5. Control Of Gene Expression

Front 1

What is an excellent example of gene expression regulation in prokaryotes?

Back 1

The Lac Operon

Front 2

What is the Lac Operon?

Back 2

An operon required for the transport and metabolism of lactose in E. coli and other enteric bacteria.

Front 3

What does the Lac operon consist of?

Back 3

-3 structural genes
-A promoter
-An operator
-A regulator gene

Front 4

What is the promoter?

Back 4

The nucleotide sequence that serves as a recogn motif for RNA Polymerase

Front 5

What is the operator?

Back 5

The nucleotide sequence that binds the repressor protein.

Front 6

What is the regulatory gene in the Lac Operon??

Back 6

The LacI gene, which encodes a REPRESSOR PROTEIN.

Front 7

In prokaryotes, where is gene regulation primarily exerted?

Back 7

At the level of TRANSCRIPTION.

Front 8

What are Inducers?

Back 8

Proteins which bind the Repressor protein and weaken its affinity for the Operator.

Front 9

What are 2 inducers for the Lac Operon?

Back 9

-Allolactose (byproduct of Beta-galactosidase
-IPTG (gratuitous inducer)

Front 10

What causes the expression of the REPRESSOR gene?

Back 10

It is upstream of the Lac Operon promoter, so it has its OWN promoter.

Front 11

What results when Repressor protein is bound to Operator?

Back 11

Transcription of the 3 structural genes is prevented.

Front 12

What are the 3 genes encoded by the Lac Operon?

Back 12

1) LacZ - B-galactosidase
2) LacY - B-galactoside permease
3) LacA - B-galactoside transacetylase

Front 13

What does B-galactoseidase do?

Back 13

-Intracellular enzyme that cleaves the disaccharide lactose -> glucose/galactose.

Front 14

What does B-galactoside permease do?

Back 14

-Membrane bound transport protein that pumps lactose into the cell

Front 15

What does B-galactoside transacetylase do?

Back 15

-Transfers an acetyl group from acetyl-CoA to B-galctosides.

Front 16

What type of expression is exhibited by the Lac I gene?

Back 16

Constitutive expression - the Repressor protein is always around, because Bacteria prefer to utilize glucose.

Front 17

How does Repressor inhibit Lac operon gene expression?
-What is essential for this?

Back 17

When bound to Operator it inhibits RNAP from binding the Promotor sequence.
-Lac levels MUST BE LOW.

Front 18

What happens when Lactose becomes available to E.coli?

Back 18

-Allolactose (lactose byproduct) binds Repressor
-Conformational change reduces affinity for Operator
-RNAP can now transcribe genes

Front 19

What is an important characteristic of the Operator site on lac operon?

Back 19

It exhibits palindromic sequences - opposite in each direction.

Front 20

What is the significance of the palindromic nature of the Operator site?

Back 20

The protein that binds it (Repressor) must do so as a DIMER.

Front 21

What is the mechanism of the Repressor protein?

Back 21

It binds DNA within the region that would be protected by RNA Pol; this excludes RNAP from binding.

Front 22

-Does Repressor bind Operator specifically or nonspcfcly?
-So where on DNA helix does it bind?

Back 22

-Very Specifically - an ARG residue binds a G-C base pair.
-Repressor binds within the major groove.

Front 23

Resulting from the specific binding btwn Operator and Repressor, in what 2 ways could the bond affinity be altered?

Back 23

1. Altering the Arg residue (to a neutral AA like Lys)
2. Mutate GC bp to AT

Front 24

Does the DNAP protected site on Lac operon have palindromic symmetry?

Back 24

No. :-)

Front 25

What is the very start of what makes enterics prefer Glucose as a carbon source?

Back 25

When Glucose=high, cAMP=low

When cAMP=high, Glucose=low

Front 26

Why is cAMP important with respect to Glucose preference?

Back 26

cAMP must be complexed to CAP in order for CAP to bind the promoter on lac operon.

Front 27

What is CAP?

Back 27

Catabolite Activator Protein

Front 28

What does CAP do?

Back 28

When bound to cAMP, it binds the Promoter on lac operon.

Front 29

What is the result of CAP-cAMP binding lac operon's Promoter region?

Back 29

It turns on the promoter and allows RNAP to bind and transcribe!

Front 30

So what 3 conditions are essential for lactose utilization?

Back 30

-Glucose must be low
-Lactose must be present
-cAMP must be high

Front 31

If CAP binds the promoter region, doesn't it get in the way of RNAP binding?

Back 31

No; it binds a region just adjacent to the promoter.

Front 32

How exactly does CAP-DNA interaction facilitate RNAP binding?

Back 32

B/c it shows 2-fold rotational symmetry, CAP-cAMP binding provides more sites for RNAP to interact w/ DNA.

Front 33

In addition to facilitating RNAP binding to Promoter site, what does CAP do?

