Principles Of The Regulation Of Gene Expression

Front 1

Why do we need to control gene expression?

Back 1

Different cell types, vary massively in size structure and function eg compare neuron to another cell
There is no loss of DNA during development therefore both cells share the same genome so these differences arise from differences in the pattern of gene expression
ALSO 1 cell may change patterns in gene expression due to changes in their environment

Front 2

3 experiments that prove theres no loss of DNA during development

Back 2

1. Nucleus of fully differentiated frog injected into a frog egg cell whos nucleus has been removed, develops into normal tadpole

2. Chromosomes have same banding pattern in the same cell of every individual
3. Differentiated pieces of plant tissue can be placed in culture and dissociated into different cells, one of these individual cells eg root cell can regenerate an entrire plant

Front 3

At what levels can gene expression be regulated?

Back 3

1. DNA processing - DNA can be re-arranged, amplified or activated
2. transcriptional control controlling how and when a gene is transcribed
3. RNA processing control controlling splicing and processing
4. RNA transport and localization control
5. Translational control
6. Protein processing: activating, deactivating or degrading proteins

Front 4

Where does the majority of gene control take place?

Back 4

The transcriptional level

Front 5

What are Cis-regulatory elements?

Back 5

regions of non-coding DNA which affects the expression of near-by genes on the same helix

Front 6

What are GRPs?

Back 6

Gene regulatory proteins bind to cis dominant recognition element to inflict either positive or negative regulation. they recognise short sections of DNA through structural motifs.

Front 7

What are master regulatory proteins?

Back 7

1 GPR which can regulate many genes in different ways eg +ve and -ve regulation. For example, Drosphila has just 30 master regulatory proteins have 300,000 genes to be expressed

Front 8

What's a good example of a master regulatory protein?

Back 8

MyoD in muscle cell development. MyoD Myf5 and Myogenin are all master regulators. Stem cells differentiate into myoblasts which differentiate into myotubules all of these processes are controlled by the 3 GPRs which turn many genes on and off during embronic development

Front 9

Describe the tryptophan operon

Back 9

5 E.coli genes code for the enzymes that manufacture the amino acid tryptophan. When tryptophan is present in the medium then bacteria no longer needs to express these genes so cuts off their production.
To bind to the operator the repressor must have 2 molecules of tryptophan bound to it. The repressor then becomes active binds to operator and prevents binding of DNA polymerase

Front 10

Describe the lac operon in response to lactose levels

Back 10

lac operon consists of 3 structural genes

lac Z= Gene for ß-galactosidase converts lactose into glucose and galactose

lac Y=Gene for ß-galactoside permease transports lactose into the cell

lac A=Gene for ß-galactoside transacetylase and a promoter, terminator, regulator and an operator. Its under both positive and -ve control

In the absence of lactose, lactose repressor binds to the operator so RNA polymerase cannot bind, no gene transcription.
In the presence of lactose allolactose binds to an allosteric site on the repressor protein causing a conformational change. As a result of this change, the repressor can no longer bind to the operator region and falls off. RNA polymerase can then bind to the promoter and transcribe the lac genes.

Front 11

Describe the lac operon in response to glucose levels

Back 11

CAP (catabolite activator protein) is a GRP for the lac operon and enables E.coli to use alternative food sources in the absence of glucose eg lactose.
the prevelance of cAMP is inversely proportional to the prevelance of glucose. Glucose conc lowers, cAMP conc rises it binds to CAP allowing CAP to bind to the DNA assisting the binding of RNApolymerase so conc of beta galactosidase increases and lactose hydrolysed to glucose and galactose. Operon is only fully upregulated in the presence of lactose and absence of glucose. Dual control prevents wasteful transcription. Although in reality much more complex i.e lac operon never completely shuts down.

Front 12

Describe control of nitrogen metabolism by E.coli

Back 12

In time of nitrogen deficiency:
1. alpha-ketoglutorate :glutamine ratio rises
2. Uridylating enzyme is activated
3. Uridylating enzyme puts UMP onto subunit II of ntrB
4. PII dissociates from ntrB
5. ntrB kinase activity is activated
6. ntrB phosphorylates ntrC (a GPR)

7. ntrC activates transcription
SO ntrC = GPR ntrB = kinase
End result is activation of gene for gluatmine synthetase which plays an essential role in the metabolism of N by catalysing the condensation of glutamate and ammonia to glutamine
The sigma factor is sigma54 and its coded for by the ntrA gene. enviromental conditions induce sigma 54 as well as ntrB and ntrc.
n is essential element of most macromolecules. glutamine is primary amine donar for aas and nucleotide biosynthetic pathways
Note: NtrC acts from a distance see notes for diagram

Front 13

What sequences do eukaryotes have that are lacking in prokaryotes?

