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| The combination of a promoter and an enhancer linked to a gene is the basic model of a human gene. The transcription of the gene is initiated and regulated by a number of different sequence-specific DNA-binding proteins, known as transcription factors. These factors bind to specific nucleotide sequences and bring about differential expression of the gene, not only during development, but also within tissues of the mature organism (Fig. 33.2). Many transcription factors act positively and promote transcription, while others act negatively and promote gene silencing. The unique pattern of
transcription factors present in the cell will determine in large part the different activities of the many genes present in the nucleus. Transcription factors are sometimes referred to as 'trans-acting' factors, to emphasize that, as soluble proteins, they can diffuse within the nucleus and act on multiple different genes on different chromosomes.
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| Transcription unit versus gene |
| Exactly what a 'gene' is has become increasingly difficult to define in recent years. The initial notion that a gene was a piece of DNA that gave rise to a single gene product - one gene, one protein - has been challenged. It is now clear that many functional products - different mRNA species or different protein products - may arise from a single region of transcribed DNA, as a result of differences either at the level of transcription or at the post-transcriptional level. Thus there is now a tendency to refer to such 'genes' as transcription units. The transcription units encapsulate, not only those parts of the gene such as the promoters, exons, and introns, classically regarded as the gene unit, but also the molecular events that modify the transcription process from the initiation of transcription to the final post-transcriptional modifications. This is a shift away from the notion of a gene being a single strand of DNA with exons and introns, to one of a gene being a complex structure that directs a dynamic process, giving rise to the final gene product or products at various stages of development of an organism. |
| There are other kinds of proteins involved in regulating transcription besides the sequence-specific transcription
factors. The so-called general transcription factors form a complex with RNApol II and this complex is necessary for the initiation of transcription. The general transcription factors are needed for the successful use of every promoter; they vary somewhat with the class of gene, being generally different for RNA polymerase I, II, and III. In eukaryotic cells, and mammalian cells in particular, the RNA polymerases cannot recognize promoter sequences themselves. It is the task of the gene-specific factors to create a local environment that can successfully attract the general factors, which in turn, attract the polymerase itself.
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| In addition, other proteins can bind to the sequence-specific transcription factors and modulate their function by repressing or activating gene expression; these factors are often called co-activators or co-repressors. Thus the overall rate of RNA transcription from a gene is the result of the complex interplay of a multitude of transcription factors, co-activators and co-repressors. Since there are thousands of these factors, there is an almost unimaginably large number of combinations that can occur on any one promoter and thus gene control can be very specific and very subtle.
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| Initiation of transcription requires binding of transcription factors to DNA
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| Figure 33.2 Regulation of gene expression by specific regulatory elements. Binding of transcription factors to a steroid response element modulates the rate of transcription of the message. Different elements have varying effects on the level of transcription, some exerting greater effects than others, and may also activate tissue-specific expression. MyoD, muscle-cell-specific transcription factor. GRE, glucocorticoid response element. The proteins are shown in a linear array for convenience, but they interact physically with one another, both because of their size and the folding of DNA. |
| For transcription to occur, transcription factors must bind to DNA. These are trans-acting factors that recognize and bind to short nucleotide sequences in the promoter. For example, a protein known as TATA-binding protein (TBP) binds to the region of the TATA box. TBP is a general transcription factor, associated with the complex of RNAPol II and a variable number of other proteins. Binding of TBP to the TATA box directs the positioning of the transcription apparatus at a fixed distance from the startpoint of transcription and thus allows RNAPol II to be positioned exactly at the site of initiation
of transcription. Once RNAPol II and a number of other transcription factors have bound to the region of the startpoint, transcription can occur. When transcription begins, many of the transcription factors required for binding and alignment of RNAPol II are released, and the polymerase travels along the DNA, forming the primary messenger RNA transcripts (sometimes called pre-mRNA).
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| Transcription factors have common structures that permit DNA binding
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| The binding of transcription factors to DNA involves a relatively small area of the transcription factor protein, which comes into close contact with the major and/or minor groove of the DNA double helix to be transcribed. The regions of these proteins that contact the DNA are called DNA-binding domains or motifs, and are highly conserved between species. There are a variety of DNA-binding domains, some of these occur in multiple transcription factors. Four common classes of DNA-binding domain are (Fig. 33.3):
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- helix-turn-helix (HTH) motif: mediates DNA binding by fitting into the major groove of the DNA helix, allowing precise alignment of the factor in relation to the DNA sequence recognized;
- helix-loop-helix (HLH) motif: promotes both DNA binding and protein dimer formation. It is believed that HLH motifs mediate mainly negative influences on gene expression;
- zinc finger: loops or fingers of amino acids
 that have a zinc ion at their core. The adjacent amino acid residues often form α-helices that make contact with the DNA in the major groove; - leucine zipper: enables the protein to form a dimer that grips the DNA double helix like a peg, by inserting into the major groove.
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| Most sequence-specific transcription factors contain at least one of these DNA-binding motifs. Transcription factors can bind to the DNA in a sequence-specific fashion because the tertiary structure of the DNA-binding site of a transcription factor enables it to interact with the exposed groups of the nucleotide bases in the major or minor groove (see Chapter 30) of the DNA double helix, and the nature of these exposed groups is base-pair specific. Although binding between amino acids and DNA is via weak hydrogen bonds, the average transcription factor has 20 or more sites of contact, which amplifies the strength and specificity of the contact.
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| Figure 33.3 The four main classes of DNA-binding domains. Leucine zippers have hydrophobic leucine residues consistently on one face of the helix, which allows two leucine zippers to align with their hydrophobic residues facing each other. HLH, helix-loop-helix; HTH, helix-turn-helix. |
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| In addition to a DNA-binding domain, sequence-specific transcription factors also have a transcription-regulatory domain that is required for their ability to modulate transcription. This domain may function in a variety of ways. It may interact directly with the RNA polymerase-general transcription factor complex, it may have indirect effects via co-activators or co-repressor proteins, or it may be involved in remodeling the chromatin and so alter the ability of the promoter to recruit other transcription factors.
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