1. EUKARYOTIC TRANSCRIPTION REGULATION
CHAPTER 28 (Genes X)
EUKARYOTIC TRANSCRIPTION REGULATION

2. Control of Gene Expression in Eukaryotes
Eukaryotic gene expression is usually controlled at the level of initiation of transcription.
the promoter
enhancers
the TATA box
silencers
GC box
Methylation
CCAAT box
(called the CAT box)
Hormonal Control

3. Thinking about Gene Regulation
Humans begin life from a single cell; all the genetic information needed to create an adult is in our genome.
Embryonic cells undergo differentiation to produce specific cell types such as muscle, nerve, and blood cells.
Different cell types are the consequence of differential gene expression.

4. A typical differentiated mammalian cell makes about 10,000 proteins from approximately 30,000 genes.
Most of these are housekeeping proteins needed to maintain all cell types.
Certain proteins can only be detected in specific cell types.
How is gene expression regulated?

5. COORDINATELY CONTROLLED GENES
PROCARYOTES
Clustered in operons
Same regulatory sites
Single mRNA molecule
Translated together
EUCARYOTES
NO operons !
Genes individually transcribed
Own promoter
Specific enhancers for each gene

7. (B) In procaryotes the production of mRNA molecules is much simpler
(B) In procaryotes the production of mRNA molecules is much simpler. The 5' end of an mRNA molecule is produced by the initiation of transcription by RNA polymerase, and the 3' end is produced by the termination of transcription. Since procaryotic cells lack a nucleus, transcription and translation take place in a common compartment. In fact, translation of a bacterial mRNA often begins before its synthesis has been completed.

8. The promoter is the RNA polymerase binding site.
Genes have regulatory DNA sequences upstream from the initiation site where transcription begins.
The promoter is the RNA polymerase binding site.
Gene regulatory proteins bind to regulatory DNA sequences and can either prevent or enhance RNA polymerase binding.
Activators in enhancer
Activators in distal promoter
Co-activators
Figure: 22.2
Title:
Several types of factors affect transcription
Caption:
Factors involved in gene expression include RNA polymerase and the basal apparatus, activators that bind directly to DNA at the promoter or at enhancers, co-activators that bind to both activators and the basal apparatus, and regulators that act on chromatin structure.

9. Independent domains bind DNA and activate transcription
DNA-binding activity and transcription-activation are carried by independent domains of an activator.
The role of the DNA-binding domain is to bring the transcription-activation domain into the vicinity of the promoter.
Figure: 22.3
Title:
An activator has independent domains
Caption:
DNA-binding and activating functions in a transcription factor may comprise independent domains of the protein.

10. Acidic activators: They have multiple negative charges
Yeast activators: GAL4 , GCN4
Herpes Simplex Virus: VP16

11. The modular structure of a gene activator protein
The modular structure of a gene activator protein. Outline of an experiment that reveals the presence of independent DNA-binding and transcription-activating domains in the yeast gene activator protein Gal4. (A) The normal activation of gene transcription produced by the Gal4 protein. (B) The chimeric gene regulatory protein requires the LexA protein DNA-binding site for its activity.

12. Figure: 22.4
Title:
Specificity for activation is determined by the DNA-binding domain
Caption:
The ability of GAL4 to activate transcription is independent of its specificity for binding DNA. When the GAL4 DNA-binding domain is replaced by the LexA DNA-binding domain, the hybrid protein can activate transcription when a LexA operator is placed near a promoter.

13. Identification of Protein-Protein Interactions by the Yeast 2-Hybrid System

14. The two hybrid techniques tests the ability of two proteins to interact by incorporating then into hybrid proteins where one has a DNA-binding domain and the other has a transcription-activating domain.
Figure: 22.6
Title:
Two hybrid assay measures protein interaction
Caption:
The two hybrid technique tests the ability of two proteins to interact by incorporating them into hybrid proteins where one has a DNA-binding domain and the other has a transcription-activating domain.

