Regulation Of Gene Expression : Eukaryotic Regulation

Front 1

Do eukaryotes have polycistronic or monocistronic mRNA?

Back 1

Monocistronic

Each gene is regulated separately

Front 2

Do eukaryotes have a specific repressor for each gene? Why?

Back 2

No

Eukaryotes have many genes so a specific repressor for each gene is not practical

Front 3

Per cell, approximately how many protein encoding genes are transcribed?

Back 3

10-20,000

Front 4

10-20,000 protein encoding genes are transcribed per eukaryotic cell. The majority of these are common (____________). Some are ________ specific.

Back 4

Housekeeping

Tissue

Front 5

What are housekeeping genes?

Give some examples.

Back 5

Gene products are needed for general cell functions

e.g metabolic enzymes (glycolysis pathway), cytoskeletal proteins (proteins for cell division)

Front 6

What is an example of a tissue specific gene?

Back 6

Globin genes in developing RBC's

Front 7

Different levels of regulation of gene expression are possible in eukaryotes. Where can gene expression be regulated?

Back 7

1. Transcription; initiation, elongation,termination

2. RNA processing

3. mRNA transport (out of nucleus)

4. RNA stability

5. Translation

Front 8

What level of gene expression is most common and important?

Back 8

Transcription; initiation, elongation, termination

Front 9

What is meant by RNA stability?

Back 9

How long mRNA hangs round for-determines how much protein you make

Front 10

What is a promoter?

Back 10

DNA sequence that initiates and regulates transcription

Front 11

Where can the promoter be found?

Back 11

Usually upstream (5') of the transcribed sequence

Front 12

What are transcription factors?

Back 12

Regulatory proteins that recognise and bind specific DNA sequences

Any protein that isn't RNA polymerase but assists in transcription

Front 13

What does the arrow denote?

What is the core promoter?

Back 13

Where transcription is going to occur

core promoter = minimum sequence needed to get some transcription

Front 14

What does RNA polymerase II do?

Back 14

Makes mRNA

Front 15

What are these?

Back 15

Typical sequences we tend to find in most of regulatory promoter sequences

Front 16

How do transcription factors bind to DNA?

Back 16

By specific amino acid chain:base interactions

(N.B interaction = hydrogen bonding)

Front 17

RNA polymerase II requires general transcription factors. What are some things that these transcription factors should do?

Back 17

Bind to all promoters

Position RNA polymerase at the promoter

Help to separate DNA strands to allow transcription to being

Allow RNA polymerase to leave promoter as transcription begins (prevents RNA polymerase getting stuck)

Front 18

Just one interaction on its own wouldn't achieve any sequence specificity-the DNA binding protein could just bind anywhere is there was an A-T base pairs. If next to that amino acid there was another amino acid that bound to a different b.p sequence, etc, then you would build up ___________ __________

Back 18

sequence specificity

Front 19

What is TBP?

Back 19

TATA binding protein

An example of a general transcription factor

Front 20

What complex is TBP part of?

Back 20

TFIID complex

Front 21

What protein binds TATA box?

Back 21

TATA binding protein

Front 22

TBP is a "_____________ factor" needed for transcription of mRNA encoding genes

Back 22

commitment

Front 23

Where does TBP bind to DNA and what does it do once it has bound here?

Back 23

Binds DNA in minor groove and bends it, this opens up the DNA and allows other proteins, including RNA polymerase, to recognise and bind promoter

Front 24

TBP is needed for transcription. Is it enough on its own?

Back 24

No

Front 25

What is required for RNA polymerase II to being transcription?

Back 25

General transcription factors

Front 26

How are transcription factors named?

Back 26

TF = transcription factor

II = related to RNA polymerase II

A/B/D related to order of discovery

Front 27

What do you know about TFIIH?

Back 27

Acts with DNA helicase

Has kinase activity, so phosphorylates part of RNA polymerase so it can leave promoter and be mobile and move along down sequence

Front 28

How do TF's activate (or repress) transcription?

Back 28

Interacting with pol II and/or general transcription factors

Modification of chromatin

Front 29

Where are transcription factors typically found?

Back 29

~ 100 bases upstream of transcription site

Front 30

What are enhancers?

Back 30

DNA sequences that regulate eukaryotic gene expression

(N.B can even be within an intron)

Front 31

Normally, how long are enhancers?

Back 31

200 bp long

Front 32

Where do enhancers function in respect to a promoter?

Back 32

Up to 50 kbp upstream of downstream of promoter

Front 33

What do enhancers contain?

Back 33

Multiple binding sites for transcription factors

Front 34

Enhancer-bound transcription factors alsointeract with the ______ _____________ ________,and/or modify ________.

