1. Chapter 14 Gene Regulation

2. Gene Regulation
Cells differ because gene expression is regulated, and only certain subsets of the total genetic information are expressed in any given cell
Gene expression involves three basic steps, each of which is regulated in many ways:
Transcribing the gene to form messenger RNA (mRNA)
Translating mRNA into protein
Activating the protein

3. Control Mechanisms Mechanisms that regulate gene expression include:
control of the amount of mRNA transcribed
rate of translation of mRNA
activity of the protein product
Control mechanisms use various signals, some originating within the cell and others coming from other cells or from the environment

4. 14.1 GENE REGULATION IN BACTERIA AND EUKARYOTES
LEARNING OBJECTIVE:
Explain why bacterial and eukaryotic cells have different mechanisms of gene regulation

5. Bacterial Gene Regulation
The main requirement of bacterial gene regulation is production of enzymes and other proteins when needed
Transcriptional-level control is the most efficient mechanism – bacteria rarely regulate enzyme levels by degrading proteins
Related genes are organized into groups that are rapidly turned on and off as units

6. Eukaryotic Gene Regulation
Because a single gene is regulated in different ways in different types of cells, eukaryotic gene regulation is complex
Transcriptional-level control predominates, but control at other levels of gene expression is also very important, especially in multicellular organisms
In many instances, pre-formed enzymes and other proteins are rapidly converted from an inactive to an active state
In multicellular organisms, each type of cell has certain genes that are active and other genes that may never be used

7. KEY CONCEPTS 14.1
Cells regulate which genes will be expressed and when
Gene regulation in bacteria is primarily in response to stimuli in the environment; gene regulation in eukaryotes helps to maintain homeostasis

8. 14.2 GENE REGULATION IN BACTERIA
LEARNING OBJECTIVES:
Define operon and explain the functions of the operator and promoter regions
Distinguish among inducible, repressible, and constitutive genes
Differentiate between positive and negative control, and show how both types of control operate in regulating the lac operon
Describe the types of posttranscriptional control in bacteria

9. Bacterial Gene Expression
The E. coli bacterium has some genes that encode proteins that are always needed (constitutive genes), and some that encode proteins that are needed only in certain conditions
Example:
E. coli living in the colon of a calf need enzymes that digest the milk sugar lactose
E. coli living in the colon of an adult cow are not exposed to milk and do not need lactose-digesting enzymes

10. Enzyme Activity Cell metabolic activity is controlled in two ways:
By regulating the activity of certain enzymes (how effectively an enzyme molecule works)
By regulating the number of enzyme molecules present in each cell
Bacteria respond rapidly to changing environmental conditions because functionally related genes are regulated together in gene complexes called operons

11. Operons in Bacteria
The DNA coding sequences for all three enzymes needed to digest lactose are linked as a unit on bacterial DNA and are controlled by a common mechanism
Each enzyme-coding sequence is a structural gene
operon
A gene complex consisting of a group of structural genes with related functions plus the closely linked DNA sequences responsible for controlling them

12. The lac Operon
The structural genes of the lactose operon (lac operon)—lacZ, lacY, and lacA—code for three enzymes
RNA polymerase binds to a promoter region upstream from the coding sequences
A single mRNA molecule is transcribed that contains the coding information for all three enzymes
mRNA synthesis is controlled by a single molecular “switch,” the operator

13. The lac Operator
The operator is a sequence of bases upstream from the first structural gene in the operon
In the absence of lactose, a repressor protein (lactose repressor) binds tightly to the operator
RNA polymerase binds to the promoter but is blocked from transcribing the protein-coding genes of the lac operon

14. The Lactose Repressor
Lactose repressor protein is encoded by a repressor gene located upstream from the promoter site
The repressor gene is a structural gene that is constitutively expressed (constantly transcribed)
In the absence of lactose, the repressor protein binds specifically to the lac operator sequence, and transcription of the lac operon is suppressed

