Chapter 19: Transcriptional Regulation in Eukaryotes

homeodomain

A class of helix-turn-helix DNA binding domains that recognizes DNA in essentially the same way as bacterial proteins. It consists of three alpha helices, of which two form the structure resembling the helix-turn-helix motif. One of these two helices is the recognition helix, which is inserted into the major groove of DNA and amino acid residues along its outer edge make specific contacts with base pairs.

eukaryotes

Initiation of transcription in ————- involves even more "regulatable" steps

insulators

Specific elements that control the actions of activators. When placed between an enhancer and a promoter, it inhibits activation of the gene by that enhancer. It does not inhibit activation of that same gene by a different enhancer, one placed downstream from the promoter; nor does it inhibit the original activator from working on a different gene. Thus, the proteins that bind them do not actively repress the promoter, nor do they inhibit the activities of the activators. Rather, they block communication between the two. These elements often bind a large zinc finger protein called CTCF. CTCF also binds cohesion, and this complex form a chromosomal look with the nearest promoter, thereby precluding enchancers distal to the insulator from forming a similar loop.

Transcription, pre-mRNA splicing, nuclear export, translation, post-translational modifications

Steps at which protein coding gene expression can be regulated in eukaryotes

heterochromatin

The silent mating type locus in yeast is composed of

euchromatin

loosely packed chromatin where genes are more readily expressed (more acetylated)

DNA methylases

Enzymes that silence transcription by methylation of DNA. Methylation of DNA can inhibit binding of proteins, including the transcriptional machinery, and thereby block gene expression. Methylation can also inhibit expression because some DNA sequences are recognized only when methylated by specific repressors then then switch off nearby genes, often recruiting histone modifying enzymes. In mammalian cells but not test

amino acid content

Instead of being characterized by structure, activating regions are grouped on the basis of ——-. Ex) the activating region of Gal4 is called an "acidic" activating region, reflecting a preponderance of acidic amino acids.

Telomeres and centromeres These regions are typically composed of repetitive sequences, and contain very few of any protein coding genes. If a gene is experimentally moved into these regions, it is typically switched off.

Regions of the chromosome associated with heterochromatin

1. Packing/unpacking DNA 2. Transcription 3. mRNA processing 4. mRNA transport 5. translation 6. protein processing 7. protein degradation

The control of gene expression in eukaryotes can occur at any step in the pathway from gene to functional protein. Which are these possible steps?

transcriptional silencing

A specialized form of repression that can spread along chromatin, switching off multiple genes without the need for each to bear binding sites for specific repressors. This process typically involves histone deacetylation on nucleosomes to form heterochromatin (a dense form of chromatin). Histone deactylases repress transcription by removing acetyl groups from the tails of histones.

enhancers

A unit of regulatory sequences in eukaryotes that can spread thousands of nucleotides from the promoter (both upstream and downstream) and can be made up of tens of thousands of regulator binding sites. A given ——- binds regulators responsible for activating the gene at a given time and place. Alternative ——— binds different groups of regulators and control expression of the same gene at different times and places in response to different signals.

Zinc finger domain

DNA binding domain that contains zinc. The zinc atom interacts with cysteine and histidine residues and serves a structural role essential for integrity of the DNA binding domain. The DNA is recognized by an alpha helix inserted into the major groove.

Gal4

Eukaryotic activator that activates transcription of the galactose genes in S. Cerevisae used for galactose metabolism. One such gene is GALI. This activator binds to four sites located upstream 275bp upstream of GALI. When bound there, in the presence of galactose, it activates transcription of the GALI gene 1000-fold. The DNA binding domain of the protein is separate from the region of the protein containing the activating region (activation domain). (Activation is not mediated by DNA binding alone. Instead, the DNA binding domain serves merely to tether the activating region to the promoter.

Remodeling and certain modifications can uncover DNA binding sites that would otherwise remain inaccessible within the nucleosome. For example, by removing or increasing the mobility of nucleosomes, remodelers are proposed to free up binding sites for regulators and for the transcriptional machinery. Similarly, the addition of acetyl groups to histone tails alters the interactions between those tails and adjacent nucleosomes. This medication is often said to "loosen" chromatin structure, freeing up sites. Adding actual groups also helps the binding of transcriptional machinery and other proteins by creating specific binding sites on nucleosomes for proteins bearing bromodomains. One component of the TFIID complex bears bromodomains and thus binds to acetylated nucleosomes better than to unacetylated nucleosomes. Thus, a gene bearing acetylated nucleosomes at its promoter will likely have a higher affinity for transcriptional machinery.