Back 34

basal transcription complex

chromatin

Front 35

What is a mediator?

Back 35

A complex of proteins that links upstream-bound transcription factors to basal complex

Front 36

What is chromatin?

Back 36

The material (DNA and protein) of chromosomes

Front 37

What are the major structural proteins of eukaryotic chromosomes?

Back 37

Histones

Front 38

Histone are DNA-binding proteins. Approximately, what is their Mr and how many amino acids are they composed of?

Back 38

Mr 20-30,000

100-135 amino acids

Front 39

Histones contain a high proportion of what kind of amino acids ( gives 2 examples) and why?

Back 39

Basic amino acids ( positive charge)

E.g Lysine and arginine

Helps binding to negative DNA

Front 40

What are the 5 histone proteins?

Back 40

H1, H2A, H2B, H3, H4

Front 41

What do we know about H2A and H2B?

Back 41

They have closely related sequences

Front 42

What is a nucleosome?

Back 42

The basic subunit of chromatin

2 molecules each of H2A, H2B, H3 and H4 (the histone octamer) with 146 bp of DNA wrapped round them (1.8 turns), plus linker DNA each side

Front 43

What is the total DNA per nucleosome?

(N.B this is DNA wrapped around each nucleosome plus linker DNA)

Back 43

200 bp

Front 44

What is the diameter of a nucleosome?

What is the diameter of DNA?

Back 44

10-11nm

2 nm

Front 45

What does this show?

Back 45

Chromatin in a low salt solution outside of cell

Front 46

What does histone 1 (H1) do?

Back 46

Binds DNA where it leaves and enters the nucleosome core

Front 47

What does Histone H1 aid?

Back 47

Further packaging of nucleosomes into a 30nm fibre (i.e helps chromatin to wrap up further)

This is thought to have the form of a solenoid (coil)

Front 48

The human chromosome in the 30 nm fibre would be 0.1 cm long. Therefore further packaging is needed. How is this achieved?

Back 48

The 30 nm fibres are folded into loops that are attached to a scaffold of non histone proteins

Front 49

What happens at M phase?

The net result is that each DNA molecules has been packaged into a mitotic chromosome that is _________ shorter than its extended length

Back 49

Looped chromatin is wound up further

50,000 x

Front 50

Chromatin is a dynamic structure.

How can transcription factors access and bind DNA?

Back 50

'Chromatin remodelling'

Looser packing

Front 51

What is meant by histone acetylation?

Back 51

An acetyl group can be added to the lysine/arginine group to make it lose its positive charge so it binds less tightly to DNA

Front 52

What is HAT?

What is HDAC?

Back 52

Histone acetyl transferase

Histone deacetylase

Front 53

Histone acetylation reduces __________ charge so histonetails bind DNA ____ tightly, allowing further __________ ________and ___ __________ to bindDNA.

Back 53

positive

less

transcription factors

RNA polymerase

Front 54

Tissue specific transcription factors bind to the promoter and enhancer sequences leads to what?

Back 54

Tissue specific gene expression

Front 55

What transcription factor is specific to developing muscle cells?

Back 55

Mao D

Front 56

The transcription factor MyoD is specific to developing muscle cells. What sequences does it bind to in the promoters and enhancers of muscle specific genes?

Back 56

'E box sequences'

Front 57

Lab cultured fibroblasts + MyoD ------> ?

Back 57

Muscle cells

Front 58

What are transcription factors?

Back 58

Proteins that bind to regulatory sequences in genes

Front 59

What does moD drive the expression of?

Back 59

Gene that we need to develop muscle

Front 60

What are some muscle specific genes that respond to MyoD?

Back 60

Muscle actin

Creatine kinase

Front 61

Where does the muscle Muscle Creatine Kinase (mMCK) gene contain E boxes?

Back 61

In its promoter and enhancer sequences

N.B this sequence will give muscle specific expression

Front 62

What is the E box sequence?

Back 62

CANNTG

N.B N can be any one of the 4 bases

Front 63

E-box sequences and promoter sequences are responsible for driving what?

Back 63

Muscle specific expression

Front 64

We can move the regulatory sequence from the mouse genome to a different type of embryo. The enhancer and promoter sequences are bound by MyoD and drive muscle specific expression of what?

Back 64

GFP

Front 65

What does this show?

Back 65

Not only do developing muscle cells start to express these muscle specific genes, but also as cell divide, the progeny of these cells retain the ability to express muscle specific genes.

Front 66

How can tissue specific gene expression be maintained when DNA replicates and cells divide?

