July 21, 2001 Lecture Notes

Figure 20.11 Building an RNA Pol II Complex

Figure 20.13 Phosphorylation of Carboxy Terminal Domain (CTD) occurs by TFIIH

• Helicase and Kinase activity of TFIIH lets polymerase clear promoter.
• Once the polymerase starts moving, TFIIH moves along with the polymerase elongation.
• The 1^st general transcription factor to bind is TFIID (TATA binding protein)

In genes with no TATA box, TFIID cannot bind like normal, but transcription initiation still occurs. TFII D binds in the vicinity of the start site through TBP-associated factors (TAF) interactions. There are different TAFs. TFIID is made up of both the TATA binding protein and TAFs.

Identity of the TAF gives specificity ® initiator (InR)

CAAT box and GC box effect efficiency at which Basal Transcription Apparatus (BTA- consists of general transcription factors and polymerase) assembles.

Two Factors that bind to these sequences:

• Upstream factors—common factors that are present in all cells all the time, usage of these is dependent on the presence of the CAAT and/or GC boxes.
• Inducible Factors— tissue and/or time specific

In all cases, the binding of factors at the CAAT and GC boxes increases the efficiency of transcription. Some elements that work in the promoter can also be contained in the enhancer.

Figure 20.16 — Mutagenesis of the b -globin promoter shows that three regions, the TATA, CAAT and GC boxes, when mutated, have a severe effect on the relative transcription level.

Figure 20.17 — Illustrates that promoters contain different combinations of elements. No one element of a promoter is essential, and most promoters use a mix-and-match approach. The number and types of promoter elements, the distance from the start sequence, and the orientation are all variable (except for TATA box and InR-which are site and orientation specific). Generally, however, the elements are within the 1^st 200 bp of the start site.

Differences from promoter:

• Position of enhancer is not fixed, the enhancer can move several kb from the start site.
• The orientation of the entire enhancer can change, as opposed to the promoter, where the orientation of the entire promoter can not change.
• Spacing of elements in the enhancer is more compact: a higher percentage of the enhancer is required for function.

Figure 20.19 — Shows how there are many bases in enhancers that, when mutated, bring transcription activity down, compare to the promoter where 3 sections were primarily responsible for a decrease in activity when mutated.

• Fixed position
• Determines where transcription starts, provides a place for BTA assembly, which defines where transcription starts.

Enhancers increase transcription efficiency, factors bind to enhancer region to activate gene expression.

More about the Enhancer

• Presence can increase transcription by 200X
• Can work while residing downstream, upstream, or even inside the transcription unit.
• Increases the rate of transcription assembly, but doesn’t increase how fast open complex is formed or when the polymerase moves away from the promoter.

Proteins bound to the enhancer can interact with proteins bound to the promoter. Transcription factors, either inducible or upstream, bound to the enhancer interact with general transcription factors at the promoter, which bind with RNA polymerase II.

A modification of this model involves the use of an intermediate protein, called a coactivator, which acts as a bridge between the transcription factors at the enhancer and the promoter.

Because there are different coactivators and TAFs, there are many combinations, and a great deal of diversity is possible.

• Found primarily in enhancers, though they can also be found in promoters.
• They cause genes to be turned on under specific conditions.
• Can be contained in multiple genes.
• Response elements can bind tissue specific factors, which are only found in certain cell types, such as in muscle cells, or they can bind tx factors that are only active under certain conditions (like stress).

Heat Shock Response:

An increase in temperature increases the expression of the heat shock genes.

• There are about 20 of these genes, and most code for chaperone proteins, which help to fold proteins, as well as to prevent them from denaturing.
• All the heat shock genes contain at least 1 heat shock element (HSE), which is recognized by the heat shock transcription factor (HSTF).
• The HSTF protein is always present, when the temperature is raised, a phosphorylating kinase puts a phosphate onto HSTF. Once phosphorylated, it can bind to DNA and cause transcription.

Metallothionein Gene:

Codes for a protein that binds to heavy metals, such as cadmium. If cell ia assaulted by heavy metals, transcription of the gene increases.

Metallothionein Response Element (MRE) — inducible factor responds to heavy metals, The MRE is a promoter element

can’t move it-needs to be nearby by tx start site to work.

Figure 21.1 — Shows the number of different response elements in the same gene

BLE — basal level elements, required for a basal level of transcription. BLEs fit the definition of enhancer elements, for they still work even if moved or have their orientation switched.

Glucocorticoid Response Element (GRE) —if the glucocorticoid receptor is bound to steroid hormone, then the glucocorticoid receptor binds GRE and you get activity from GRE.

Again-

Upstream factors can bind to enhancer elements as well as promoter elements. The cis-acting sequence elements that bind upstream factors can work independently. Upstream factors are present in all cells at all times. But not all genes have the same binding sites for upstream factors.

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If an element isn’t an upstream factor or a general transcription factor, it is an inducible factor, which is a factor that is not present in every cell and/or not always active.

1) Synthesize it — common in tissue specific factors, synthesized in one type of cell and not in others.

2) Post translational modification — protein is there, but needs to be changed to be active. Most common example is phosphorylation (and dephosphorylation also occurs).

