Gene Expression and Cell Decisions Flashcards
Identical twins have the same DNA and different people have different DNA, is this the same for cells?
No, different cells all have the same DNA
Who did the experiment and what was it that showed cells from the same organism possess the same DNA and hence changes in cells must be due to the way the DNA is expressed?
John Gurdon took a nucleus from somatic cells of a Xenopus tadpole. And placed in enucleated oocyte. Modified egg grew into a frog - differentiated cells still contain full set of genetic info required for development
What is an experimental method to analyse the cause of a change in phenotype?
Complementation tests: look for mutants in which the process has gone wrong, gather the mutants and organise into complementation group. Will only complement if mutations in different gene so can tell you if they map to one gene or more and about dominance relationships without actually knowing what gene product is doing
What is a conditional mutation?
One that only mutated under certain conditions edge temperature sensitive mutations
What is the difference between cis and trans mutations?
Cis act only on the chromosome in which they reside, trans act on a different chromosome
Why does E.coli prefer to metabolise glucose?
Lactose is a disaccharide that must first be cleaved to glucose and galactose
Who uncovered the notion of the lac operon and how?
What was he basis to suggest co-linearity of the genes lac Z, Y and A?
What was the dominance relationship of each of the genes lacZ, Y, A and I?
Jack Monod and F. Jacob by isolating mutations that would affect the growth of e.coli on lactose and analysing mutations (complementation, cis/trans, dominance)
Polarity - the mutations of Z affect Y and A and the mutations of Y affect A (a polar mutation affects the expression of upstream or downstream genes) however, in general most of the mutations in these three genes affects only the gene they are in, making the cis mutations
Mutations in any of these genes are recessive as presence of the other functioning allele will still result in functioning gene product
How are the lac operon genes transcribed?
As part of the same unique mRNA so they will also be translated in series therefore
What helps to confer specificity of the operon?
The binding site of the repressor molecule is composed of a duplication of the 5bp binding site of the recognition alpha helix of the repressor, so requires a dimer of the repressor.
being made up of two parts reduces the chances of a binding site being elsewhere in the genome as the 5bp binding sequence of the helix alone would be to likely to appear elsewhere
How do mutations in the I gene differ to those in the lacZ, Y or A genes?
Mutations in the lac I gene are trans rather than cis as they alter the expression on the other three genes
How do mutations in the O gene (operator) differ?
Where is P in relation to O?
Not affecting a protein product but a binding site
Two types of mutation:
Constitutive (cause X,Y and A to be always expressed even in absence of lactose)
Superrepressor - no expression under any conditions
They are cis acting and dominant
p is just upstream of O suggesting mutations in O operate by affecting activity of RNA polymerase
How does the superrepressor O mutation prevent expression?
The repressor molecule binds so tight it won’t come off
What ensures glucose will always be metabolised first even though both may be present?
CAP = catabolise activating protein which binds cAMP causing the DNA binding site on CAP to rotate 60 degrees, increasing affinity for DNA by allowing CAP to fit into the major groove without steric clashes. By binding DNA CAP bends DNA, stabilising the binding of RNA polymerase.
cAMP levels are kept low in the presence of glucose, when glucose levels low cAMP rises to promote operon transcription, aided by the fact that the inducer molecule prevents repression
How does the repressor molecules binding in dimers aid the system?
Through cooperativity: when one repressor on its own bind it can unbind quite easily but when another binds nearby (on the repeat) binding is stabilised
By stabilising binding fewer repressor molecules are required to regulate the system, increasing specificity and sensitivity of the system
What further aids this cooperativity?
What is the result?
The are an additional two operator regions, one either side of O1 but far away. They increase cooperativity by binding repressor and through looping of DNA to fix repression in place .
Results in one functional molecule of repressor per cell which has consequences
What do we call the use of proteins like CAP to regulate binding of proteins like RNA pol?
Regulated recruitment
What is the significance of having only one functional repressor per cell?
Gene leakage - the repressor continually binds and unbinds, when unbound there is no other molecule to do its job so some expression is allowed (leakage) this allows some molecules of permeate to be made so that when lactose eventually is present it can get into the cell to induce the system (via allolactose)
Allows positive feedback loop
How are feedback loops used in maintenance of the genetic system?
