9.4

While, strictly speaking, the term [blank_start]promoter[blank_end] refers to the DNA sequence that determines where a polymerase initiates transcription, the term is often used to refer to both a promoter and its associated [blank_start]promoter[blank_end]-[blank_start]proximal[blank_end] control elements.

There is flexibility in the spacing of promoter-proximal elements and the promoter.

As noted earlier, transcription from many eukaryotic promoters can be stimulated by control elements located thousands of base pairs away from the transcription start site. Such long-distance transcription-control elements, referred to as [blank_start]enhancers[blank_end], are common in eukaryotic genomes but fairly rare in [blank_start]bacterial[blank_end] genomes.

The general consensus now is that a spectrum of control elements regulates transcription by RNA polymerase [blank_start]II[blank_end]. At one extreme are [blank_start]enhancers[blank_end], which can stimulate transcription from a promoter tens of thousands of base pairs away. At the other extreme are [blank_start]promoter[blank_end]-[blank_start]proximal[blank_end] elements, such as the upstream elements controlling the HSV-I tk gene, which lose their influence when moved 30–50 bp farther from the promoter.

About 70 percent of mammalian genes are expressed from [blank_start]CpG[blank_end] island promoters, usually at much lower levels than genes with [blank_start]TATA[blank_end] box promoters.

The various transcription-control elements found in eukaryotic DNA are binding sites for regulatory proteins called [blank_start]transcription factors[blank_end].

In this approach, a DNA regulatory element that has been identified by the kinds of mutational analyses described above is used to identify [blank_start]cognate[blank_end] proteins—those proteins that bind specifically to it. Two common techniques for detecting such cognate proteins are [blank_start]DNase I[blank_end] footprinting and the [blank_start]electrophoreticmobility shift assay[blank_end].

Like activators, most eukaryotic repressors are modular proteins that have two functional domains: a [blank_start]DNA-binding[blank_end] domain and a [blank_start]repression[blank_end] domain.

Many bacterial repressors are dimeric proteins in which an α helix from each monomer inserts into the major groove in the DNA helix and makes multiple, specific interactions with the atoms there (Figure 9-29). This α helix is referred to as the [blank_start]recognition helix[blank_end] or [blank_start]sequence-readinghelix[blank_end] because most of the amino acid side chains that contact bases in the DNA extend from this helix.

The recognition helix, which protrudes from the surface of a bacterial repressor, is usually supported in the protein structure in part by [blank_start]hydrophobic[blank_end] interactions with a second α helix just N-terminal to it. This entire structural element, which is present in many bacterial repressors, is called a [blank_start]helix[blank_end]-[blank_start]turn[blank_end] [blank_start]helix[blank_end] [blank_start]motif[blank_end].

Many eukaryotic transcription factors that function during development contain a conserved 60-residue DNA-binding motif, called a [blank_start]homeodomain[blank_end], that is similar to the helix-turn-helix motif of bacterial repressors.

The [blank_start]C2H2[blank_end] zinc finger is the most [blank_start]common[blank_end] DNA-binding motif encoded in the human genome and the genomes of other mammals.

A second type of zinc-finger structure, designated the [blank_start]C4[blank_end] zinc finger (because it has four conserved cysteines in contact with the Zn2+), is found in some 50 human transcription factors.

Another structural motif present in the DNA-binding domains of a large class of transcription factors contains the hydrophobic amino acid [blank_start]leucine[blank_end] at every [blank_start]seventh[blank_end] position in the sequence. These proteins bind to DNA as [blank_start]dimers[blank_end], and mutagenesis of the leucines showed that they were required for [blank_start]dimerization[blank_end]. Consequently, the name leucine [blank_start]zipper[blank_end] was coined to denote this structural motif of a coiled coil of two α helixes.

The DNA-binding domain of another class of dimeric transcription factors contains a structural motif that is very similar to the basiczipper motif except that a nonhelical loop of the polypeptide chain separates two α-helical regions in each monomer (Figure 9-30d). Termed a [blank_start]basic[blank_end] [blank_start]helix[blank_end]-[blank_start]loop[blank_end]-[blank_start]helix[blank_end] (bHLH), this motif was predicted from the amino acid sequences of these proteins, which contain an N-terminal α helix with basic residues that interact with DNA, a middle loop region, and a C-terminal region, with hydrophobic amino acids spaced at intervals characteristic of an amphipathic α helix, that dimerizes into a coiled coil. As with basic-zipper proteins, different bHLH proteins can form heterodimers.

Biophysical studies indicate that acidic activation domains have an unstructured, random-coil, intrinsically disordered conformation. These domains stimulate transcription when they are bound to a protein [blank_start]co-activator[blank_end]

Multiple different transcription factors can interact with each other to influence gene-control.

Analysis of the roughly 50-bp enhancer that regulates expression of β-interferon, an important protein in defense against viral infections in vertebrates, provides a good example of the structure of the DNA-binding domains of several transcription factors bound to the several transcription-factor-binding sites that constitute an enhancer (Figure 9-34). The term [blank_start]enhanceosome[blank_end] has been coined to describe such large DNA-protein complexes that assemble from transcription factors as they bind to the multiple binding sites in an enhancer.

This tolerance for variable spacing between binding sites for specific transcription factors, and between promoter binding sites for the general transcription factors and for Pol II, probably contributed to rapid evolution of gene control in eukaryotes.

[blank_start]Promoters[blank_end] direct binding of RNA polymerase II to DNA, determine the site of [blank_start]transcription[blank_end] initiation, and influence the [blank_start]frequency[blank_end] of transcription initiation.

[blank_start]Promoter[blank_end]-[blank_start]proximal[blank_end] elements occur within about 200 bp of a start site. Several such elements, containing 6–10 bp, may help regulate a particular gene.

[blank_start]Enhancers[blank_end], which contain multiple short control elements, may be located from 200 bp to tens of kilobases upstream or downstream from a promoter, within an intron, or downstream from the final exon of a gene.

Promoter-proximal elements and enhancers are often celltype- specific, functioning only in specific differentiated cell types.

[blank_start]Transcription factors[blank_end], which activate or repress transcription, bind to promoter-proximal regulatory elements and enhancers in eukaryotic DNA.

Transcription activators and repressors are generally modular proteins containing a single DNA-binding domain and one or a few activation domains (for activators) or repression domains (for repressors). The different domains are frequently linked by rigid, intrinsically ordered polypeptide regions

Activation and repression domains in transcription factors exhibit a variety of amino acid sequences and threedimensional structures. In general, these functional domains interact with [blank_start]co-activators[blank_end] or [blank_start]co-repressors[blank_end], which are critical to the ability of transcription factors to modulate gene expression.

The transcription-control regions of most genes contain binding sites for multiple transcription factors. Transcription of such genes varies depending on the particular repertoire of transcription factors that are expressed and activated in a particular cell at a particular time.

Binding of multiple transcription factors to multiple sites in an enhancer forms a DNA-protein complex called an [blank_start]enhanceosome[blank_end]