Plant Physiol, October 2000, Vol. 124, pp. 491-494

SCIENTIFIC CORRESPONDENCE

Several Features of the GT-Factor Trihelix Domain Resemble Those of the Myb DNA-Binding Domain

Graduate School of Agricultural Sciences, Nagoya University, Nagoya 464-8601, Japan

	ARTICLE

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It is generally believed that GT-factors, DNA-binding proteins with specificity for GT-elements, are present only in plants. However, the current study shows that several features of the GT-factor trihelix domain resemble those of the Myb DNA-binding domain. The GT-element was first identified as a regulatory component of a light up-regulated element (Box II or GT-1 binding site), which is present within the upstream region of pea rbcS-3A (for review, see Zhou, 1999). Subsequently, sequences similar to the GT-1 binding site were found in the regulatory DNA sequences of many plant genes. Many analyses have shown that GT elements have diverse and important functions.

Affinity screening using GT-1 binding sites has isolated the GT-binding factor, named GT-1a or B2F (Gilmartin et al., 1992; Perisic and Lam, 1992). These studies have postulated that three putative -helices (trihelix domain) in GT-1a might be involved in DNA binding. Subsequent deletion analyses of GT-1a have shown that the trihelix domain of GT-1a is certainly involved in DNA binding (Hiratsuka et al., 1994; Lam, 1995). GT-2 was the first GT-factor to be isolated (Dehesh et al., 1990) and contains two separate trihelix domains (Ni et al., 1996). Each trihelix domain is involved in DNA binding. Additional members of this family have been isolated from the cDNA library (Smalle et al., 1998) or predicted from the genome sequence of Arabidopsis (Zhou, 1999). To date, GT-factors have been found only in plants.

The Myb gene was originally identified as the oncogene (for review, see Introna et al., 1994). The c-Myb proto-oncogene has an important role in controlling the proliferation and differentiation of cells. The DNA-binding domain consists of three imperfect tandem repeats of 51 to 52 amino acids (R1, R2, and R3 from the N terminus). The solution structure of a specific DNA complex of the Myb DNA-binding domain has been determined (Ogata et al., 1994). Each R domain contains three helices. A hydrophobic core that includes three regularly spaced Trp residues maintains the trihelix structure (Ogata et al., 1992). The three regularly spaced Trp residues are characteristic of Myb proteins. More than 100 Myb genes have been found in Arabidopsis (Kranz et al., 1998). Myb proteins from animals have three Myb motifs (R1, R2, and R3), whereas most plant Myb proteins have two Myb motifs corresponding to R2 and R3 and forming the R2R3 Myb gene family.

The proposed trihelix structure of GT-factors somewhat resembles the solution structure of the Myb DNA-binding domain. I therefore performed the sequence similarity search using the UCSC SAM-T98 program (http://www.cse.ucsc.edu/research/compbio/HMM-apps/T98-query.html) with default parameters (Karplus et al., 1998). This method uses an iterative hidden-Markov model. The proposed trihelix domain (from 66-153) of tobacco GT-1a (Gilmartin et al., 1992) was used as a query. The SAM-T98 search detected many Myb proteins (e.g. mouse c-Myb; E-value = 7.9 × 10¹⁸). This result shows that amino acid sequences of GT-factors and Myb proteins are related. A multiple alignment of homologous proteins was constructed based on the SAM-T98 alignment, as shown in Figure 1. In addition to the GT-factors (shown in red), the alignment includes several intermediate proteins (shown in black) between GT-factors and Myb proteins. Furthermore, the alignment includes maize myb protein C1 (Paz-Ares et al., 1987) and mouse c-Myb protein (shown in blue). The alignment shows that many residues are well conserved between GT-factors and Myb proteins.

View larger version (97K): [in this window] [in a new window]   Figure 1.   Multiple alignment of GT-factors, Myb proteins, and other related proteins. Proteins predicted from the genome sequence are listed. In the first two columns, GT-factors and Myb proteins are shown in red and blue, respectively. Seventy percent consensus amino acid sequence is calculated using http://www.bork.embl-heidelberg.de/Alignment/consensus.html. Seventy percent identical amino acid residues are shown in yellow. Seventy percent consensus amino acid residues are colored: h, hydrophobic residues (A, C, F, G, H, I, K, L, M, R, T, V, W, and Y are shown in pink); s, small residues (A, C, D, G, N, P, S, T, and V are shown in blue); p, polar residues (C, D, E, H, K, N, Q, R, S, and T are shown in green); +, positively charged residues (H, K, and R are shown in dark gray); a, aromatic residues (F, H, W, and Y are shown in sky blue); l, aliphatic residues (I, L, and V are shown in red); and c, charged residues (D, E, H, K, and R are shown in light gray). -Helix regions of mouse c-Myb are underlined. Arrowheads indicate three regularly spaced Trp residues. The numbers of residues at gaps in the alignment are indicated. The left column indicates species abbreviations as follows: Nt, tobacco; At, Arabidopsis; Os, rice; Zm, maize; and Mm, Mus musculus. The next column indicates protein name or accession number. N, Amino-terminal trihelix domain; C, carboxyl-terminal trihelix domain.

The protein sequence-structure alignment, an approach also known as protein threading, provided convincing evidence that the GT-factor trihelix domain had a fold similar to that of the Myb DNA-binding domain. The GenTHREADER program (Jones, 1999; http://globin.bio.warwick.ac.uk/psiform.html) is able to more clearly discriminate between true and false positives than were the threading methods developed previously. A search using the same query predicted that the GT-factor trihelix domain contained a region that had a fold similar to those of the Myb DNA-binding domains (e.g. c-Myb R1 domain [probability of correct match, P = 0.943], c-Myb R2 domain [P = 0.930], and c-Myb R3 domain [P = 0.829]). Furthermore, running a trihelix domain of GT-1a through a fold-recognition server (http://www.cs.bgu.ac.il/~bioinbgu/) supported the predicted protein sequence-structure alignment (data not shown).

In Figure 1, -helix regions of the mouse c-Myb DNA-binding domain are underlined based upon the solution structure (Ogata et al., 1992, 1994, 1995). The residues of Myb -helix regions are well conserved between GT-factors and Myb proteins. Myb DNA-binding domains have three regularly spaced Trp residues in each R domain (Introna et al., 1994). The three-dimensional structure of the each R domain (Ogata et al., 1992, 1995) has shown that the Trp residues form the hydrophobic core. In addition, the structure of the DNA-Myb complex has shown that second Trp residues of R2 and R3 are involved in the contacts with the phosphate backbones (Ogata et al., 1994). The regularly spaced Trp residues are also found in the trihelix domain of GT-factors (Fig. 1, arrowheads). Thus the trihelix domain of GT-factors contains the main features of Myb proteins.

Although the important residues involved in DNA binding are well conserved between GT-factors and Myb proteins, the GT-factors have relatively longer intervening sequences between two helices. Probably, GT-factors arose in evolution from Myb proteins, and the insertions of gaps between two helices resulted in different recognition sequences between GT-factors and Myb proteins.

	FOOTNOTES

Received April 17, 2000; accepted June 6, 2000.

¹ This work was supported by grants from the Japanese Ministry of Education, Science, Sports and Culture.

^* E-mail nagano{at}agr.nagoya-u.ac.jp; fax 81-52-789-4296.

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