Permease allows some lactose in, which induces the system so you get more permeate and more lactose in…positive feedback that supports economy of system
What is a bacteriophage?
What plaques can form on infection?
What is the result of a bacteriophage persisting within the cell?
Can lytic plaques give rise to a lysogenic plaque?
A bacterial parasite that, when it infects a cell, will either kill the cell in the process of making many replicates of itself, or persist in the cell by integrating its DNA into the bacterial chromosome
If the bacteriophage just kills in a lytic cycle a clear plaque called a lytic plaque forms
If is sometimes kills and sometimes persists a turbid plaque forms
The infected bacterium gives rise to a lysogen not which is resistant to further infection
No but you can induce lysis of a lysogenic plaque using UV light
What is a complementation group?
A set of mutations that don’t complement one another make up a complementation group
What are the four complementation groups that determine the plaques formed?
cI, cII, cIII and cro
What is cI and what happens if you mutate it?
What about cII and cIII?
What about cro?
A repressor of lysis so if cI is mutated lambda will always lyse
cII and cIII help to establish lysogeny but aren’t required for its maintenance hence when mutated a lysogenous, turbid plaque can possibly form
Cro represses lysogeny so wen mutated yields lysogens
What four principles of regulation does the lac operon illustrate?
Regulation of local concentration through confinement or polymerisation (repressor molecule dimers and tetramers resulting in single molecule in cell)
Cooperativity
Regulated recruitment
Network organisation
What experiment suggest cII and cIII are only required for estaishment of lysogenous plaques and not maintenance?
Temperature sensitive mutations were induced in cI, cII and cIII plaques, as temperature was raised from thirty to forty degrees, cI plaques lysed but cII and cIII plaques did not; only needed for establishment of plaque not maintenance
What is a lambda vir mutant?
What kind of mutation are these?
A lambda that is unable to produce lysogenic, turbid plaques because of a mutation in cII or cIII which affects the production or binding of a repressor molecule
Cis mutation
What is required for the lambda lytic system?
A high level control region determining the sequence of events
A genetic program (sequence of transcriptional and translational events that lead to physiological changes and new programmes through activation f a subset of genes)
What does the genetic program of the lambda lytic cycle do?
What is the program mediated by?
Building of a new phage and lysis of a bacterium
Proteins - decision whether to lyse or not is determined by interaction of proteins with the DNA. In the case of lambda cro, cI, cII and cIII
The operative region (where the operator is) is the control region
What is the cro protein (which represses lysogeny) made up of?
cI ?
How do they function?
How do they bind DNA?
66 amino acids folded into Simple globular domain that binds DNA
Two globules linked by a hinge of 40 amino acids. One globule is an amino domain and the other s carbonyl domain. The amino globule binds DNA (17 bp operator region) the carboxyl region binds to another copy of itself l
Both cI and cro function as dimers using duplicated binding sites on the DNA (specificity and reducing local concentration by polymerisation). Interactions between the carboxyl domains allow dimers to form.
Monomers and dimers are in equilibrium for cI but as concentration increases the proportion of dimer repressors increases (control of local concentration of repressor by polymerisation which allows greater specificity and control). Cro repressors are almost always dimers
In cI the amino domains bind the operator region in major groove of DNA using one face of DNA binding helix. Cro binds along the same face of DNA as the cI repressor
What is the operator region made up of?
What are the promoters?
Three operators: Or1, Or2 and Or3, which overlap with two promoters. Each operator site is 17 base pairs
Promoter for repressor maintenance (Prm) and promoter for right (Pr).
Cro and cI bind to the operator regions to regulate the activity of the two promoters which are adjacent but do not overlap
What happens if Prm is more active?
If Pr is more active?
More of the repressor for lysis cI is made and the phage integrates into the bacterial chromosome to persist
If Pr is more active more cro is synthesised and the bacteria will lyse. Activation of Pr leads to expression of lytic genes which help the phage to reproduce then kill the cell
What determines which will be more active?
Battle between cI and cro for binding sites on the three operators, binding is dependent on relative concentration of the two proteins and the differing affinities of each of the operators for either protein
What does cI binding to Or2 do? What is this fm example of?
