Eukaryotic Transcription Factors Flashcards
What are the 3 phases of transcription?
Initiation
Elongation
Termination
How many subunits make up RNA pol II?
12
From what do RNA polymerases and its accessory factors take their cues about where to stop and start?
Sequences within the DNA.
There is a code written into DNA that tells RNA pol and its factor how to interpret where transcription units (genes) are located/begin etc.
E.g. TATA box and polyadenylation signal (AATAAA/AAUAAA).
What is the default state of gene transcription in eukaryotes?
OFF
RNA pols won’t form a PIC on a given isolated promoter without being explicitly told to do so - even with the 44 GTFs in place, RNA pol still won’t do anything.
Which is the only nuclear polymerase to recieve signals directly from the outside world instructing it to begin transcribing?
RNA pol III recieves signals directly from the outside world instructing the transcription of tRNA-encoding genes.
With which nuclear RNA polymerase is most eukaryotic gene regulation associated with?
RNA pol II responsible for transcription of protein-coding genes.
What is the approximate length of gene regulatory motifs/cis-acting motifs?
5-15bp.
What name is given to the cluster of gene regulatory motifs found close to the promoter in higher eukaryotic protein-coding genes?
Upstream promoter element (UPE).
Define the terms upstream, downstream and intergenic?
Upstream = everything in the region in front of the TSS. Downstream = everything after the mRNA (NOT everything after the TSS). Intergenic = everything within the gene codign region itself.
Give some examples of gene regulatory motifs found in higher eukaryotics particularly humans?
UPEs, enhancer, silencer, locus control region, insulator.
What is the function of most gene regulatory motifs?
These motifs are binding sites for sequence specific DNA-binding proteins: transcription factors.
What are transcription factors?
Small proteins that bind to gene regulatory motifs in DNA via sequence-specific recognition.
What is the function of transcription factors?
Transcription factors sense signals from inside or outside the cell and then communicate with RNA pol II to influence the initiation or elongation of transcription.
Transcription factors by binding to DNA are recruiting co-activators/co-repressors.
Give an analogy to demonstrate how different TFs can regulate one gene?
Transcription factors bind different motifs and therefore, each TF can relay a different signal. Jointly, they ‘decide’ on the activitiy of the gene and therefore, transcription regulation can be thought of as a parliamentary demoncracy - the sum of yes (transcription activation) and no (transcripiton repression) votes determines the outcome (transcription).
Are most transcription factors activators or repressors?
Activators.
Describe the general structure of a transcription factor?
Broadly speaking, transcription factors have two really distinct structural domains, the effector/activation domain and the DNA-binding domain.
How is transcription factor structure affected by its gene sequence?
Transcription factor modularity is reflected in the primary structure of the protein/the gene sequence.
If you look at the sequence, you will often find the effector domain is encoded in one bit of primary sequence, then there will be a gap then the DNA-binding domain is encoded in another region.
How are transcription factors classified into families?
Transcription factors are classified into families according to the structure of their DNA-binding domains.
There are different kinds of protein folds that are able to bind to DNA and recognise sequences.
How many transcription factors make up the repertoire of human transcription factors?
The human transcripiton factor repertoire consists of 1600 different proteins.
What is the most common DNA-binding domain type in the repertoire of human transcription factors?
Zinc finger (750 human TFs have a zinc finger DNA-binding domain).
Why are zinc finger DNA-binding domains thought to be the most common DNA-binding domain in mammals?
Zinc finger domains themselves are modular with combination of fingers decoding different short nucleotide sequences.
From an evolutionary perspective zinc fingers represent a wonderful mix-and-match DNA-binding toolkit for transcription factor design.
In being modular, you can take fingers out of a gene,mix and match them and evolution seems to do this.
Each finger seems to recognise 2bp sequence so 3 fingers can recognise a unique 6bp sequence which can be changed by swapping that finger out in evolution.
In being modular, zinc fingers allow evoltuion to explore different sequences.
How many transcription factors make up the repertoire of yeast transcription factors?
209
What is the function of the DNA-binding domain of a transcription factor?
Interacts with the gene regulatory DNA motifs.
What is the function of the effector domain of a transcription factor?
Effector domains make protein-protein contacts with other factors - the effector domain can be viewed as an interface for protein interactions.
They are often targets for signals - they transduce external signals/cues by directly communicating with the DNA-binding domain directly.
Effector domains may have a catalytic activity.
Describe the regulation of the metallothionein (MT) gene?
Heavy metals (e.g. Cu, Cd, Zn and Hg) are toxic as ions in free solution.
Cells fight back by producing metallothioneins - small proteins that chelate heavy metal ions preventing their toxicity.
MT genes are relatively simple, they have a promoter, a protein-codign region, a relatively simple upstream promoter element.
The MT genes are sensing two related cues and integrating that information.
External signals are transduced regarding the presence of heavy metal ions and tissue stress.
The MT UPE drives transcriptional acitvation of the gene when toxic metal ions are present and when the cell is stressed.
USF, AP2 and SP1 are consistutively bound to the MT UPE, GR and Mtf1 bind only in response to signals.
GR (glucocorticoid receptor) is expressed in most cells but normally remains as an inactive monomer in the cytoplasm.
In a case of tissue stress, stress hormones like cortisol directly bind to the GR causing its release from cytoplasmic partners Hsp70/90 and FKBP 52 allowing it to dimerise and translocate to the nucleus where it can bind its motifs (glucorticoid response elements) and regulate transcription.
The GRE is therefore a regulatory region to respond to cellular stress.