15. Turing On Transcription
Lactose “turns on” (induces ) transcription of the lac operon
The lactose repressor protein contains a second functional region (an allosteric site) separate from its DNA-binding site
In the presence of lactose, the allosteric site binds to the inducer (allolactose) which inactivates the repressor protein
RNA polymerase is able to actively transcribes the structural genes of the operon

16. The lac Operon

17. Precursor of repressor protein
lac operon
Repressor gene
Promoter
Operator
lacZ
lacY
lacA
DNA
Transcription
Repressor protein
mRNA
Precursor of repressor protein
Figure 14.2: The lac operon.
Translation
Ribosome
(a) Lactose absent. In the absence of lactose, a repressor protein, encoded by an adjacent gene, binds to a region known as the operator, thereby blocking transcription of the structural genes.
Fig. 14-2a, p. 310

18. Precursor of repressor protein
lac operon
Repressor gene
Promoter
lacZ
Operator
lacA
lacY
DNA
Repressor protein
Precursor of repressor protein
Translation
Transcription
mRNA
Ribosome
Figure 14.2: The lac operon.
(a) Lactose absent. In the absence of lactose, a repressor protein, encoded by an adjacent gene, binds to a region known as the operator, thereby blocking transcription of the structural genes.
Stepped Art
Fig. 14-2a, p. 310

19. Repressor gene RNA polymerase
lac operon
Repressor gene
Promoter Operator
lacZ lacY lacA
RNA polymerase
Transcription
mRNA
mRNA
Inducer (allolactose)
Translation
Transacetylase
Figure 14.2: The lac operon.
Lactose permease
Repressor protein (inactive)
β -galactosidase
Enzymes for lactose metabolism
(b) Lactose present. When lactose is present, it is converted to allolactose, which binds to the repressor at an allosteric site, altering the structure of the protein so it no longer binds to the operator. As a result, RNA polymerase is able to transcribe the structural genes.
Fig. 14-2b, p. 310

20. Repressor gene RNA polymerase
lac operon
Repressor gene
RNA polymerase
Promoter Operator
lacZ lacY lacA
mRNA
Inducer (allolactose)
Repressor protein (inactive)
Transcription
mRNA
Transacetylase
Translation
Lactose permease
β -galactosidase
Enzymes for lactose metabolism
Figure 14.2: The lac operon.
(b) Lactose present. When lactose is present, it is converted to allolactose, which binds to the repressor at an allosteric site, altering the structure of the protein so it no longer binds to the operator. As a result, RNA polymerase is able to transcribe the structural genes.
Stepped Art
Fig. 14-2b, p. 310

21. Inducible Genes The lac operon an inducible operon
A repressor usually controls an inducible gene or operon by keeping it “turned off ”
The presence of an inducer inactivates the repressor, permitting the gene or operon to be transcribed
Inducible genes or operons usually code for enzymes that break down molecules (catabolic reactions)
This saves the energy cost of making enzymes when no substrates are available

22. Repressible Genes
Repressible operons and genes are usually “turned on” –they are turned off only under certain conditions
Repressible genes usually code for enzymes that synthesize essential biological molecules from simpler materials (anabolic reactions)
The molecular signal for regulating these genes usually is the end product of the anabolic pathway

23. The trp Operon
The tryptophan operon (trp operon) is an example of a repressible system
A repressor gene codes for a repressor protein which is synthesized in an inactive form (it can’t bind to the operator region of the trp operon)
When tryptophan binds to the repressor, the repressor is able to binds to the operator, which switches the operon off – transcription is blocked

24. The trp Operon

25. Repressor gene RNA polymerase
trp operon
Repressor gene
Promoter Operator
trpE trpD trpC trpB trpA
DNA
RNA polymerase
Transcription
mRNA
mRNA
Translation
Repressor protein (inactive)
Enzymes of the tryptophan biosynthetic pathway
Figure 14.4: The trp operon.
Tryptophan
(a) Low intracellular tryptophan levels. Repressor protein is unable to prevent transcription because it cannot bind to the operator.
Fig. 14-4a, p. 312