In addition to direct recruitment of transcriptional machinery, activators also recruit nucleosome modifiers that help the transcriptional machinery bind at the promoter or initiate transcription. How do these modifications help activate a gene?

In eukaryotes, and activator might work in one of three ways 1. Recruitment of nucleosome modifiers and remodelers to "open" the promoter 2. Recruitment of general transcription factor complexes (e.g mediator) 3. Recruitment of protein complexes that stimulate Pol II initiation and elongation (e.g. pTEFb/SEC complex).

In bacteria, activators work by recruiting RNA polymerase to the promoter. How do activators work in eukaryotes?

activating regions

In contrast to DNA binding domains of activators , ————— do not always have well defined structures. They have been shown to form helical structures when interacting with their targets within the transcriptional machinery, but it is believed that these structure are induced by that binding. The lack of defined structure is consistent with the idea that ————— are adhesive surfaces capable of interacting with several other protein surfaces.

These domains can be located on different regions of the same activator, or they can be carried on different polypeptides that form a complex on the DNA. As long as these polypeptides interact, and the activating region is thereby tethered to the DNA near the region to be activated. (Yeast two-hybrid system for protein-protein interaction screening and testing)

In eukaryotic activators, DNA binding domains and activating domains are separable. Explain.

Three proteins encoding by the genes encoding regulators of silencing SIR2, SIR3 and SIR4 form a complex that associates with silent chromatin. One of these proteins, Sir2, is a histone deacetylase. The silencing complex is recruited to the telomere by a DNA binding protein that recognizes the telomere's repeated sequences (Rap1). Recruitment of Sirs triggers local deacetylation of histone tails by the Sir2 component of that complex. The deacetylated histones are in turn bound by Sir 3 and Sir4, recruiting more silencing complex. And thus the local deacetylation readily spreads along the chromatin in a self perpetuating manner, producing an extended region of heterochromatin.

Silencing in yeast is mediated by deacetylation and methylation of histones. Explain the example of how telomeres are silenced.

Helix-loop-helix motif

Structural motif similar to the leucine zipper that involves a long alpha helix involved in both DNA recognition and in combination with second shorter alpha helix, dimerization.

Leucine Zipper Motif

Structural motif that combines dimerization and DNA binding surfaces within a single structural unit. Two long alpha helices form a pincer like structure that grips the DNA, with each alpha helix inserting into the major groove half a turn apart. Dimerization is mediated by another region within those same alpha helices: in this region, they form a short stretch of coiled coil, wherein the two helices are held together by hydrophobic interactions between appropriately spaced leucine (or other hydrophobic) residues.

Yeast have less signal integration than multicellular organisms and their genes have less extensive regulatory sequences than those of multicellular eukaryotes. Unlike higher eukaryotes, yeast also lack "action at a distance". Yeast regulatory sequences are usually located within a few hundred base pairs of their promoters.

The expansion of regulatory sequences (the increase in the number of binding sites for regulators at a typical gene) is the most striking in multicellular organisms. This reflects the more extensive signal integration found in those organisms (the tendency for more signals to regulate a given gene). But not all eukaryotes have extensive signal integration. Explain.

1. The genome of a eukaryote is wrapped in proteins called histones to form nucleosomes. Thus, the transcriptional machinery is presented with a partially concealed substrate. This condition reduces the expression of many genes in the absence of regulatory proteins. Eukaryotic cells also contain several enzymes that rearrange or chemically modify histones. These modifications alter histones in ways that affect how easily the transcriptional machinery and DNA binding proteins can bing and operate. 2. In eukaryotes, more regulatory proteins control a typical gene (as reflected in the number and arrangement of regulator binding sites associated with that gene). These binding sites are often more numerous than in bacteria and positioned further from the start of transcription.

There are additional features of eukaryotic cells and genes that complicate the actions of regulatory proteins (activators and repressors) during transcription. What are these two major additional complexities called?

1. histone acetyltransferases (HATs): add chemical groups (acetyl groups) to the tails of histones 2. SWI/SNF: displace or "remodel" the nucleosomes using ATP dependent activity

Two types of nucleosome modifiers

Promoters are regulated by activators that recruit and repressors that block RNA polymerase

Typical transcriptional control paradigm for bacteria