Back 33

Upregulate RNA Polymerase.

Front 34

What is the consensus sequence for the bacterial -10 Promoter?

Back 34

TATAAT

Front 35

What is the effect of:
+Glu
+Lac

Back 35

Glucose is high so cAMP is low; no CAP can bind to allow RNAP binding to promoter.

Front 36

What is the effect of:
+Glu
-Lac

Back 36

-Glu is high, cAMP is low, so no CAP is bound.
-No Lac present, so no inducer to loosen repressor.

Front 37

What is the effect of:
-Glu
-Lac

Back 37

-cAMP is high so CAP can bind
-Lac is not present so no inducer to loosen repressor.

Front 38

What is the effect of:
-Glu
+Lac

Back 38

-Glu is low, cAMP is high, CAP can bind to allow RNAP to bind Promoter (TATAAT)
-Lac is present, Inducer is present to loosen Repressor
-RNAP can bind and transcribe

Front 39

What are 2 types of Negative Regulation via Ligands?

Back 39

1. Ligand binding repressor removes it and turns gene on.
2. Ligand binding repressor keeps it on the gene; removal of ligand turns gene on.

Front 40

What is negative regulation?

Back 40

The repression of genes via a repressor protein - a ligand can be used as either an inducer or helper.

Front 41

What is positive regulation?

Back 41

The activation of a gene via binding of an activator protein

Front 42

What type of regulation is exhibited by CAP?

Back 42

Positive - where binding of the ligand (cAMP) facilitates binding of the regulatory protein and turns the gene on.

Front 43

What type of regulation is exhibited by LacI?

Back 43

Negative - ligand binding causes repressor to unbind.

Front 44

What are 2 major differences between Euk and Prok gene expression?

Back 44

1. Proks are polycistronic, Euks are monocistronic.
2. Txn/Tsln in Proks is coupled; in Euks, uncoupled.

Front 45

How is the SPACING (dstnc from reg protein-binding sites to Promoter regions) different btwn Euks/Proks?

Back 45

Prokaryotes: CAP must be very close - addition of even 5 nt can result in failure to txn.
Euks - distance is not as important - may be 500-1000s of nt btwn them.

Front 46

List 4 major areas of Eukaryotic Gene expression:

Back 46

1. Basal Txn Machinery
2. Regulating Proteins (activators/repressors)
3. Coactivators (mediators)
4. Insulators

Front 47

What is the basal transcriptional machinery made of?

Back 47

-RNA Pol II
-General Transcription factors
-TATA binding protein

Front 48

How is it that Regulating proteins can act at a distance from the promoter?

Back 48

DNA can wrap around to bring them within close proximity.

Front 49

What are Coactivators?

Back 49

Mediating proteins that link the basal txn apparatus to upstream regulatory proteins.

Front 50

What are insulators?

Back 50

Sequences of DNA that protect the basal txn machinery to prevent the regulatory proteins from talking to a different polymerase on adjacent genes.

Front 51

What is the order of assembly of the basal txn machinery?
(6 steps)

Back 51

1. TBP - TATAA binding protein
2. Other factors
3. Unphosphorylated RNAP II
4. Lose some factors
5. Phosphorylate CTD of RNAPII
6. Txnl elongation occurs!

Front 52

What is the CTD of RNA Pol II?

Back 52

C-terminal domain - its phosphorylation is essential for activity.

Front 53

What are the 4 major types of Transcription Factors in eukaryotes?

Back 53

1. Helix-turn-helix proteins
2. Zinc finger proteins
3. Leucine zipper proteins
4. Helix-loop-helix proteins

Front 54

What is the structural feature of a helix-turn-helix protein?

Back 54

2 alpha helices, separated by B-turn "Recognition helix" which fits into major groove.

Front 55

What is the structural feature of a Zinc finger protein?

Back 55

Zinc finger proteins contain ZINC bound to Cys and His side chains.

Front 56

What is the structural feature of leucine zipper proteins?

Back 56

2 alpha helices:
-one w/ basic residues for DNA binding
-one w/ regularly spaced Leu for dimerization.

Front 57

What is the structural feature of helix-loop-helix proteins?

Back 57

DNA-binding alpha helix, 2 dimerization helices separated by nonhelical loops.

Front 58

What are 2 examples of helix-turn-helix proteins?

Back 58

1. Homeodomain proteins (regulators in embryonic devo)
2. Most Prokaryotic repressors

Front 59

What are good examples of Zinc finger proteins?

Back 59

Steroid and Thyroid hormone receptors

Front 60

What are some examples of Leucine zipper proteins?

Back 60

-C/EBP (gene activator in liver)
-c-Myc, c-Fox, c-Jun (growth regulators, protooncogene products)

Front 61

What are 3 areas of trnscrption that euk proteins involved?