26. trpE trpD trpC trpB trpA
trp operon
Repressor gene
Promoter Operator
trpE trpD trpC trpB trpA
DNA
Active repressor – corepressor complex
mRNA
Inactive
repressor protein
Figure 14.4: The trp operon.
Tryptophan (corepressor)
(b) High intracellular tryptophan levels. The amino acid tryptophan binds to an allosteric site on the repressor protein, changing its conformation. The resulting active form of the repressor binds to the operator region, blocking transcription of the operon until tryptophan is again required by the cell.
Fig. 14-4b, p. 312

27. Negative Regulators and Positive Regulators
The lac and trp operons are examples of negative control, a regulatory mechanism in which the DNA binding regulatory protein is a repressor that turns off transcription of the gene
Positive control is regulation by activator proteins that bind to DNA and thereby stimulate the transcription of a gene
The lac operon is controlled by both a negative regulator (the lactose repressor) and a positively acting activator protein

28. Positive Control of the lac Operon
A DNA sequence near the promoter binds another regulatory protein, the catabolite activator protein (CAP)
When activated, CAP stimulates transcription of the lac operon and several other bacterial operons
In its active form, CAP is bound by an allosteric site to cyclic AMP (cAMP)
cAMP levels increase as cells become depleted of glucose

29. Positive Control (cont.)
Activated CAP binds to the CAP binding site near the lac operon promoter
Binding strengthens the affinity of the promoter region for RNA polymerase – so the rate of transcriptional initiation accelerates in the presence of lactose
The lac operon is fully active only if lactose is available and intracellular glucose levels are low

30. Positive Control of the lac Operon

31. RNA polymerase – binding RNA polymerase binds poorly
Promoter
RNA polymerase – binding
site
CAP- binding site
Repressor gene
Operator
lacZ lacY lacA
DNA
mRNA
RNA polymerase binds poorly
CAP (inactive)
Figure 14.5: Positive control of the lac operon.
Allolactose
Repressor
protein (inactive)
(a) Lactose high, glucose high, cAMP low. When glucose levels are high, cAMP is low. CAP is in an inactive form and cannot stimulate transcription. Transcription occurs at a low level or not at all.
Fig. 14-5a, p. 313

32. RNA polymerase – binding RNA polymerase binds poorly
Promoter
RNA polymerase – binding
site
CAP- binding site
Repressor gene
lacZ lacY lacA
Operator
DNA
RNA polymerase binds poorly
CAP (inactive)
mRNA
Allolactose
Repressor
protein (inactive)
Figure 14.5: Positive control of the lac operon.
(a) Lactose high, glucose high, cAMP low. When glucose levels are high, cAMP is low. CAP is in an inactive form and cannot stimulate transcription. Transcription occurs at a low level or not at all.
Stepped Art
Fig. 14-5a, p. 313

33. RNA polymerase – binding
Promoter
RNA polymerase – binding
site
Repressor gene
CAP- binding site
Operator
lacZ lacY lacA
DNA
Transcription
RNA polymerase binds efficiently
mRNA
mRNA
CAP
Translation
Galactoside transacetylase
cAMP
Figure 14.5: Positive control of the lac operon.
Lactose permease
Allolactose
Repressor protein (inactive)
β -galactosidase
Enzymes for lactose metabolism
(b) Lactose high, glucose low, cAMP high. When glucose concentrations are low, each CAP polypeptide has cAMP bound to its allosteric site. The active form of CAP binds to the DNA sequence, and transcription becomes activated.
Fig. 14-5b, p. 313