The DNA-binding domain of Mtf1 cannot form a functional structure without the presence of heavy metal ions, when present, the DNA-binding domain folds correctly and the TF translocates to the nucleus and can bind its motifs the metal responsive element (MRE).
Which of these transcription factors are constitutively bound to the MT promoter and which bind in response to signals: USF, AP2, SP1, GR, Mtf1?
USF, AP2 and SP1 are constitutively bound to the MT promoter.
GR and Mtf1 bind in response to external signals.
What are the 3 strategies by which eukaryotic transcription factors were discovered and characterised?
- Biochemistry.
- Genetics.
- Genomics.
What are in vitro reporters?
DNA constructs which work in a test tube.
What are in vivo reporters?
DNA constructs carried on plasmids or integrated into a genome which work within a host cell.
What is the difference between in vitro and in vivo reporters?
In vitro reporters are DNA constructs which work in a test tube, in vivo reporters are DNA constructs carried on plasmids or integrated into a genome which work within a host cell.
What is meant by the term reporter gene?
A reporter gene is something with a codign region that ultimately produces a transcript/protein that can be easily scored e.g. it mays a colour or something measurable in the lab.
Then using recombinant DNA technology, this ‘reporter gene’ is stuck next to a promoter of interest to allow the study of a single/group of transcription factors.
What was the main method of transcription factor discovery during the 20th century?
Biochemistry was used to purify and reconstitute mRNA transcription using in vitro reporters.
Why is biochemistry not really used for transcription factor discovery anymore?
It is really difficult and laborious.
It requires growing cells, making a nuclear extract, using columns to purify indivdiual proteins and idenitfy new transcription factors that way.
How were RNA pol II, the GTs and many activating transcription factors identified?
A standard reporter that measures the incorporation of radioactivity into an RNA, was generated by cloning a reporter sequence next to a promoter and UPE and adding a combination of purified proteins to this DNA
Any RNA synthesis from the promoter is captured usign radiolabelled nucleotide incorporation and hte resulting transcript is detected after gel electrophoresis.
This type of biochemistry is difficult and expensive!
What are the advantages of yeast as a model eukaryote?
Yeast are hugely powerful for genetic engineering.
Yeast exhibit simple propagation via standard microbial techniques meaning you can easily grow them on Petri dishes.
We can have complete control over yeast genetics, it is easy to mutagenise them, you can grow the cells as haploid, you can look at meiosis and mating.
Yeast are amenable to genetic manipulation techniques meaning you can put circular plasmids into them, you can easily replace genes via recombination, you can easily integrate bits of DNA into their genome.
How does the LacZ reporter work?
When the LacZ reporter is transcribed, cells turn blue.
The LacZ+ phenotype is blue yeast when cells are grown on X-GAL substrate on a petri dish.
This gives a very powerful blue, white colour selection.
What are the main steps of a yeast transcription factor genetic screen?
- Make mutations that inactivate the reporter, select mutants according to phenotype on petri dishes.
We grow some blue (LacZ+) yeast and mutagenise them and select the white yeast and hopefully they contain mutations in transcription factors.
Then you perform complementation cloning. - Make a gene library from normal yeast.
Take a normal yeast and take every single normal gene from a yeast cell and put them onto plasmids and make a gene library.
Then take the white mutant and add the library. - Introduce the library into mutant yeast and look for complementation.
You simply look for colonies where the reporter is reactivated again so yeast go from white to blue and you pick that mutant and look to the plasmid inside it. - Purify and sequence the complementing plasmid to identify the gene encoded - probably a transcription factor.
What was the first zinc finger protein to ever be isolated?
SWI5.
Can yeast genetics be used to identify human TFs?
Yes! This is why they are such powerful tools.
How can yeast genetics be used to identify human TFs?
Because eukaryotic TFs are modular in structure, domains can be swapped using recombinant DNA technology to generate hybrid factors.
E.g. you could use this technology to attach a huamn effector domain to a yeast DNA-binding domain.
Because the basic mechanisms of eukaryotic trnascription are conserved, human effector domains will often activate transcription in yeast whilst tethered to DNA by a yeast DNA-binding domain.
The production of hybrid transcription factors using recombinant DNA methods provides the basis of one-hybrid genetic screens.
What are the main steps to producing a yeast one hybrid screen?
- Make an in vivo reporter.
Make a knockout mutation in a gene encoding a yeast transcription factor which activates via a motif in reporter gene.
So, we have a binding motif which is the ‘bait’ motif which acts as a trap. - Make a one-hybrid plasmid library.
Human protein-coding fragments are cloned next to the DNA-binding domain of our transcription factor to create a library of plasmids with expression hybrid fusion proteins.
Effectively, you’re putting random bits of human gene which make random bits of human protein next to a yeast DNA-binding domain, in the majority of cases, nothing happens, we see no reporter activation.
However, one of these many plasmids will happen to be a human TF effector domain which becomes attached to the yeast DNA-binding domain and can now potentially activate transcription. - When present in the reporter strain, hybrid transcription factors with bind the bait motif via the DNA-binding domian and the prey effector domain (human) will activate the reporter.
- If you can clone a novel human effector domain, some simple plasmid sequencing will allow you to identify entire transcription factors from the genome sequence.
You isolate the plasmid, sequence the bit of human DNA that activate dthe reporter and go back to the human genome and potentially, a new transcription factor has been identified.
What is EMSA?
Electrophoretic mobility shift assays are a general method for exploring interactions between DNA sequences and DNA binding proteins.
What kind of gel is used in an EMSA?
EMSAs use ‘native’ polyacrylamide gels to gently separate moelcules according to size/molecular weight under physiological-like conditions.
What is the function of EMSAs?
EMSA allows visualisation of protein:DNA interaction.