3) Binding of hormone — the hormone receptor is present, as is the hormone receptor response element, but the presence of the hormone is needed in order for the receptor to interact with the response element. An example of this is the glucocorticoid receptor, which needs the presence of the hormone in order to interact with the GRE.

4) Release of an inhibitor — when an inhibitor is bound to a transcription factor, the transcription factor loses the ability to bind to DNA. In order to activate the transcription factor, the inhibitor needs to be degraded.

Figure 21.2 Control of regulatory transcription factor activity.

Modular in nature, most transcription factors contain different domains that do different jobs. Each domain acts independently.

Types of domains

® DNA binding domain

® Transcription activation domain

® Protein-protein interaction domain

In lab, the system of the UAS, Gal4, PI, AP3, the activation domain, and the basal transcription apparatus illustrate one way in which these differing domains can interact.

Another 2-hybrid system uses the DNA binding domain of the bacterial LexA protein and the transcription activation domain of VP16 from herpes virus. This shows that the DNA binding domain and transcription activation domain DO NOT need to come from the same protein. The DNA binding domain needs to get the activation domain into the right place so it can interact with the BTA (basal transcription apparatus).

See handout for illustrations of these DNA binding domains

1) Zinc finger — binds a molecule of Zn⁺⁺

• Classic Zinc Finger has two each of histidines and cysteines, all four are needed to hold onto an atom of Zn⁺⁺.
• Usually, it exists in multiple fingers.
• One side of each finger forms an a -helix, which interacts with the double helix of DNA.
• Zinc fingers have a certain amount of conservation, depending on the finger, they can recognize different DNA sequences.
• Zn fingers not only bind to DNA, but also bind protein-protein (see below).

2) Steroid Hormone Receptors (SHR) — another type of Zn finger.

• SHR proteins only to DNA when bound with a hormone.
• It is C₂C₂, so it has a total of 4 cysteines.
• Most have two fingers, with each finger having a different job. One finger works with dimerization; the other finger is involved with DNA binding.
• Receptors work as dimers.
• All steroid hormones have a 3^rd domain that binds the hormone. When the hormone binds, it changes the activity of the steroid.

The hormone can serve to activate or deactivate the SHR:

In the Glucocorticoid Receptor, if the hormone binding domain is deleted and the the hormone cannot bind, the steroid becomes constitutively active.

In the Estrogen Receptor, if the hormone-binding domain is deleted, the receptor can bind to DNA, but it can no longer activate transcription, for it needs hormone binding for activation.

3) Homeodomain (also called helix-turn-helix) — provides cell fate specificity, tells cells what to become.

• 3 a -helices
• Homeodomain is responsible for binding to DNA
• Experiments suggest that DNA recognition lies within the homeodomain. Different homeodomains recognize different sequences
• The homeodomain is similar to the l repressor.

In Drosophila, mutant AntP genes cause the growth of legs where antenna should be, this is caused by a failure to express the protein at the right time.

Figure 21.12, Helix 3 of the homeodomain.

Another motif is the helix-loop-helix:

• Involved with protein-protein interactions.
• Contains 2 amphipathic helices separated by a loop.
• Important in dimerization.
• Forms homo- and heterodimers.
• Amphipathic helices are often connected to a section of basic (positively charged) amino acids, these basic amino acids are able to interact with negatively charged DNA.

4) Leucine Zipper-

• Zippers itself into protein-protein reactions, provides a dimerization motif.
• Every 7^th amino acid is a leucine, so every 2 turns you have a leucine.
• Amphipathic helix, the two hydrophobic faces come together
• Usually, there is a basic domain that is involved in DNA binding

Chromatin Remodeling-

In eukaryotes, there isn’t naked DNA, it is packaged in nucleosomes. Transcription factors must find their sequences in chromatin. Translational positioning is the DNA sequence’s position relative to the nucleosome, rotational positioning concerns which faces of the DNA are facing toward the histone and which are facing away.

In a general sense, histones are transcription repressors.

Experiments:

#1: Test tube with plasmid DNA + RNA pol II + general transcription factors ® transcription

#2: Test tube with plasmid DNA + histones, incubate, then add RNA pol II and GTF ® no transcription

#3: Test tube with plasmid DNA + TFII D, then histones, then RNA pol II and GTF ® transcription

At first, it was thought to be a race between TFII D and the histones to bind at the TATA box during S phase. If TFII D were first, the gene would be expressed, if a histone got there first, it would not be expressed. This doesn’t make much sense and is not the case.

Instead, nucleosomes are moved around or even removed in some cases. It is an energy (ATP) dependent process.

SWI/SNF complex-

• from yeast, 2 X 10 ⁶ daltons
• has ATPase activity
• basic role is chromatin remodeling, causing nucleosomes to change position

Acetylation:

Histone acetylation is an important way to change the manner in which DNA is binding with the histone. It reduces the positive charge on the amino acids in the histone, which decreases the attraction between DNA and the histone, causing the DNA to be held less tightly. With the DNA less tightly bound, it is more accessible to transcription.

Increase in acetylation = increase in transcription

Certain Coactivators have Histone Acetylase Transferase activity (HAT).

If a transcription factor at an enhancer can recruit a coactivator with HAT, the DNA near the promoter can be loosened around the histone and transcription activity will increase.