34. RNA polymerase – binding
Promoter
RNA polymerase – binding
site
Repressor gene
CAP- binding site
Operator
lacZ lacY lacA
DNA
RNA polymerase binds efficiently
Transcription
mRNA
CAP
cAMP
mRNA
Repressor protein (inactive)
Allolactose
Lactose permease
Translation
Enzymes for lactose metabolism
Galactoside transacetylase
β -galactosidase
Figure 14.5: Positive control of the lac operon.
(b) Lactose high, glucose low, cAMP high. When glucose concentrations are low, each CAP polypeptide has cAMP bound to its allosteric site. The active form of CAP binds to the DNA sequence, and transcription becomes activated.
Stepped Art
Fig. 14-5b, p. 313

35. Binding of CAP to DNA

36. Transcriptional Control in Bacteria
Table 14-1, p. 314

37. Transcription of Constitutive Genes
Constitutive genes are continuously transcribed, but not all are transcribed at the same rate
Promoter elements control transcription rate
“Strong” promoters bind RNA polymerase more frequently and transcribe more mRNA molecules than those with “weak” promoters

38. Posttranscriptional Regulation in Bacteria
Posttranscriptional controls are regulatory mechanisms that occur after transcription
Translational controls regulate the rate at which an mRNA molecule is translated
Posttranslational controls act as switches that activate or inactivate one or more existing enzymes
Example: feedback inhibition

39. KEY CONCEPTS 14.2
Gene regulation in bacteria occurs primarily at the level of transcription; regulation of transcription can be either positive or negative

40. 14.3 GENE REGULATION IN EUKARYOTIC CELLS
LEARNING OBJECTIVES:
Discuss the structure of a typical eukaryotic gene and the DNA elements involved in regulating that gene
Give examples of some of the ways eukaryotic DNA-binding proteins bind to DNA
Illustrate how a change in chromosome structure may affect the activity of a gene
Explain how a gene in a multicellular organism may produce different products in different types of cells
Identify some of the types of regulatory controls that operate in eukaryotes after mature mRNA is formed

41. Gene Regulation in Eukaryotes
Eukaryotes have transcriptional, posttranscriptional, translational, and posttranslational gene controls that allow individual cells, tissues, and organs to function
Eukaryotic genes are not typically arranged in operon-like clusters – each eukaryotic gene has specific regulatory sequences that control transcription
Some genes are constitutive, others are inducible

42. Inducible Genes
Some inducible genes respond to environmental threats such as heavy-metal ingestion, viral infection, and heat shock
Example: Some heat-shock proteins are molecular chaperones that help proteins fold into their proper shape
Some genes are inducible only during certain periods in the life of the organism (controlled by temporal regulation)
Some genes are under tissue-specific regulation

43. Chromosome Organization
Chromosome organization affects expression of some genes
In multicellular eukaryotes, only a subset of genes in any specific type of cell is active – in many cases, inactivated genes are irreversibly dormant
Some inactive genes lie in highly compacted heterochromatin, such as the inactive X chromosomes in female mammals (Barr body)

44. Chromosome Organization (cont.)
Active genes are associated with euchromatin, which is loosely packed and allows interaction with transcription factors and other regulatory proteins
Chromatin can be changed between heterochromatin and euchromatin by chemically modifying histones, the proteins that associate with DNA to form nucleosomes

45. Regulation of Chromatin

46. Gene Inactivation by DNA Methylation
Once a gene has been turned off by some other means, DNA methylation ensures it will remain inactive
Enzymes add methyl groups to certain cytosine nucleotides in DNA, which blocks transcription
DNA methylation can be maintained for multiple generations, as in genomic imprinting (parental imprinting), in which expression of certain genes is determined by whether the allele is inherited from the female or the male parent

47. Genomic Imprinting and Epigenetics
Methylation provides a mechanism for epigenetic inheritance (changes in how a gene is expressed)
New phenotypic traits can arise from epigenetic changes despite the fact that the nucleotide sequence of the gene itself has not changed
Tumor suppressor genes that inhibit cell division (and cancer) are sometimes inactivated epigenetically, resulting in cancer

