1. REGULATION OF GENE EXPRESSION IN EUKARYOTES

2. INTRODUCTION
Eukaryotic organisms have many benefits from regulating their genes
For example
They can respond to changes in nutrient availability
They can respond to environmental stresses
In plants and animals, multicellularity and a more complex cell structure, also demand a much greater level of gene expression
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

3. INTRODUCTION Gene regulation is necessary to ensure
1. Expression of genes in an accurate pattern during the various developmental stages of the life cycle
Some genes are only expressed during embryonic stages, whereas others are only expressed in the adult
2. Differences among distinct cell types
Nerve and muscle cells look so different because of gene regulation rather than differences in DNA content
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

5. REGULATORY TRANSCRIPTION FACTORS
Transcription factors are proteins that influence the ability of RNA polymerase to transcribe a given gene
There are two main types
General transcription factors
Required for the binding of the RNA pol to the core promoter and its progression to the elongation stage
Are necessary for basal transcription
Regulatory transcription factors
Serve to regulate the rate of transcription of nearby genes
They influence the ability of RNA pol to begin transcription of a particular gene
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

6. Regulatory transcription factors recognize cis regulatory elements located near the core promoter
These sequences are known as control elements or regulatory elements
The binding of these proteins to these elements, affects the transcription of an associated gene
A regulatory protein that increases the rate of transcription is termed an activator
The sequence it binds is called an enhancer
A regulatory protein that decreases the rate of transcription is termed a repressor
The sequence it binds is called a silencer
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

7. Copyright ©The McGraw-Hill Companies, Inc
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

8. Most Eukaryotic genes are regulated by many factors
This is known as combinatorial control
Common factors contributing to combinatorial control are:
One or more activator proteins may stimulate transcription
One or more repressor proteins may inhibit transcription
Activators and repressors may be modulated by:
binding of small effector molecules
protein-protein interactions
covalent modifications
Regulatory proteins may alter nucleosomes near the promoter
DNA methylation may inhibit transcription
prevent binding of an activator protein
recruiting proteins that compact the chromatin
Various combinations of these factors can contribute to the regulation of a single gene
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

9. Structural Features of Regulatory Transcription Factors
Transcription factor proteins contain regions, called domains, that have specific functions
One domain could be for DNA-binding
Another could provide a binding site for effector molecules
A motif is a domain or portion of it that has a very similar structure in many different proteins
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

10. Enhancers and Silencers
The binding of a transcription factor to an enhancer increases the rate of transcription
This up-regulation can be 10- to 1,000-fold
The binding of a transcription factor to a silencer decreases the rate of transcription
This is called down-regulation
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

11. Enhancers and Silencers
Many response elements are orientation independent or bidirectional
They can function in the forward or reverse orientation
Many response elements are orientation independent or bidirectional. They
can function in the forward or reverse orientation.
5’-GATA-3’ 5’-TATC-3’
3’-CTAT-5’ 3’-ATAG-5’
Most response elements are located within a few hundred nucleotides upstream of the promoter
However, some are found at various other sites
Several thousand nucleotides away
Downstream from the promoter
Even within introns!
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

12. TFIID and Mediator
Most regulatory transcription factors do not bind directly to RNA polymerase
Three common interactions that communicate the effects of regulatory transcription factors are
1. TFIID-direct or through coactivators
2. Mediator
3. recruiting proteins that affect nucleosome composition
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

13. TFIID ON
OFF
Activator
protein
Coactivator
Repressor
protein
TFIID
Coding sequence
Coding sequence
Core
promoter
Enhancer
Core
promoter
Silencer
The activator/coactivator complex recruits TFIID to the core promoter
and/or activates its function. Transcription will be activated.
The repressor protein inhibits the binding of TFIID to the core promoter
or inhibits its function. Transcription is repressed.
(a) Transcriptional activation via TFIID
(b) Transcriptional repression via TFIID
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

14. Transcriptional activator stimulates the function of mediator
Core
promoter ON
Core
promoter
OFF
Mediator
Mediator
TFIID
TFIID
RNA polymerase
and general
transcription
factors
RNA polymerase
and general transcription
factors
Coding sequence
Coding sequence
Activator protein
Repressor protein
Enhancer
Silencer
The activator protein interacts with mediator. This enables
RNA polymerase to form a preinitiation complex that can
proceed to the elongation phase of transcription.
The repressor protein interacts with mediator so
that transcription is repressed.
(a) Transcriptional activation via mediator
(b) Transcriptional repression via mediator
Transcriptional activator stimulates the function of mediator
This enables RNA pol to form a preinitiation complex
It then proceeds to the elongation phase of transcription
Transcriptional repressor inhibits the function of mediator
Transcription is repressed
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