15. Yeast Two Hybrid Assay Yeast Gal4 Transcription factor
Zinc Finger DNA binding domain (DBD)
Transcriptional Activation Domain (AD) AD
DBD
Gene X ON
promoter

16. Yeast Two Hybrid Assay Bait AD Prey DBD OFF OFF ON
The bait: cDNA of the protein you
want to find partners for.
Reporter Gene
OFF
promoter AD
Prey
The prey: from random cDNA’s which
encodes for various proteins.
Do any interact with the bait?
Reporter Gene
OFF
promoter
Reporter Gene ON
promoter
Yeast cells turn blue

17. Yeast Two Hybrid Assay
Uetz, 2001

18. Activators interact with the basal apparatus
DNA-binding domain determines the specificity of activators for the target promoter or enhancer.
DNA-binding domain is responsible for localizing a transcription-activating domain in the proximity of the basal appapratus.
An activator that works directly has a DNA-binding domain and an activating domain.
An activator does not have an activating domain may work by binding a coactivator that has an activating domain.
Several factors in the basal apparatus are targets with which activators or coactivators interact.

19. Figure: 22.7
Title:
An activator may use a coactivator
Caption:
An activator may bind a coactivator that contacts the basal apparatus.
An activator may bind a co-activator that contacts the basal apparatus.

20. Figure: 22.8
Title:
Activators contact the basal apparatus
Caption:
Activators may work at different stages of initiation, by contacting the TAFs of TFIID or contacting TFIIB.
Activators may work at different stages of initiation, by contacting the TAFs of TFIID or contacting TFIIB.

21. How does an activator stimulate transcription?
Two models:
The recruitment model: Activators sole effect is to increase the concentration of RNA polymerases to the promoter.
Alternative model: Activator induces some change in the transcriptional complex, i.e. conformational change in enzyme, increases its efficiency.

22. Figure: 22.9
Title:
RNA polymerase exists as a holoenzyme
Caption:
RNA polymerase exists as a holoenzyme containing many activators.
RNA polymerase may be associated with various alternative sets of transcription factors in the form of a holoenzyme complex.

23. A model for the action of some eukaryotic transcriptional activators
A model for the action of some eukaryotic transcriptional activators. The gene activator protein, bound to DNA in the rough vicinity of the promoter, facilitates the assembly of some of the general transcription factors. Although some activator proteins may be dedicated to particular steps in the pathway for transcription initiation many seem to be capable of acting at several steps.

24. Some Promoter Binding Proteins are Repressors
Repression is usually achieved by affecting chromatin structure, but there are repressors that act by binding to specific promoters and function like trans-acting (such as bacterial repressors) to block transcription.
The global repressor NC2/Dr1/DRAP1, is a heterodimer that binds to TBP to prevent it from interacting with other components of the basal apparatus.

25. Figure: 22.10
Title:
The CAAT box can be controlled by a repressor
Caption:
A transcription complex involves recognition of several elements in the sea urchin H2B promoter in testis. Binding of the CAAT displacement factor in embryo prevents the CAAT-binding factor from binding, so an active complex cannot form.
In embryo
A transcription complex involves recognition of several elements in the sea urchin H2B promoter in testis. Binding of the CAAT displacement factor (CDP) in embryo prevents the CAAT-binding factor from binding, so an active complex cannot form.

26. Take away message:
The function of a protein in binding to a known promoter element cannot be assumed: It may be activator, a repressor, or even irrelevant to gene transcription.

27. Transcription factors are activated in several ways
The activators are classified according to their
The activity of an inducible activator may be regulated by several ways

28. Figure: 22.12
Title:
Transcription factors are activated in several ways
Caption:
The activity of a regulatory transcription factor may be controlled by synthesis of protein, covalent modification of protein, ligand binding, or binding of inhibitors that sequester the protein or affect its ability to bind to DNA.

29. Oncogenes that code for transcription factors have mutations that inactivate transcription (v-erbA and possibly v-rel) or that activate transcription (v-jun and v-fos).

30. Gene Regulatory Proteins Bind to DNA
Transcription is controlled by proteins binding to regulatory DNA sequences.
Promotor includes
RNA polymerase binding site
initiation site
Regulatory DNA sequences bound by gene regulatory proteins
some short – simple gene switches
some long and complex (eukaryotic)
molecular microprocessors which respond to a variety of signals, integrate them, and determine the rate of transcription.
Expression depends on;
cell type
its environment
its age
extracellular signals
Edge of bases

32. Gene Regulatory Proteins Bind to DNA
Gene regulatory proteins insert into the major groove, contacts and binds (non-covalently) to the edges of the bases (about 20 interaction), usually without disrupting the hydrogen bonds.
This binding is very strong and very specific for the nucleotide sequence. Frequently DNA-binding proteins contain alpha-helices bind in pairs - dimerization - which doubles the contact area, increasing the strength and specificity of the interaction.

