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

doi:10.1074/jbc.272.5.2889

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*

From the Centro Nacional de Biotecnología-CSIC, Campus Cantoblanco, Carretera de Colmenar Km 15.5, Madrid 28049, Spain

^§ To whom correspondence should be addressed. Tel.: 341-5854504; Fax: 341-5854506; E-mail: jpazares{at}samba.cnb.uam.es

↵^‡ Recipient of a predoctoral fellowship from the Comunidad Autónoma de Madrid. Present address: Dept. of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia, PA 19104-6018.

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.

Footnotes

↵* This research was supported by the European Community (contract-BIO2-CT93-0101). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

MBSI and MBSII

Myb.Ph3 binding sites; types I and II; respectively

PCR

polymerase chain reaction

PAGE

polyacrylamide gel electrophoresis

EMSA

electrophoretic mobility shift assay.
↵² I. Romero, A. Fuertes, M. J. Benito, A. Leyva, and J. Paz-Ares, manuscript in preparation.
- Received August 1, 1996.