binds to MBSI, and Am305 from Antirrhinum only binds to MBSII. DNA binding studies with hybrids of these proteins pointed to the N-terminal repeat (R2) as the most involved in determining binding to MBSI and/or MBSII, although some influence of the C- terminal repeat (R3) was also evident. Furthermore, a single residue substitution (Leu71 → Glu) in MYB.Ph3 changed its specificity to that of c-MYB, and c-MYB with the reciprocal substitution (Glu132 → Leu) essentially gained the MYB.Ph3 specificity. Molecular modeling and DNA binding studies with site-specific MYB.Ph3 mutants strongly supported the notion that the drastic changes in DNA binding specificity caused by the Leu → Glu substitution reflect the fact that certain residues influence this property both directly, through base contacts, and indirectly, through interactions with other base-contacting residues, and that a single residue may establish alternative base contacts in different targets. Additionally, differential effects of mutations at non-base-contacting residues in MYB.Ph3 and c-MYB were observed, reflecting the importance of protein context on DNA binding properties of MYB proteins.