Regulatory Sequences Control Gene Expression
Enhancer and Silencer Elements Conserved Sequences in Eukaryotic Promoters Trans-Acting Factors Control Gene Expression Cloning A Plant Trans-Acting Factor Regulatory Genes As Trans-Acting Factors |
Cloning A Plant Transcription FactorHistones are DNA binding proteins that are part of the chromatin structure. These are encoded by a family of genes and one member of the family is called H3. As with many histone promoters they contain two conserved sequence motifs.Hexamer Motif - ACGTCA Octamer Motif - CGCGGCAT These two sequeces are located close to each other in the H3 promoter.
. .-173. . . . . . . . . . . . .-161 This promoter was analyzed by the gel retardation assay to determine 1) whether a specific nuclear protein binds to a promoter fragment that contains these sequences, and if so 2) does the protein bind to both motifs, one or neither [Plant Cell Physiol 30:107 (1989)]. The following are the results that the authors obtained from these experiments.
Now that we know that protein binds to the histone promoter and that the binding is specific for the hexamer and that the protein is not a zinc finger protein, we next want to clone the gene for that protein and determine its molecular expression. A cloning strategy has been developed that takes advantage of the fact that an interaction occurs between the promoter and the trans-acting factor. The cloning vector that is used for these studies is lambda GT 11. This is a cDNA cloning factor that can be used for protein screening. Clones can be induced to make fusion proteins between the gene and the portion of beta- galactosidase that is in the vector. If these fusion proteins are in correct frame then the clone can be selected by antibody screening. The same strategy is used to clone trans-acting factors. Instead of using an antibody as a probe, a radiolabelled fragment of the promoter that binds to the factor is used. The same type of interaction that is used for gel retardation assay is used except you are looking for binding to a clone. This type of screening was used to clone the trans-acting factor that bind to the H3 hexamer sequence. This gene was called HBP-1. For these experiments, the H3 promoter fragment was used as the probe. The following features of the clone were determined. [Science 245:965 (1989)]
The HBP-1 Gene Encodes a Leucine Zipper ProteinThe leucine zipper structure of the HBP-1 protein that binds to the promoter of the histone genes is a structure that is conserved in certain mammalian and fungal trans-acting factors. A comparison of these proteins has suggested a model for how they bind DNA. It is known that a dimer is formed between two leucine zipper proteins. This occurs in the region of the leucine residues. In a single protein the leucine zipper region forms an alpha-helix. When two proteins interact, they form a coiled alpha-helix region in the leucine zipper region.NH2-terminal to the leucine zipper motif are two regions consisting of basic amino acid. These regions seem to have the ability to bind to DNA. This binding occurs in the major groove and is facilitated by the presence on an invariant asparagine residue. This residue allows for a localized break in the alpha-helix structure of the basic region and permits the basic region to continue to bind in the major groove. Several of the sequences in the DNA where the leucine zipper proteins bind have a dyad symmetry. It is suggested that this sequence is recognized by the amino acid six to seven amino acids NH2-terminal to the leucine zipper. This is the amino acid that begins the basic region that interacts directly with the DNA. Why is a dimer a better structure for recognizing a specific DNA binding site. If it is assumed that the dimer acts as a single structure and as such would double the contact area with the DNA and thus square the energy of affinity between the protein and the DNA. This would then mean that far more dilute concentrations of the protein would be needed to generate the specific binding. Copyright © 1998. Phillip McClean |