35. There are many types of DNA binding domains
The activators are classified according to their DNA binding domain.
Zinc Finger
Steroid Receptors
Helix-turn-helix motif
Homeodomain
Helix-loop-helix
Leucine zippers
Members of the same group have sequence variations of a specific motif that confer specificity for individual target sites.

36. DNA binding zinc-finger motifs
One or more zinc atoms are added to the structure.
It is found in in a cluster with additional zinc fingers, arranged one after another, the a helix of each contact the major groove of the DNA, foring a continous strech of a helices along the groove.
Two a helices are packed together with zinc atoms. Mostly found in the large family of intracellular receptor proteins (dimers).
Cys2/His2 Finger
Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X3-His

37. Figure: 22.13
Title:
Zinc fingers are based on Cys2His2Zn++
Caption:
Transcription factor SP1 has a series of three zinc fingers, each with a characteristic pattern of cysteine and histidine residues that constitute the zinc-binding site.
Transcription factor SP1 has a series of three zinc fingers each with a characteristic pattern of cysteine and histidine residues that constitute the zinc-binding site.

38. ~23 aa
7-8 aa
Figure: 22.14
Title:
The Zn finger is a DNA binding motif
Caption:
Zinc fingers may form a-helices that insert into the major groove, associated with b-sheets on the other side.
Zinc fingers may form a-helices that insert into the major groove, associated with b-sheets on the other site.
Cys2/His2 finger

39. DNA binding by a zinc finger motif: The zinc-finger motif is composed of an alpha helix and a beta sheet.
Each zinc-finger is held together by a molecule of zinc.

42. A dimer of the zinc finger domain of the intracellular receptor family bound bound to its specific DNA sequence. (e.g., glucocorticoid receptor)

43. b-sheet can also recognized DNA:
i.e. bacterial met repressor. It is a dimeric protein. Each dimers b sheet can bind to the major goorve of the DNA.

44. DNA binding by a zinc finger protein: This protein recognizes DNA using three zinc fingers of the cys-cys-his-his type arranged as direct repeats.

45. Sequence specific interactions between different six fingers and their DNA recognition sequences.

47. Steroid receptors have several independent domains
All the receptors have independent domains for DNA binding and hormone binding.

48. Figure: 22.15
Title:
A variety of hydrophobic ligands activate transcription factors
Caption:
Several types of hydrophobic small molecules activate transcription factors.

49. Figure: 22.16
Title:
Ligand-gated receptors share structural features
Caption:
Receptors for many steroid and thyroid hormones have a similar organization, with an individual N-terminal region, conserved DNA-binding region, and a C-terminal hormone-binding region.

50. Steroid receptors have zinc fingers
Figure: 22.17
Title:
Specific amino acids control binding and spacing
Caption:
The first finger of a steroid receptor controls which DNA sequence is bound (positions shown in red); the second finger controls spacing between the sequences (positions shown in blue).
The first finger of a steroid receptor controls which DNA sequence is bound (red);
The second finger controls spacing between the sequences (blue).

51. Figure: 22.18
Title:
A swap identifies two critical amino acids
Caption:
Discrimination between GRE and ERE target sequences is determined by two amino acids at the base of the first zinc finger in the receptor.
Discrimination between GRE and ERE target sequences is determined by two amino acids at the base of the first zinc finger in the receptor.
Cys-X2-Cys-X13-Cys-X2-Cys

52. Figure: 22.19
Title:
Ligands activate steroid receptors
Caption:
Glucocorticoids regulate gene transcription by causing their receptor to bind to an enhancer whose action is needed for promoter function.

53. Each receptor recognizes response elements that are related to a consensus.
These response elements have a characteristic feature:
Each consensus consists of two short repeats (or half sites). The receptors bind as a multimer, so that each half of the consensus is contacted by one subunit.
The response elements for the various receptors may be either palindromes or direct repeats in which the half sites are separated by 0-4 bp whose sequence is irrelevant.

54. Figure: 22.20
Title:
Head to head homodimer binds palindrome
Caption:
Response elements formed from the palindromic half site TGTTCT are recognized by several different receptors depending on the spacing between the half sites.
Response elements formed from the palindromic half site TGTTCT are recognized by several different receptors depending on the spacing between the half sites.