15. Modulation of Regulatory Transcription Factor Functions
There are three common ways that the function of regulatory transcription factors can be affected
1. Binding of a small effector molecule
2. Protein-protein interactions
3. Covalent modification
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

16. The transcription factor can now bind to DNA
Formation of homodimers and heterodimers
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

17. Steroid Hormones and Regulatory Transcription Factors
Regulatory transcription factors that respond to steroid hormones are termed steroid receptors
The hormone actually binds to the transcription factor
The ultimate action of a steroid hormone is to affect gene transcription
Steroid hormones are produced by endocrine glands
Secreted into the bloodstream
Then taken up by cells that respond to the hormone
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

18. These changes range from a few nucleosomes to large scale changes
CHROMATIN REMODELING
ATP-dependent chromatin remodeling refers to dynamic changes in chromatin structure
These changes range from a few nucleosomes to large scale changes
Carried out by diverse multiprotein machines
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

19. Chromatin Structure
The three-dimensional packing of chromatin is an important parameter affecting gene expression
Chromatin is a very dynamic structure that can alternate between two conformations
Closed conformation
Chromatin is very tightly packed
Transcription may be difficult or impossible
Open conformation
Chromatin is accessible to transcription factors
Transcription can take place
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

20. One way is through ATP-dependent chromatin remodeling.
Nucleosomes have been shown to change position in cells that normally express a particular gene compared with cells in which the gene is inactive
A key role of some transcriptional activators is to orchestrate changes in chromatin structure
One way is through ATP-dependent chromatin remodeling.
Energy of ATP hydrolysis is used to drive change of nucleosomes
Makes the DNA more or less amenable to transcription
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

21. These effects may significantly alter gene expression
ATP-dependent chromatin remodeling
The energy of ATP is used to alter the structure of nucleosomes and thus make the DNA more accessible
These effects may significantly alter gene expression
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

22. These effects may significantly alter gene expression
ATP-dependent chromatin remodeling
ATP-dependent
chromatin-remodeling
complex
Histone octamers are removed.
(b) Histone eviction
ATP-dependent
chromatin-remodeling
complex
Variant histones
(c) Replacement with variant histones
These effects may significantly alter gene expression
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

23. There are five histone genes
H1, H2A, H2B, H3 and H4
Human genome contains over 70 histone genes
Most encode standard histones
a few have altered amino acid sequences
Some variants are incorporated into a subset of nucleosomes to create specialized chromatin
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

24. ac 5
Lys ac p p
Amino-terminal tail ac
Lys
Ser 15 ac
Ser
Lys 20
Lys ac ac 1
Lys
Lys 15 5 10 20
H2B
H2A
Globular domain ac p ac
Lys 10
Lys m ac m
Ser
Lys ac m
Arg
Arg m 15 m 5
Lys
Lys
Lys ac ac
Ser 20 5 m
Lys
Lys H4 ac 15 20
Lys
Ser H3 10
(a) Examples of histone modifications
Core histone
protein
COCH3
Histone
acetyltransferase
COCH3
Histone
deacetylase
COCH3
Acetyl
group
DNA is less tightly bound
to the histone proteins
(b) Effect of acetylation
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

25. A common pattern of nucleosome organization-2 -1
NFR +1 +3 +3
NFR
Nucleosome
positions:
DNA
Transcriptional start site
Transcriptional termination site
A nucleosome-free region (NFR) is found at the beginning and end of many
genes. Nucleosomes tend to be precisely positioned near the beginning and
end of a gene, but are less regularly distributed elsewhere.
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

26. DNA Methylation
DNA methylation is a change in chromatin structure that silences gene expression
Carried out by the enzyme DNA methyltransferase
It is common in some eukaryotic species, but not all
Yeast and Drosophila have little DNA methylation
Vertebrates and plants have abundant DNA methylation
In mammals, ~ 2 to 7% of the DNA is methylated
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

27. Only one strand is methylated Both strands are methylated

28. DNA methylation usually inhibits the transcription of eukaryotic genes
Especially when it occurs in the vicinity of the promoter
In vertebrates and plants, many genes contain CpG islands near their promoters
These CpG islands are 1,000 to 2,000 nucleotides long
Contain high number of CpG sites
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