At Or3?
At Or1?
How does cro bind?
CI binds preferentially to Or2
Prevents RNA polymerase for Pr from binding preventing cro being made
Helps its own polymerase bind to Prm to promote expression of cI. The Prm polymerase is recruited because it is held there not only by contacts with DNA but by protein-protein interaction with repressor. Repression of Pr and activation of Prm works because Or2 is closer to Pr than Prm
Negative regulation (the repressor action of cI preventing Pr expression) and positive regulation promoting cI synthesis
Binding at Or3 results in cro expression:
By binding here cI can’t recruit its own polymerase because it is too far and can’t prevent Pr from binding so Pr does bind and more cro is made.
No expression of cI or cro - cI may bind at Or3 in which case it blocks Pr and can’t activate Prm because it is too far away
Cro binds to O in a fashion that blocks and helps to determine the binding of cI
How do the affinities of the operator regions for cI and cro differ?
What is the binding state in a lysogenous cell?
Or1>Or2>Or3 for cI
The reverse for cro
In a lysogenous cell, the majority of the time repressor sate bound to Or1 and Or2 but rarely at Or3
What is a difference between how the polymerases activate Pr and Prm?
What helps to make the system like a genetic switch?
The RNA polymerase for Pr will bind and begin transcription without the aid of a positive regulator whereas Prm requires an activator protein (cI repressor) to recruit polymerase
Note the two polymerases can never be bound at the same time (mutually exclusive) like a switch
Gave up writing stuff on here for this lecture so need to finish the lecture
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What does the biochemical system utilising galactose in yeast provide?
What is different with eukaryotes?
Insight into how the prokaryotic principles of DNA regulation are applied in eukaryotes
They have additional factors like nucleus (compartmentation), DNA packing and proteins, additional components to assembly of transcriptional regulatory machinery, sexual reproduction, adds multiple layers of complexity (more computational power) but also multiple additional layers at which regulation can occur
How did they carry out survey of genes required for growth of yeast on galactose?
In the same way as the lac operon, use mutations that affect ability to grow on galactose, explore nature of mutations/genes with complementation tests, temperature sensitive, loss of function, gain of function mutations etc. analyse results and epistasis to determine number of genes, nature cis trans, dominant etc
Can eventually develop generic circuit required
What does epistasis in this process allow us to do?
Order of gene function and functional relationships can be determined
If a and b are two different mutant phenotypes and double mutant ab has b phenotype, taken as meaning b is downstream/regulated by A
How is regulation of galactose utilisation in yeast different to lactose in E.coli?
In lac operon genes are linear and expressed as such. In yeast the galactose genes are not linear, they are spread throughout the genome - genes are coordinated in a regulated manner
What are the two main components of the yeast galactose circuit?
What are the regulated by?
What is this region like in the lac operon?
How is it different?
Structural genes- encode enzymes and molecules for actually metabolism of galactose (GAL1, GAL10, GAL7 and GAL8)
Regulatory genes- encode proteins for regulation of structural genes (GAL4, 80, 3 and 11)
Regulatory box = region called UASgal (upstream activating sequence for gal) present in all genes involved in growth on galactose. There are about 300 of these sequences in the genome -many genes under coordinated regulation
Like CAP binding site
Unlike in prokaryotes, the regulatory regions are very far away, and distinct from the promoter region
What binds UASgal?
Where is UASgal?
What does this DNA blinding molecule resemble?
Gal4 encoded by the GAL4 gene (note capitals) the main regulatory protein. GAL4 has C6 zinc cluster which interacts with DNA
In the promoter region
The cl repressors
What can be used to investigate expression of a gene/gene regulation?
What did looking at the regulatory region of the GAL1 gene reveal?
What is found further downstream?
What does the repressor do?
Reporters eg attach GFP through gene constructs (like first plants prac)
400bp region required for GAL1 expression, within which is a UASgal4 which contains four binding sites for Gal4
Binding site for repressor Mig1
Represses GAL1 expression in presence of glucose, ensures galactose genes only expressed when galactose is around in absence of other sugars
Why isn’t Mig1 like a bacterial repressor?
