RESEARCH PAPER |
Structural relationships between diverse cis-acting elements are critical for the functional properties of a rbcS minimal light regulatory unit
1Departamento de Ingeniería Genética de Plantas, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional – Unidad Irapuato, Apartado Postal 629, Irapuato, Guanajuato 36500, Mexico
2Laboratorio Nacional de Genómica para la Biodiversidad, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional – Unidad Irapuato, Apartado Postal 629, Irapuato, Guanajuato 36500, Mexico
To whom correspondence should be addressed: E-mail: lherrera{at}ira.cinvestav.mx
Light-regulation of photosynthesis-associated nuclear genes is mediated by multipartite cis-regulatory units located in their promoter regions. The combination, spacing, and relative orientation of transcription factor binding sites in these units influences the assembly of specific multiprotein complexes that control gene expression. In this work, the functional architecture of the conserved modular array 5 (CMA5), a previously characterized minimal light-regulatory unit of rbcS gene promoters, has been analysed. With the aim of defining the sequences of CMA5 that, besides the I- and G-box elements, are essential for CMA5 responsiveness to light and chloroplast-derived signals, a series of mutations affecting the sequences flanking these elements was performed. It was found that an I-box associated module, named IbAM5, is essential for CMA5 functionality and is able to bind nuclear proteins in vitro. The spacing requirements of the I- and G-box elements in achieving adequate combinatorial interaction were also studied as well as the effect of interchanging the relative position of these elements in the native rbcS promoter arrangement. The results show that helical phasing and distance between the I- and G-box motifs are critical to determine the functionality and transcriptional strength of CMA5. Furthermore, it is shown that the relative position of the IbAM5/I-box composite element and the G-box element is not critical for the enhancer activity of CMA5, as long as the proper distance between them is maintained. Taken together, these results suggest that the light-responsive, plastid-dependent activity of CMA5 requires the synergistic interaction of several DNA-binding transcription factors assembled in a higher-order nucleoprotein complex.
Key words: Enhancer element, gene regulation, light regulation, nuclear–plastid signalling, photosynthesis genes, promoter architecture
* Present address: Department of Molecular and Structural Biochemistry, North Carolina State University, 360 Partners III, Centennial Campus, 851 Main Campus Drive, Raleigh, NC 27606, USA.
Present address: Colegio de Postgraduados, Campus Campeche, Calle Nicaragua 91 Tercer piso entre Tamaulipas y Circuito Baluartes Col. Santa Ana, Campeche, Campeche 24050, Mexico.
División de Biología Molecular, Instituto Potosino de Investigaciones Científicas y Tecnológicas, Camino a la Presa San José 2055, Lomas Cuarta Sección, San Luis Potosí, San Luis Potosí 78216, Mexico.
Received 26 September 2007; Accepted 5 November 2007