Back 61

1. DNA binding
2. Protein-protein interaction
3. Transcriptional activation

Front 62

How do helix-turn-helix proteins interact with DNA?

Back 62

They bind palindromic sequences as a dimer by fitting into the major groove.

Front 63

What exactly on the helix-turn-helix proteins binds the major groove?

Back 63

2 alpha helices.

Front 64

Describe the Zinc Finger Motif

Back 64

-2 Cys and 2 His bind Zinc in a tetrahedral coordination.
-Holds an alpha helix so that its basic Arginine residues optimally bind major groove.

Front 65

What are 2 examples of the zinc finger motif again?

Back 65

-Steroid hormone receptors
-Thyroid hormone receptors

Front 66

Describe the Leucine zipper motif

Back 66

-Leucine zippers are a dimer of basic proteins - result of protein-protein interaction.
-The dimer binds a palindromic DNA sequence, for its regulatory function.

Front 67

What does the leucine zipper consist of?

Back 67

A heptad repeat of leucine resideus - which allows interaction of 2 alpha helices.

Front 68

Explain the concept of Helix-loop-helix proteins:

Back 68

Amphipathic molecules that BIND another protein to make a dimer; The DNA-binding dimer is often heterodimeric.

Front 69

Again, give 3 examples of helix-loop-helix proteins:

Back 69

MyoD
Myc
Max

Front 70

How exactly do transcription factors regulate gene expression?

Back 70

TF's have different domains:
-DNA binding domains
-Protein-protein or ligand binding domains

Front 71

Give 3 examples of characteristic activation domains in TF's:

Back 71

1. Acidic domains
2. Glutamine-rich domains
3. Proline-rich domains

Front 72

How could the activity of a putative activator be studied?

Back 72

By fusing it to a known DNA binding domain (tether)

Front 73

What is the difference between euchromatin and heterochromatin?

Back 73

Heterochromatin = highly condensed.
Euchromatin = relatively diffuse

Front 74

Which type of chromatin is more active; euchromatin or hetero?

Back 74

Euchromatin.

Front 75

Where in chickens is the globin gene more active, and how would you know?

Back 75

More active in blood cells, not in the oviduct; can tell because it is nuclease sensitive.

Front 76

Where in chickens is the ovalbumin gene more active?

Back 76

Active in the oviduct, not in blood cells.

Front 77

How does methylation affect chromatin?

Back 77

Methylation causes condensing and deactivation of genes.

Front 78

List 4 possible ways that Negative TF's may act:

Back 78

1. Bind DNA to block a POS factor from binding.
2. Bind a POS TF to prevint it from binding - or speed its degradation
3. Inhibit binding/assembly of preinitiation complex
4. Recruit histone deacetylase

Front 79

What is the term for
-Highly condensed chromatin
-Relatively diffuse chromatin

Back 79

Condensed = herochromatin

Diffuse = euchromatin

Front 80

What is the activity level of heterochromatin?

Back 80

Transcriptionally Inactive

Front 81

What type of protein is H1 generally described as?

Back 81

A linker protein - it binds the linker regions of nucleosomal DNA.

Front 82

Which kind of chromatin are H1 proteins generally associated with?

Back 82

Condensed - Heterochromatin - they hold it together.

Front 83

How do Methylation and Acetylation change chromatin?

Back 83

Acetylation opens it up; Methylation closes it down.

Front 84

What 3 chemical modifications are associated with active chromatin?

Back 84

-Acetylation
-Demethylation
-Sensitivity to nuclease

Front 85

What is a syndrome associated with histone modification?

Back 85

Rubinstein-Taybi

Front 86

Why is it that methylated DNA is generally inactive?

Back 86

Proteins recognize the methyls, and prevent binding of transcription factors.

Front 87

So what is associated with cancerous tissue?

Back 87

Hypomethylated DNA

Front 88

What are insulators?

Back 88

sequences that can block enhancer promoter interaction

Front 89

What are insulators important in?

Back 89

Gene therapy - blocking aberrant activation of genes.

Front 90

How are insulators proposed to work?

Back 90

By providing a binding site for proteins (CCCTC-binding factor) which lead to the recruitment of chromatin remodeling enzymes.

Front 91

What is a disease that has been associated with loss of insulator sequences?

Back 91

Myotonic muscular dystrophy

Front 92

What is the problem in myotonic muscular dystrophy?

Back 92

Insulator sequence loss results in aberrent activation of the DMPK gene by a nearby enhancer.

Front 93

What is a Barr Body?

Back 93

The inactive X chromosome in female somatic cells.

Front 94

How do cells know which X chromosome to turn off?

Back 94

They don't; it's random

Front 95

What is seen in women heterozygous for anhidrotic ectodermal dysplasia?

Back 95

Patches of skin w/out sweat glands(mutant active), and Patches of normal skin (mutant inactive).