It doesn’t occlude he binding of polymerase itself, but by recruiting proteins that regulate chromatin structure and interactions of other regulatory proteins
It takes advantage of compartmentalisation, in absence of glucose it is phosphorylated in cytoplasm allowing expression of gal genes, presence of glucose promotes dephosphorylation so it can enter nucleus, bind and recruit repressive complexes
Differences to prokaryotes:
Multiple regulatory (UAS) sites
These are a long way away from promoter
Way in which repressor works
Exploits nuclear compartmentalisation
These features are typical of eukaryotic regulatory regions
How is gal circuit negatively regulated?
What is Gal3 and how does it work?
What is NADP?
What can Gal4 do when free?
Mig1 repressor and
Gal80 binds to domain on Gal4 (Gal4 is bound to UASgal in DNA as dimer) preventing interactions that promote transcriptional activation. Gal80 shuttles between cytoplasm and nucleus
Gal3 = cytoplasmic shuttle protein that binds galactose and ATP, altering its structure so it can bind Gal80 in the presence of NADP in the cytoplasm. This masks the NLS of Gal80 so it can’t enter the nucleus and bind Gal4
Thus Gal3 is positive regulator of Gal4
Can be considered as second messenger metabolic sensor
Recruit members of mediator complex which recruits RNA complex. Mediator complex consists of 20 proteins and is conserved from yeasts to humans
Gal11 = element of the mediator complex that interacts with Gal4 to provide a bridge aiding recruitment of RNA pol. Gal11 is part of mediator complex that binds RNA pol II
What principles of regulation are employed in gal expression and in common with prokaryotes?
Have regulated recruitment
Positive and negative regulation
Cooperative binding (dimers and mediators)
Dimerisation
Modularity of transcription factors and combinatorial approach
What is the structure of Gal4 and how does it relate to function?
What is the importance of its structure?
What was an experiment to investigate modularity?
What do these kind of experiments allow?
Multiple domains, one for DNA binding, one for dimerisation, one activation domain which is bound by Gal80. Transcriptional activation part is made up of two domains, domain I and domain II. Domain II is the one that interacts with Gal80
MODULARITY - means each of these domains will work on their own and means there are many combinations of DNA binding domains and activation domains - enables diversification of regulation to be more complex
Replaced DNA binding domain with LexA (a bacterial transcriptional regulator) and added the LexA binding site into the yeast genome near GAL1. GAL1 was activated (ie the activation and dimerisation domains of Gal4 worked when combined with different DNA binding domain)
Investigation into the functions of different domains
Is modulation general to all eukaryote transcription factors?
How can this be used to provide specificity?
What can combining different domains be used to create?
Yes, all have modular TFs, three domains, activation domain (or repressor domain for repressors), DNA binding domain and dimerisation/interaction domain
Work independently of one another (eg activation domain will work for any DNA binding domain) but come together to provide unique code (multiple ways in which different units can be combined so specific to genes)
TFs, homeodomains, leucine zippers, zinc fingers, helix-loop-helix etc emerge from these different combinations
Very large repertoire - 400 or so different eukaryotic TFs through combinatorial approach drive expression of over 30 000 genes
What is particular about the activation and repression domains?
What is an experiment to show interactions between TFs and basal transcriptional machinery are universal?
They interact with wide range of transcriptional machinery eg other TFs and polymerases
The machinery from one organism works in another - already seen how Gal4 activator domain attached to bacterial LexA works in yeast (different Kingdom) also took Gal4 protein from yeast and expressed in Drosophila.
To do this they put a UASgal upstream of a lacZ gene in Drosophila. LacZ was expressed in the pattern of Gal4
Universal molecular language for transcription
What is Bicoid and what is its structure?
A TF in Drosophila with a homeodomain (DNA binding motif) at its N terminus and an activation domain at its C terminus
Need to do rest of lecture three about Bicoid
Band
What are proposed structures of the transcriptional activation domain?
Unstructured acidic blobs that interact with their targets by ionic interactions - supported by removal of residues reducing activity gradually
OR
Acidic activation domains form alpha helices (with acid residues along one face) likely secondary structure = anti parallel beta sheet
What is a useful method to follow gene expression?
Analysis of mRNA through microarray