55. The receptors fall into two groups:
1) GR, MR, AR, PR receptors all form homodimers. They recognize half sites, TGTTCT consensus sequence, arranged as palindormes. The spacing between the elements determins the type of element.
2) T3R, VDR, RAR, 9-cis retinoic acid (RXR) receptors form heterodimers, which recognize half elements with the sequence TGACCT.
1bp - RXR - RXR
3bp - VDR - RXR
4bp - T3R RXR
5bp - RAR - RXR
They can also form homodimers, which recognize palindromes.

56. Figure: 22.21
Title:
Heterodimer binds direct repeats
Caption:
Response elements with the direct repeat TGACCT are recognized by heterodimers of which one member is RXR.
Response elements with direct repeats TGACCT are recognized by heterodimers of which one member is RXR.

57. Figure: 22.22
Title:
Repression prevails in absence of ligand
Caption:
TR and RAR bind the SMRT corepressor in the absence of ligand. The promoter is not expressed. When SMRT is displaced by binding of ligand, the receptor binds a coactivator complex. This leads to activation of transcription by the basal apparatus.

58. Homeodomain proteins:
Homeotic selector genes play a critical role in Drosophila development.
Several homeotic selector genes contain almost identical streches of 60 amino acids that defines this class of proteins called “homeodomain”.
Homeodomain proteins are present virtually all eucaryotic organisms.
The heli-turn-helix motif in homeodomains is always surrounded by the same structure suggesting that the motif is always presented to DNA in the same way.

59. Figure: 22.23
Title:
The homeodomain is a discrete module
Caption:
The homeodomain may be the sole DNA-binding motif in a transcriptional regulator or may be combined with other motifs. It represents a discrete (60 residue) part of the protein.

60. Figure: 22.24
Title:
The homeodomain is a module of 60 amino acids
Caption:
The homeodomain of the Antennapedia gene represents the major group of genes containing homeoboxes in Drosophila; engrailed (en) represents another type of homeotic gene; and the mammalian factor Oct-2 represents a distantly related group of transcription factors. The homeodomain is conventionally numbered from 1 to 60. It starts with the N-terminal arm, and the three helical regions occupy residues 10-22, 28-38, and Amino acids in red are conserved in all three examples.

61. Figure: 22.25
Title:
The homeodomain has 3 a-helices
Caption:
Helix 3 of the homeodomain binds in the major groove of DNA, with helices 1 and 2 lying outside the double helix. Helix 3 contacts both the phosphate backbone and specific bases. The N-terminal arm lies in the minor groove, and makes additional contacts.

62. A homeodomain bound to its specific DNA sequence.
In the homeodomain, three alpha helices interact with the DNA. Helix 2 and 3 closely resembles the helix-turn-helix motif.
Asparagine in helix number 3 binds to adenine in the DNA

63. Comparison of homeodomains from two organisms separated by more than a billion years of evolution. The Yeast a2 protein (green) and Drosophila engrailed protein (red). Black dots mark sites with identical amino acids. Orange dots indicate the position of a three amino acid insert in the a2 protein.

64. Helix-loop-helix proteins interact by combinatorial association
Helix-loop-helix proteins have a motif of amino acids that comprises two amphipathic -helices of residues separated by a loop.
The helices are responsible for dimer formation.
bHLH proteins have a basic sequence adjacent to the HLH motif that is responsible for binding to DNA.
Class A bHLH proteins are ubiquitously expressed. Class B bHLH proteins are tissue specific.
A class B protein usually forms a heterodimer with a class A protein.
HLH proteins that lack the basic region prevent a bHLH partner in a heterodimer from binding to DNA.
HLH proteins form combinatorial associations that may be changed during development by the addition or removal of specific proteins

65. HLH can make both homo and heterodimers
Flexible loop
A helix-loop-helix dimer bound to DNA. The two monomers are held together in a four-helix bundle.: each monomer contributes two a helices connected by a felixible loop of protein (red). A specific DNA sequence is bound by the two a helices that are project from the four-helix bundle.
HLH can make both homo and heterodimers

66. An inhibitory regulation by truncated HLH proteins.
Lacks DNA binding domain
An inhibitory regulation by truncated HLH proteins.
HLH motif is responsible both dimerization and DNA binding.
It recognizes a symmetric DNA sequence.