Back 66

DNA methylation

Front 67

Which base in mammalian DNA is extensively methylated?

Back 67

Cytosine

Front 68

What is a consequence of having methyl groups on cytosine?

Back 68

A gene will not be expressed

Front 69

Methylcytosine is associated with what?

Back 69

Transcriptional inactivation

Front 70

Why does methylation of cytosine prevent transcription of a gene?

Back 70

Methylation may prevent transcription factors from binding at promoters

Methylated DNA can be bound by specific proteins that form complexes with histone deactylases

N.B histone deacetylases are enzymes that modify chromatin and shut it down and make it not be expressed as it is bound so tightly to histone protein that transcription factors cannot access promoter

Front 71

Methylation allows stability of cell lineage. DNA methyltransferase enzymes recognise methylated C in template strand and so what do they do in the replicated strand?

Back 71

Methylate C

Front 72

Eukaryotic DNA methylation is a different phenomenon from prokaryotic DNA methylation. How?

Back 72

Prokaryotic DNA methylation 'marks' the template strand to allow DNA repair and protects cell's own DNA from digestion by restriction enzymes

Front 73

Gene expression is regulated in response to signals from outside the cell. Describe how steroid hormone receptors can act as transcription factors.

Back 73

Hormone bound receptors binds to DNA

Receptor sits in cytosol until hormone enters cell. When hormone binds to the receptors, its conformation changes so it moves into the nucleus. The receptor recognises and binds specific DNA sequences and activates their expression in response to the hormone (cortisol).

Front 74

Many mammalian genes are transcriptionally activated by cAMP. What do they therefore have in there promoter sequence?

Back 74

The cAMP-responsive element

N.B this is like the Ebox in muscle promoters that binds to myo D

Front 75

What is a transcription factor (a cAMP-responsive element binding protein) that is activated by cAMP?

Back 75

CREB

Front 76

What is an example of a cAMP responsive gene?

Back 76

Carboxykinase

Front 77

What does PEP catalyse the formation of?

Back 77

Phosphoenol pyruvate from oxaloacetate in gluconeogenesis

Front 78

CREB is a transcription factor activated by hormone signalling via protein kinase A. Describe this sequence of events.

Back 78

N.B example hormone is adrenaline but can also be glucagon (with a glucagon receptor)

Front 79

What does the phosphorylate CREB help to do?

Back 79

Recruit RNA polymerase II and histone acetyl transferase to its target genes

Front 80

In response to a decrease in blood glucose, there is increased glycogen degradation, decreased glycogen synthesis and increase gluconeogenesis. Deci

Back 80

Front 81

Which of these is the slowest response and why?

Back 81

Increase gluconeogenesis

You are making enzymes rather than activating them

Front 82

What are the different levels of regulation of gene expression in eukaryotes?

Back 82

Front 83

RNA interference (RNAi) regulates eukaryotic gene expression at the post-transcription level. What can the regulatory RNAs be?

Back 83

Exogenous in origin (small interfering RNAs or siRNAs) e.g from viruses or transposons

Endogenous in origin (micro RNAs or miRNAs)

>1% of human genome

Aprox. 1/3 of all human genes are targeted

Front 84

What are siRNAs produced from?

Back 84

Double-stranded foreign RNAs as a defence mechanism

Front 85

Describe how siRNAs are produced from double stranded RNA

Back 85

Front 86

What happens when the foreign double stranded siRNA complexes with RISC protein?

Back 86

One strand is removed (passenger strand) and the other one remains as the guide strand.

The guide strand+RISC proteins go looking for cDNA.

Front 87

What is a dicer?

Back 87

A nuclease enzyme that chops RNA into smaller bits

Front 88

What does RISC stand for?

Back 88

RNA-induced silencing complex

Front 89

Describe what happens to each of the strands of RNA when it interacts with the RISC protein.

Back 89

One strand ( the passenger stand) of the siRNA is removed by ribonuclease digestion

The remaining guide strand binds to its complementary target RNA

Front 90

miRNAs target complementary mRNAs for destruction as part of normal gene regulation. Describe how.

Back 90

Front 91

What happens when the RISC complex finds an extensive match?

Back 91

Front 92

What happens when the RISC complex finds a less extensive match?

Back 92

Less exact match prevents ribosome from being able to translate RNA efficiently

Eventually RNA is broken down as it is not being translated

Front 93

miRNAs could be used for therapies therapeutically and experimentally e.g in cancer cells to stop them expressing particular genes. True or false.

Back 93

True

Front 94

What are miRNAs transcribed from?

Back 94

Part of our genome that doesn't encode proteins