The Dr1/DRAP1 complex.

Dr1 is a particularly interesting transcriptional regulator for several reasons. It is a general repressor, negatively affecting the transcription of many Pol II genes. It is also active in repressing Pol III genes, and as such it is one of the very few proteins, TBP being another, that has been implicated in the function of more than one of the three eucaryotic polymerases. An essential gene in yeast, overexpression of Dr1 results in yeast lethality, as would be expected from a general transcriptional repressor.

Dr1 works most efficiently in conjunction with its corepressor DRAP1. These two proteins, Dr1 and DRAP1, form a very tight complex and the formation of this complex is dependent on the presence of histone fold motifs in the n-termini of both proteins (Figure 1). The activity of both Dr1 and the Dr1/DRAP1 complex is correlated completely with their ability to bind TBP and the TBP/TATA complex as measured by gel shift analysis and in vivo and in vitro transcription assays.

Some, but not all, transcriptional activators overcome Dr1-mediated transcriptional repression in vivo, indicating that Dr1 mediated repression can be regulated specifically. Immunodetection experiments during brain development show that Dr1 and DRAP1 are differentially expressed in different cells, and expression of both proteins correlated with cells that were post mitotic and highly differentiated, consistent with its role in general transcriptional repression. Both Dr1 and DRAP1 have been cloned from humans and yeast.

Essentially the entire polypeptide chain of Dr1 is necessary for DRAP1 mediated transcriptional repression. A 20 amino acid helical region from residue 80-100 is important for TBP binding, and another helical stretch of 20 amino acids is important for transcriptional repression. The activities of the various domains of Dr1 and DRAP1 are summarized in Figure 1.

The determination of several crystal structures are planned. The structure of the protein alone will be pursued in all cases to understand any conformational changes necessitated by binding to the TBP/TATA complex. Since it is not known what protein constructs will produce crystals from the beginning, and it is often easier to produce well diffracting crystals of individual protein domains, the problem will be attacked simultaneously on several fronts.

The Msx-1/TBP/TATA complex.

Msx-1 is another potent transcriptional repressor that acts by binding to the TBP/TATA complex. The mechanism of repression of this protein is distinct from that of Dr1/DRAP1 however, because it does not abrogate the formation of the TBP/TFIIA/TATA complex or the TBP/TFIIA/TFIIB/TATA complex, but instead represses a later step in transcription initiation.

Msx-1 is a homeodomain containing protein and binds specifically to DNA via this homeodomain. Because Msx-1 represses transcription by binding to the TBP/TATA complex, but does so with a mechanism that is distinct from Dr1/DRAP1,we are interested in determing the structure of the Msx-1/TBP/TATA complex as well. We are also interested in investigating the homeodomain portion of Msx-1 bound to its DNA target to understand the specificity of this interaction. 


HOME