67. Figure: 22.26
Title:
HLH proteins have two helical regions
Caption:
All HLH proteins have regions corresponding to helix 1 and helix 2, separated by a loop of residues. Basic HLH proteins have a region with conserved positive charges immediately adjacent to helix 1.

68. An HLH dimer in which both subunits are of the bHLH type can bind DNA, but a dimer in which one subunit lacks the basic region cannot bind.
Figure: 22.27
Title:
HLH proteins form two sorts of dimers
Caption:
An HLH dimer in which both subunits are of the bHLH type can bind DNA, but a dimer in which one subunit lacks the basic region cannot bind DNA.
Formation of muscle cells is triggered by several bHLH proteins, including MyoD as a heterodimer MyoD-E12 or MyoD-E47, rather than a MyoD homodimer. Before myogenesis begins, the Id protein may bind to MyoD and or E12 or E47 to form hterodimers that can not bind to DNA. So removal of Id could be the trigger that releases MyoD to initiate myogenesis.

69. Leucine zippers are involved in dimer formation
The leucine zipper is an amphipathic helix that dimerizes.
The zipper is adjacent to a basic region that binds DNA.
Dimerization forms the bZIP motif in which the two basic regions symmetrically bind inverted repeats in DNA.

70. In the leucine zipper motif, two alpha helices interact with each other to allow the DNA the DNA binding domain of their protein to bind to DNA.
The two alpha helices may be from different proteins.

72. Heterodimerization of leucine zipper proteins can alter their DNA-binding soecificity.
Two different monomers can combine to form a heterodimer, which now recognizesa hybrid DNA sequence.

73. Figure: 22.28
Title:
Leucine zippers dimerize
Caption:
The basic regions of the bZIP motif are held together by the dimerization at the adjacent zipper region when the hydrophobic faces of two leucine zippers interact in parallel orientation.

74. Types of DNA binding proteins DNA and RNA polymerase repair enzymes
structural proteins
transcription factors
DNA binding motifs
zinc fingers
leucine zippers
helix-turn-helix
helix-loop-helix

75. It is not yet possible to accurately predict the DNA sequences recognized by all gene regulatory proteins.
Is there a simple amino acid-base pair recognition code: e.g., is a G-C base pair always contacted by a particular amino acid side chain?
Although certain amino acid-base interactions appear much frequently than others the answer is NO…
One of the most common protein-DNA interactions

76. Protein surface virtually any shape and chemistry can be made from just 20 different amino acids, and a gene regulatory protein uses different combinations of these to create a surface that is precisely complementary to a particular DNA sequence. We know that the same base pair can be thereby be recognized in many ways depending on its context.

77. Sequence specific interactions between different six fingers and their DNA recognition sequences.

79. HOW GENETIC SWITCHES WORK?

80. An operon = a cluster of bacterial or viral genes transcribed from a single promoter.
A sequence of DNA within the promotor is recognized by regulatory proteins - the operator.

81. Repressor protein switch genes on or off
These regulators allow fast response to the environment because they are always present in the cell - constitutively expressed
Is recognized well by RNA polymerase.
Bacteria in your gut after you eat a steak
Regulatory proteins like these are allosteric.

82. The binding of tryptophan to the tryptophan repressor protein changes the conformation of the repressor. It is an helix-turn-helix protein.

83. Addition of an “inducing” ligand can turn on a gene either by removing a gene repressor protein from the DNA or by causing a gene activator protein to bind.

84. Activator proteins act on promoters that do not bind RNA polymerase on their own. These promoters are made fully functional by the addition of a bound activator protein which is also allosteric, activated by binding another molecule at a site different from the DNA binding site.
Example: CAP has to bind cyclic AMP (an intracellular signaling molecule) to bind DNA. Genes regulated by CAP are switched on in response to increases in cAMP, which is triggered by signals received by cell membrane receptors.

85. A model for gene activation from an enhancer
General transcription factors usually can not efficiently initiate transcription alone.
Enhancers can be thousands of base pairs away, either upstream or downstream and can either increase or decrease transcription.
“Action at a distance”

86. Summary of gene activation and regulation in procaryotes and eucaryotes.

87. The gene control region of a typical eucaryotic gene.

88. Gene Regulatory sequences of a typical eucaryotic cell
Regulated by combinations of proteins over up to 50,000 nucleotide pairs
Combinatorial control can be positive or negative