29. In housekeeping genes
The CpG islands are unmethylated
Genes tend to be expressed in most cell types
In tissue-specific genes
The expression of these genes may be silenced by the methylation of CpG islands
Methylation may change binding of transcription factors
Methyl-CpG-binding proteins may recruit factors that lead to compaction of the chromatin
DNA Methylation is Heritable

30. Transcriptional silencing via methylation
Transcriptional activator binds to unmethylated DNA
This would inhibit the initiation of transcription
Transcriptional silencing via methylation
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

31. Transcriptional silencing via methylation
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

32. INSULATORS
Since eukaryotic gene regulation can occur over long distances, it is important to limit regulation to one particular gene, but not to neighboring genes
Insulators are segments of DNA that insulates a gene from the regulatory effects of other genes
Some act as barriers to chromatin remodeling
Others block the effects of enhancers
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

33. (a) Insulators as a barrier to changes in chromatin structure
Proteins that bind
to insulators ac ac ac ac ac ac ac ac
Nonacetylated
DNA
Nonacetylated
DNA
Gene within an
acetylated region
of DNA
Insulator
Insulator
(a) Insulators as a barrier to changes in chromatin structure
Protein bound
to an insulator
The insulator prevents
the enhancer for gene A
from activating the
expression of gene B.
Gene A
Enhancer
Gene B
Insulator
(b) Insulator that blocks the effects of a neighboring enhancer
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

34. REGULATION OF RNA PROCESSING AND TRANSLATION
In eukaryotic species, it is common for gene expression to be regulated at the RNA level
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

35. Alternative Splicing
One very important biological advantage of introns in eukaryotes is the phenomenon of alternative splicing
Alternative splicing refers to the phenomenon that pre-mRNA can be spliced in more than one way
In most cases, large sections of the coding regions are the same resulting in two alternative versions of a protein that have similar functions
Nevertheless, there will be enough differences in amino acid sequences to provide each protein with its own unique characteristics
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

36. Alternative Splicing
The degree of splicing and alternative splicing varies greatly among different species
Baker’s yeast contains about 6,300 genes
~ 300 (i.e., 5%) encode mRNAs that are spliced
Only a few of these 300 have been shown to be alternatively spliced
Humans contain ~ 25,000 genes
Most of these encode mRNAs that are spliced
It is estimated that about 70% are alternatively spliced
Note: Certain mRNAs can be alternatively spliced to produce dozens of different mRNAs
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

37. Alternative Splicing
Alternative splicing for a gene that encodes a-tropomyosin
This protein functions in the regulation of cell contraction
It is found in
Smooth muscle cells (uterus and small intestine)
Striated muscle cells (cardiac and skeletal muscle)
Also in many types of nonmuscle cells at low levels
The different cells of a multicellular organism regulate their contraction in subtly different ways
One way to accomplish this is to produce different forms of a-tropomyosin by alternative splicing
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

38. Alternative ways that the rat a-tropomyosin pre-mRNA can be spliced
Found in the mature mRNA from all cell types
Not found in all mature mRNAs
These alternatively spliced versions of a-tropomyosin vary in function to meet the needs of the cell type in which they are found
Alternative ways that the rat a-tropomyosin pre-mRNA can be spliced

39. Alternative Splicing Alternative splicing is not a random event
The specific pattern of splicing is regulated in a given cell
It involves proteins known as splicing factors
These play a key role in the choice of splice sites
One example of splicing factors is the SR proteins
At their C-terminal end, they have a domain that is rich in serine (S) and arginine (R)
It is involved in protein-protein recognition
At their N-terminal end, they have an RNA-binding domain
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

40. This can occur in two ways
The spliceosome recognizes the 5’ and 3’ splice sites and removes the intervening intron
Splicing factors modulate the ability of spliceosomes to recognize or choose the splice sites
This can occur in two ways
1. Some splicing factors inhibit the ability of a spliceosome to recognize a splice site
2. Some splicing factors enhance the ability of a spliceosome to recognize a splice site
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

41. The role of splicing factors during alternative splicing
Known as exon skipping
The role of splicing factors during alternative splicing
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

42. The role of splicing factors during alternative splicing
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

43. Stability of mRNA The stability of eukaryotic mRNA varies considerably
Several minutes to several days or even months
The stability of mRNA can be regulated so that its half-life is shortened or lengthened
This will greatly influence the mRNA concentration
And consequently gene expression
Factors that can affect mRNA stability include
1. Length of the polyA tail
2. Destabilizing elements
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