Back 13

Upstream promotor complex = DNA that must be in a fixed position relative to the start of transcription, it can include TATA but excludes promoter
Enchancer = distant, non-fixed postiion upstream or downstream binding sites for GRPs activates transcription from any promotor linked to it

Front 14

What experiments were carried out to find GPR binding domains?

Back 14

1. Mutational analysis - took enchancer for beta-globulin which is 108bp long and mutated every 4th base giving 27 mutant forms. Insert into a plasmid with beta-globulin promoter and a reporter gene and test see which mutation blocks binding of GPR, map activity
2. Footprinting: label enchancer with 32P on one end, do limited digestion with DNAse 1 with cuts every base to give a ladder of fragments. Add nuclear extract and, parts of DNA bound to GPR will be protected from endonucleoase
3. Gel retardation: run DNA + nuclear extract on agrose gel, there will be motility shift of DNA+protein (won't move as far) isolate this and send to lab to identify GRP

Front 15

How do GPRs at enchancers and upstream promoter complex act combinationally?

Back 15

GAL4 in yeast bound at enchancer and flips over to help load transcription factors at TATA box

Front 16

How many domains do GRPs have?

Back 16

2, shown by domain swap experiments.
1. DNA binding domain most important
2. Activator domain - can turn on promoters which it is next to. Eg, mammalian steriod hormone family (glucocorticoids) hormones enter cell, bind to cytoplasmic receptor, complex enters nucleus and binds to GRE (glucocorticoid receptor element) at the enchancer. If no hormone is around then an inhibitor is bound. When hormone is around conf change releases inhibitor therefore these GRPs have 3 binding domains: Hormone, DNA, activating domain.

Front 17

how do GRPs bind?

Back 17

recognise a DNA sequwnce because their surfaced are highly complimentary. Makes a series of contacts with the DNA including H bonds, ionic bonds and hydrophobic interactions.
Although each interaction is weak the 20 or so formed add together to ensure the binding domain is highly specific and v strong

Front 18

What are the 4 types of DNA binding domains?

Back 18

Helix-turn-helix
C2H2 zinc finger
Leucine Zipper
Helix-loop-helix

Front 19

Describe helix-turn-helix

Back 19

one of simplist and most common motifs found in 100s of DNA binding proteins
3 alpha helices. The N-terminal binds to the minor groove of DNA. The C-terminal helix binds to minor groove. Helix 1&2 lie outside the DNA. eg. the products of homebox genes (for development in animals eg Drospila)

Front 20

C2H2 zinc finger

Back 20

cystein and histidine co-ordinate a zinc atom creating finger-like projections which project into DNA eg transcription factoer SP1 has a series of 3 zinc fingers

Front 21

Leucine Zipper

Back 21

2 alpha helices, one from each monomer. Every 7a.a there is hydrophobic side chain (usually leucine) which is responsible for holding dimers together. beyond dimer interface the 2 helices seperate into Y shape with very basic (postively charged amino acids) which binds to DNA. Eg: AP-1

Front 22

Helix-loop-helix

Back 22

NOT to be confused with helix turn helix this motif is structurally similar to the leucine zipper. Dimerise at helices, can be a heterodimer or a homo dimer, Consists of short alpha helix connected by a loop to a longer alpha helix with basic regions that bind to DNA
Eg master gene regulators such as MyoD

Front 23

Intro into DNA re-arrangements

Back 23

It is clear that differentiation in higher eukaryotes usually occurs without changes to DNA. In contrast some prokaryotes carry out DNA re-arrangements to activate or inactivate specific genes. This pattern of gene regulation can be stabley inherited to all progeny of the cell lone in contrast to simple, transient changes eg typ operon

Front 24

Describe Phase Variation

Back 24

Happens in Salmonella bacteria.