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What is the default state of transcription from a gene?



Which RNA pol is signalled directly to initiate at its promoter?



How is RNA pol II signalled to initiate at its promoter?

Short gene regulatory sequence motifs that occur in proximity to RNA pol II transcription units direct gene regulation


What is the cluster of gene-regulatory motifs found close to the promoter in higher eukaryotic protein-coding genes called?

The Upstream Promoter Element (UPE)


What is the role of a transcription factor?

To sense signals from inside or outside the cell and communicate with RNA pol II to influence the initiation or elongation of transcription


What are the two types of transcription factors?

Activators and repressors


Describe the structure of a eukaryotic transcription factor.

Modular: containing a DNA-binding domain and an effector domain


How are TFs classified into families?

TFs are classified into families according to the structure of their DNA-binding domains


What is the most common family of TFs in mammals?

Zinc-finger DNA-binding domains (they themselves are modular with combinations of fingers decoding different short nucleotide sequences)


What are the three different functions of effector domains?

1) Transduce signals
2) Mediate protein-protein interactions with other factors
3) Enzyme activity


How is the gene metallothionein regulated?

The MT UPE drives transcriptional activation of the gene when toxic metal ions are present in the cell and when the cell is stressed (cell produces metallothionein proteins to chelate heavy metal ions such as Cu, Cd, Zn and Hg and prevent toxicity).
USF, AP2 and SP1 are bound constitutively.
GR and Mtf1 bind in response to signals.
The glucocorticoid receptor (GR) is expressed in most cells but normally remains as an inactive monomer in the cytoplasm, stress hormones such as cortisol bind the factor causing it to release from its cytoplasmic parters, dimerise and translocate to the nucleus where it binds its motif and regulates transcription.
The Metal-responsive TF (Mtf1) is a zinc finger TF. The DBD of Mtf1 only fold properly and forms a functional structure in the presence of heavy metal ions.


How do we discover and characterise TFs?

1) Biochemistry
2) Genetics
3) Genomics


What are the advantages of using yeast as a model eukaryote?

1) Simple propagation via standard microbiological techniques: petri dishes etc.
2) Complete control of genetics: easy mutation, can grow cells as haploid, mating and meiosis too
3) Genetic manipulation techniques: plasmids, easy gene replacement via recombination
4) Functional genomics: easy/cheap because genome is small and simple


What has been the most powerful genetic system to explore eukaryotic transcription using in vivo reporters?

Budding yeast


How is a yeast transcription factor genetic screen carried out?

1) Make mutations that inactivate the reporter; select mutants according to phenotype on petri dishes
2) Create a gene library from normal yeast (cut the yeast genome into fragments with a restriction nuclease, and ligate into yeast plasmids)
3) Introduce library into mutant yeast and look for complementation (a plasmid containing a normal version of the mutated gene complements the original mutation)
4) Isolate and sequence the plasmid = clone of TF


What is a hybrid transcription factor?

The modular nature of TFs means that domains can be swapped between TFs using recombinant DNA methods to create hybrid factors. Since the basic mechanisms of eukaryotic transcription are conserved, human effector domains will often activate transcription in yeast whilst tethered to DNA by a yeast DBD, allowing us to identify TFs in species other than yeast.


What are the steps of a yeast one hybrid screen

1) Make in vivo reporter
2) A KO mutation is created in a gene encoding a yeast TF which would normally activate via a motif in the reporter gene
3) Make a one-hybrid plasmid library: human protein-coding DNA fragments are cloned next to the DBD of our TF to create a library of plasmids expressing hybrid fusion proteins - most hybrids will have no ED-like function however ones with a TF effector domain will and these will bind the BAIT motif via the DBD and the prey ED will activate the reporter


What is an EMSA?

Electromobility shift assays are a general method for exploring interactions between DNA sequences and DNA binding proteins. They use "native" polyacrylamide gels to separate molecules according to size/molecular weight under physiological-like conditions


What is an EMSA probe?

A short fragment of DNA, containing one or more putative binding motifs for a TF or another DNA-binding entity (probe must be labelled with either a radiolabel or a fluorescent dye)


What sort of things can you find out using an EMSA?

1) TF:DNA sequence specificity
2) TF:DNA sequence affinity
3) TF:TF competition
4) TF:TF co-operativity


If you have a TF but don't really understand the motif it binds to what can you use?

Systematic evolution of ligands through exponential enrichment (SELEX): uses the EMSA principle to isolate a DNA motif together with the protein it binds


Describe the steps of SELEX.

1) Synthesise a DNA probe containing a region of random sequences that is flanked by two know sequences to which we can apply PCR (we are making a probe that is a mixture of all possible DNA sequences)
2) Add DNA-binding protein of interest and a label
3) Separate any probe and TF complex by EMSA, affinity columns or magnetic beads
4) Purify the protein and DNA complex from the gel but cutting it out of the gel
5) PCR the DNA to make more of a new less random/"motif-enriched" probe
6) Add the TF to the new probe mix
7) Repeat 3-6
8) Sequence the enriched probe mix to discover the motif expressed as a sequence logo/position weight matrix


What divisions of genomics are needed to understand gene-regulatory motifs at the genomic level?

Basic genomics, comparative genomics and functional genomics


What can basic genomics tell us?

If we have characterised a motif and its TF interactions based on reporters, EMSA etc. at one gene we can use genome sequence to spot similar motifs elsewhere


What is comparative genomics?

The study of differences and similarities in genome sequence and organisation within and between species


What do regions of genome similarity between species imply?

Evolutionary constraint and therefore function


What are conserved non-coding elements (CNEs) and what is their significance to development?

Gene regulatory sequences upstream of a gene that are conserved. CNEs change during evolution as alteration in gene regulation drives developmental innovation


What are HARs?

Human accelerated regions: CNEs uniquely present or absent in the human genome relative to other primates


What technology is described as being at the heart of functional genomics?



Describe the steps of NGS.

1) Isolate cells
2) Make genomic DNA
3) Fragment gDNA prepare adaptor library
4) NGS
5) Assemble the sequences into the correct order to reconstruct the chromosome sequence