44. 1. Length of the polyA tail
Most newly made mRNA have a polyA tail that is about 200 nucleotides long
It is recognized by polyA-binding protein
Which binds to the polyA tail and enhances stability
As an mRNA ages, its polyA tail is shortened by the action of cellular nucleases
The polyA-binding protein can no longer bind if the polyA tail is less than 10 to 30 adenosines long
The mRNA will then be rapidly degraded by exo- and endonucleases
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

45. 5’-untranslated region 3’-untranslated region
2. Destabilizing elements
Found especially in mRNAs that have short half-lives
These elements can be found anywhere on the mRNA
However, they are most common at the 3’ end between the stop codon and the polyA tail
AU-rich element (ARE)
Recognized and bound by cellular proteins
These proteins influence mRNA degradation
5’-untranslated region
3’-untranslated region
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

46. RNA Interference Mediated by Micro RNAs
microRNAs (miRNAs) cause RNA interference
encoded by genes in eukaryotic organisms
genes do not encode a protein
give rise to small RNA molecules, typically 21 to 23 nucleotides
Silence expression of specific mRNAs
In humans, approximately 200 genes encoding miRNAs have been identified
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

47. Mechanism of RNA interference

48. Initiation Factors and the Rate of Translation
Modulation of translation initiation factors is widely used to control fundamental cellular processes
Under certain conditions, it is advantageous for a cell to stop synthesizing proteins
Viral infection
So that the virus cannot manufacture viral proteins
Starvation
So that the cell conserves resources
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

49. Iron Assimilation and Translation
Regulation of iron assimilation provides an example how the translation of specific mRNAs is modulated
Iron is an essential element for the survival of living organisms
It is required for the function of many different enzymes
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

50. Protein that carries iron through the bloodstream
A hollow spherical protein
Prevents toxic buildup of too much iron in the cell
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

51. Iron is a vital yet potentially toxic substance
So mammalian cells have evolved an interesting way to regulate iron assimilation
An RNA-binding protein known as the iron regulatory protein (IRP) plays a key role
It influences both the ferritin mRNA and the transferrin receptor mRNA
This protein binds to a regulatory element within the mRNA known as the iron response element (IRE)
IRE is found in the 5’-UTR in ferritin mRNA
And in the 3’-UTR in transferrin receptor mRNA
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

52. (a) Regulation of ferritin mRNA
Ferritin translation is inhibited by low iron, but not by high iron
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

53. (b) Regulation of transferrin receptor mRNA
Increased stability of mRNA means more translation
mRNA is degraded and cannot be translated
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

54. Transcriptional Activation
Silent: Many genes are flanked by nucleosome-free regions (NFR)
and well-positioned nucleosomes. -2 -1
NFR +1 +2
NFR
Enhancer
Transcriptional
start site
Transcriptional
termination site
Binding of activators:
Activator proteins bind to enhancer
sequences. The enhancers may
be close to the transcriptional start
site (as shown here) or they may
be far away.
Activator -2 -1 +1 +2
Enhancer
Chromatin remodeling and
histone modification:
Activator proteins recruit chromatin
remodeling complexes, such as
histone modifying
enzymes such as histone
acetyltransferase. Nucleosomes
may be moved, and histones may
be evicted. Some histones are
subjected to covalent modification,
such as acetylation.
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

55. Transcriptional Activation
Histone acetyltransferase -2 +2 AC AC AC
SWI/
SNF AC AC
Formation of the pre-initiation
complex:
General transcription factors and
RNA polymerase II are able to bind
to the core promoter and form a
pre-initiation complex. -2 +2 AC AC AC AC AC
Elongation:
During elongation, histones ahead
of the open complex are covalently
modified by acetylation and evicted or
partially displaced. Behind the
open complex, histones are
deacetylated and become tightly
bound to the DNA.
Pre-initiation complex
Deacetylated histones -2 -1 +1 +2
Pre-mRNA AC AC AC AC
Open complex
Evicted histone
proteins
Chaperone
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

56. Steroid Hormones and Regulatory Transcription Factors
Cells respond to steroid hormones in different ways
Glucocorticoids
These influence nutrient metabolism in most cells
They promote glucose utilization, fat mobilization and protein breakdown
Gonadocorticoids
These include estrogen and testosterone
They influence the growth and function of the gonads
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display