The TCP domain is a DNA-binding domain present in plant transcription factors that modulate different processes. In the present study, we show that Arabidopsis class I TCP proteins are able to interact with a dyad-symmetric sequence composed of two GTGGG half-sites. TCP20 establishes symmetric interactions with the 5' half of each strand, whereas TCP11 interacts mainly with the 3' half. SELEX (systematic evolution of ligands by exponential enrichment) experiments with TCP15 and TCP20 indicated that these proteins have similar, although not identical, DNA-binding preferences and are able to interact with non-palindromic binding sites of the type GTGGGNCCNN. TCP11 shows a different DNA-binding specificity, with a preference for the sequence GTGGGCCNNN. The distinct DNA-binding properties of TCP11 are due to the presence of a threonine residue at position 15 of the TCP domain, a position that is occupied by an arginine residue in most TCP proteins. TCP11 also forms heterodimers with TCP15 that have increased DNA-binding efficiency. The expression in plants of a repressor form of TCP11 demonstrated that this protein is a developmental regulator that influences the growth of leaves, stems and petioles, and pollen development. The results suggest that changes in DNA-binding preferences may be one of the mechanisms through which class I TCP proteins achieve functional specificity.

TCP proteins are plant transcription factors that contain the TCP domain, a conserved domain involved in DNA binding and dimerization (1). The N-terminal portion of the TCP domain is enriched in basic amino acids and is followed by a region that is predicted to contain two amphipathic ␣-helices connected by a disordered loop (2). These features give the TCP domain a resemblance with the bHLH 5 domain present in eukaryotic transcription factors. The basic region, however, differentiates these two structures because this region is longer and contains helix-breaking amino acids in the TCP domain. This makes theoretical predictions about the nature of its contacts with DNA rather inaccurate when bHLH domain-DNA complex structures are used as templates.A broad separation of TCP domains can be made based on amino acid similarities. This produces two main classes of TCP domains that also differ in the number of residues of the basic region because class II proteins contain a 4-amino acid insertion in this region (1, 2). The function of most TCP proteins studied to date is associated with the regulation of different developmental processes in plants (3-11). However, other functions have also been proposed, such as the coordination of mitochondrial biogenesis (12-14), regulation of the circadian clock (15), control of jasmonic acid biosynthesis (16), and determination of the embryonic growth potential in seeds (17). The fact that there are Ͼ20 different TCP proteins in most angiosperm species raises the question of whether there is a high degree of redundancy or different proteins perform different functions and, if the latter case is correct, the additional question is what is the basis for specificity. Studies using mutants and plants overexpressing native or modified forms of TCP proteins have suggested that partial redundancy overlaps with specific functions of different TCP proteins (8,9,16,18).One of the sources of functional specificity may be the existence of different DNA binding preferences among TCP proteins. Previous studies have provided consensus DNA sequences preferentially bound by different class I and class II proteins that, with the sole exception of Arabidopsis TCP11, can be described as GTGGGNCC for class I and GTGGNCCC for class II (11,16,19). Because these kinds of study have only been performed with a limited number of proteins, it is not known whether these consensus sequences apply to all members of each class or not.Studies on the molecular basis of DNA binding specificity of TCP proteins will help to understand how different TCP proteins perform their function and eventually construct a code linking the presence of certain residues to the DNA binding preferences of the respective proteins. In this work, we have studied the DNA binding properties of the class I TCP protein TCP16 from Arabidopsis and determined that it has a preference for a class II binding site. We show that the identity of residue 11 of the class I TCP domain and the equivalent residue 15 of the class II domain is an importan...