Transcription factors

polyA tail

Happens at the end of mRNA in eukaryotes but not prokaryotes and isn't encoded in the DNA

Splicing is specific to..

eukaryotes

What does a typical eukaryotic genome include?

exons, introns, and potentially regulatory regions

Regulatory regions

Don't code but alter how the gene is expressed

Introns

Parts of the genes removed during splicing

Exons

parts of the gene that encode for the protein

Cis-regulatory elements

regions of DNA involved in gene regulation. Contain information on when, where, how, and at what level the DNA should be expressed.

Cis-regulatory elements include

promotors, enhancers, and silencers

Trans-acting factors

Usually proteins that bind in the cis-regulatory elements to regulate gene transcription. Often transcription factors.

Transcription factors

Trans-acting factors that bind directly to DNA or associate with complexes of protein bound to DNA. These alter the activity of Cis-regulatory elements.

Proteins binding DNA

Proteins do not bind DNA the same way DNA or RNA does. proteins very rarely separate the two strands of DNA. In the majority of cases, things bind to the major groove.

What does transcription of a protein coding gene require?

binding of RNA polymerase II

RNA polymerase

forms complexes with proteins called general transcription factors to initiate transcription.

Where do general transcription factors form

TATA box

TATA box

region of DNA around 30 base pairs upstream of the transcription start site and forms part of the promotor.

Why is the TATA box called that?

Has conserved sequences of DNA bases: T-A-T-A

Examples of functions of transcription factors

recognition of the TATA box sequence in the promotor, recruitment of RNA polymerase II, and to unwind the DNA helix.

TBP (TATA binding protein)

binds to TATA box at the minor groove. Causes an 80 degree bend which begins to separate DNA. RNA polymerase can then get in

Why is binding at the minor groove rare?

It is information poor

The lower the number of hydrogen bonds between T-A pairs than G-C pairs...

Makes it easier to separate the DNA strands at these positions

General transcription factors

Aid RNA polymerase II in its function. Help position RNA polymerase II correctly at the promotor and aid in pulling apart the two strands of DNA to allow transcription to begin, and release RNA polymerase from the promotor to start its elongation mode.

Why are general transcription factors "general"?

they function/ are found near nearly all promotors.

What do general transcription factors consist of?

A set of interactive proteins denoted arbitrarily as TFIIA to TFIID

Do general transcription factors all do the same thing?

No, they work together to do the general machinery required to turn the gene on.

TFIIH

Uses ATP, unwinds DNA exposing template strands, phosphorylates polymerase II

Specific transcription factors

Specific to certain functions. They influence the activity of the transcription initiation complex.

Where do specific transcription factors bind

bind to regions in the enhancer and silencer regions. By controlling when and where specific transcription factors are active, the cell is able to alter which genes are active.

Enhancers

Bound by proteins that activate transcription of the nearby gene. They can be found at either end of the gene.

Silencers

Bind repressor proteins that inhibit transcription.

Binding motif

region a specific transcription factor binds to

Why is the length of the binding motif important?

If too short it appears everywhere such as with the TATA box, if too long it can get too complex.

How long is the region that specific transcription factors bind to?

6-12 DNA base pairs

Why is the Estrogen receptor unusual?

DNA binds to it directly

What do specific transcription factors do after binding these locations?

recruit other proteins

What do most transcription factors contain?

A DNA binding domain, an activator or repressor protein, and a domain for interactions with other proteins.

How do specific transcription factors influence over long distances (Eg. enhancers and silencers several thousand base pairs from each other)?

Forming loops to bring them back into proximity with the promotor.

Mediator complex

made of many different subunits. The different parts of the mediator complex are different in different cells. How enhancers and silencers interact with the transcription initiation complex.

Gene regulatory hierarchies

Transcription factors form hierarchies. A transcription factor will regulate the expression of multiple downstream targets, including other specific transcription factors. This means transcription factors regulate cascades of gene expression.

As we get deeper into tissues into the human body...

oxygen gets to cells less easily

Hypoxia

Lack of oxygen getting to a cell

How does the body combat deeper tissues getting less oxygen?

The body has developed the ability to sense hypoxia and stimulate the growth of blood vessels.

HIF1A

A transcription factor named HIF for hypoxia-inducible factor.

HIF1A in normoxic conditions

HIF1A is modified by another protein called proline hydroxylase which marks HIF1A in a way which is recognised by protein pVHL that targets HIF1A for degradation. Once degraded HIF1A is unable to activate genes.

HIF1A in low oxygen conditions

Proline hydroxylase is unable to mark HIF1A (as this requires oxygen) and HIF1A is not degraded. Now that HIF1A is stable, it binds DNA near genes like VEGF and activates them.

VEGF

Signalling molecule that causes the development of blood vessels

Central dogma

DNA makes RNA, RNA makes protein

Ways in which we can discuss gene expression

By checking the amount of protein made, or the amount of mRNA made. Protein and mRNA levels don't always match so checking both is often essential for research.

Challenge with using RNA in molecular biology

It is unstable and breaks down

How to overcome the problem with using RNA in molecular biology?

Convert mRNA to cDNA

How to convert RNA into DNA

Taking reverse transcriptases from viruses which helps us turn RNA to DNA. The reverse transcriptase cannot bind from scratch so we need a primer to prime it and make an mRNA cDNA construct.

What primer do we use to bind the Poly-A tail?

A poly-T tail primer

RNAase H

enzyme that breaks down RNA and has a specificity for the RNA-DNA hybrid.

RT-PCR

Heat cDNA to around 95 degrees to separate the strands then for our transcript of interest find the area we want to amplify. pick primers then do cycles of PCR to amplify this.

End point PCR

Fixed number of cycles. This is important because then you can then run it on a gel.

Molecular weight ladder

Lets us confirm the PCR product is the expected size. Acts as a control. The brightness of the band reflects how much DNA has stopped.

What is the gel typically stained with?

a fluorescence DNA binding gel