Transcriptional Regulation of Eukaryotic Gene Expression I

Yup, still rambling...

Describe gene control regions found in eukaryotes, e.g. promoters, proximal promoter elements, enhancer elements

Describe eukaryotic general transcription factors, mediators, chromatin remodelling complexes

Promoters

Promoters are basically sequences on the DNA that tell the transcription factors and polymerase where to bind. One common promoter sequence is the TATA box, so called because it has lots of T and A residues. One of the main proteins that binds here is TBP (TATA-binding protein), which is a subunit of a larger transcription factor called TFIID (the TF stands for "transcription factor," the II refers to RNA Polymerase II and the D stands for "distortion" as it "distorts" the helix). Aside from TFIID, there are other transcription factors that bind here, such as TFIIH which has some helicase activity (hence the H for "helicase"). Most of these general transcription factors (GTFs) dissociate when elongation begins.

Proximal promoter elements

Proximal promoter elements are regions near the promoter that also help to stimulate transcription. They do this via the proteins that bind to them.

Enhancer elements

Enhancers are kind of like proximal promoters in that they help to stimulate transcription, but they do their job from a distance. They can be thousands of base pairs away, whereas proximal promoter elements are only 100-200 base pairs away.

Mediators

Mediators are large complexes with roughly 30 subunits. They help to mediate transcription by "bringing together" all of the transcription factors at the promoter, proximal promoter elements and enhancer elements. They also bind RNA Polymerase II. (I'll cover the other RNA Polymerases in a later post.)

Chromatin remodelling complexes

As you should know by now, DNA doesn't just exist as a big long double helix in the nucleus- it has to be packaged well in order to fit. DNA is wound around other proteins such as histones. The complex of DNA and protein is known as chromatin. How tightly or loosely the chromatin is packaged can affect transcription: it is harder for other proteins to bind to DNA when it is tightly packaged. Chromatin remodelling complexes can influence transcription by influencing the packaging of the DNA.

Describe how some eukaryotic gene activator proteins change chromatin structure

I've just mentioned chromatin remodelling complexes, which change interactions between the DNA and histones. (Histones are basically sets of 8 proteins- an octamer- bound together. DNA can wrap around them, like thread around a spool.) As I said before, they can make chromatin more tightly packaged (heterochromatin) or more loosely packaged (euchromatin). The former decreases transcription, whereas the latter process increases transcription.

Aside from chromatin remodelling complexes, there are other proteins that can affect chromatin structure. One of these is histone chaperones, which can add or remove histones. Histone-modifying enzymes can also change groups on the histone proteins (i.e. modify the side chains of their amino acids). All of these can make the DNA more or less accessible to the transcription machinery (i.e. all of the proteins that regulate transcription). Alternatively, the acetyl or methyl groups may provide markers that transcription factors can bind to.

Describe eukaryotic gene repressor protein operation

There are many ways in which gene repressor proteins can work:

1. Competitive DNA binding. In this case, the repressor protein binds to the same spot that an activator would bind. This prevents the activator from binding and hence prevents transcription.
2. Masking the activation surface. In this case, the repressor protein binds to the activator protein, stopping it from binding to the mediator protein (or whatever other protein it may need to bind to in order to activate transcription).
3. Direct interaction with the general transcription factors. This is kind of similar to #2, but with other proteins such as mediators. This action can also prevent activator proteins from binding to the mediators.
4. Recruitment of chromatin remodelling complexes. Essentially the repressor just harnesses the action of the chromatin remodelling complex to package the DNA so that it cannot be accessed by transcription factors.
5. Recruitment of histone deacetylases. Deacetylation of histones generally leads to reduced transcription.
6. Recruitment of histone methyl transferase. The methylation state of histones can also affect transcription.

Describe cooperative binding of eukaryotic gene regulatory proteins and how this affects gene expression

Cooperative binding of eukaryotic gene regulatory proteins is somewhat like combinatorial control in prokaryotes- see my previous post. Sometimes particular combinations of proteins may attract coactivators or corepressors, each of which obviously has a different outcome on transcription.

Describe techniques/technologies used to identify gene regulatory regions and proteins

There are several different techniques and technologies that can be used to figure out the location of potential gene regulatory regions.

The first one, deletion analysis, is sometimes known more colloquially as "protein bashing" as it is a bit cruder than other methods. The first step in this method is to isolate an area upstream of the gene, as this is the area that usually contains regulatory sequences. The next step is to use restriction enzymes to cut it to give you strands of different lengths- known here as a 5'-deletion series (as it is the area upstream of the 5' end). These can then be ligated into plasmid vectors in E. coli in order to amplify and isolate the plasmids. These plasmids also contain a "reporter gene" with products that can be measured. After cultured cells have been transfected with plasmids, gene expression can be measured. Regulatory regions can be identified from there.

The next method is called DNA footprinting. Again, this involves isolating the upstream region of a gene and cleaving it with enzymes. Before cleavage, however, proteins are allowed to bind. Digestion will not occur where proteins are bound. Proteins are then removed and fragments are run through a gel (I've mentioned electrophoresis on an earlier post) and, if regulatory regions are present, there should be some areas where no cleavage is observed (as noted by some short fragments, then suddenly some longer fragments- with nothing in between). This is known as a DNA "footprint." The region can then be isolated, and the sequence can be determined.

The third and final method that I'm going to talk about is Electrophoretic Mobility Shift Assay, which thankfully has an acronym- EMSA. It's somewhat similar to DNA footprinting, except that the proteins are not removed before running through the gel. (DNA without proteins attached is also run through the gel as a control.) DNA attached to proteins does not travel as far as DNA that isn't attached to anything. Also, the larger the attached proteins, the slower the DNA will run through the gel. This method therefore gives indications of where proteins are and how large they are.

After EMSA is complete, the regulatory proteins can be identified through another process. The cell extract is fractionated in column chromatography, where different proteins pass through the column at different rates and can therefore be eluted separately. There's a couple more steps after this- namely, running aliquots of the eluted fractions through gel again and then going back to the fractions that produced a shift to purify the proteins- but I'm still not quite clear on why these are necessary. I'll have to get back to you another time on this one.