48. Epigenetic Variation in the agouti Gene
Figure 14.9: Epigenetic variation in the agouti gene in mice.
The DNA of these mice is almost identical, yet their coat colors and body shapes vary significantly. These mice differ in the presence or absence of epigenetic “marks,” such as methyl groups on DNA in the region of a gene known as agouti.
Fig. 14-9, p. 316

49. Increasing the Number of Copies of a Gene
A single gene may not provide enough copies of its mRNA to meet the cell’s needs
Genes required only by a small group of cells may be selectively replicated by gene amplification

50. Drosophila chorion gene
Gene amplification by repeated DNA replication of chorion gene region
Figure 14.10: Regulation of transcription: gene amplification.
In Drosophila, multiple replications of a small region of the chromosome amplify the chorion (eggshell) protein genes. Replication is initiated at a discrete chromosome origin of replication (top pink box) for each copy of the gene that is produced. Replication is randomly terminated, resulting in a series of forked structures in the chromosome.
Chorion gene in ovarian cell
Fig , p. 317

51. Functional Promoter Elements
Transcription requires a base pair where transcription begins (transcription initiation site or start site) plus a sequence of bases to which RNA polymerase binds (promoter)
Certain promoter elements have regulatory functions and facilitate expression of the gene – mutations in these elements reduce the rate of transcription
Example: The TATA box, located about 25 to 30 base pairs upstream from the transcription initiation site

52. Enhancers and Silencers
Enhancers and silencers regulate a gene on the same DNA molecule from very long distances
enhancers
DNA sequences that help form an active transcription initiation complex
silencers
DNA sequences that can decrease transcription

53. Elements That Regulate Transcription
Eukaryotic regulatory elements include the promoter and various enhancers and silencers

54. TATA box DNA double helix Upstream Downstream Silencer Enhancer
Promoter
Gene
Figure 14.11: Regulation of transcription: eukaryotic elements.
Eukaryotic regulatory elements include the promoter and various enhancers and silencers. The promoter elements help determine the basic level of transcription that normally occurs, whereas the enhancers (and silencers) have the potential to greatly increase (or decrease) that rate of transcription. (For simplicity, the DNA double helix is represented as a rod.)
Fig , p. 317

55. Transcription Factors
Many DNA-binding proteins that regulate transcription (transcription factors) have been identified in humans
Each eukaryotic transcription factor has a DNA-binding domain plus at least one other domain that is either an activator or a repressor of transcription for a given gene

56. Transcription Factors (cont.)
Transcription in eukaryotes requires multiple regulatory proteins that are bound to different parts of the promoter
The general transcriptional machinery binds to the TATA box of the promoter, and is required for RNA polymerase to bind
An activator has at least two functional domains:
A DNA recognition site that binds to an enhancer
An activation site that contacts the target in the general transcriptional machinery

57. Stimulation of Transcription by an Enhancer

58. Enhancer Target proteins RNA polymerase TATA box DNA
Figure 14.13: Regulation of transcription: the stimulation of transcription by an enhancer.
(a) Low rate of transcription. This gene is transcribed at a basic level when the general transcriptional machinery, including RNA polymerase, is bound to the promoter.
Fig a, p. 319

59. Activator (transcription factor) TATA box DNA
Enhancer
Activator (transcription factor)
TATA box
DNA
(b) High rate of transcription. A transcription factor that functions as an activator binds to an enhancer. The intervening DNA forms a loop, allowing the transcription factor to contact one or more target proteins in the general transcriptional machinery, thereby increasing the rate of transcription. This diagram is highly simplified, and many more target proteins than the two shown are involved.
Figure 14.13: Regulation of transcription: the stimulation of transcription by an enhancer.
Fig b, p. 319

60. Posttranscriptional Control
Eukaryotic mRNA molecules are capped, polyadenylated (addition of a poly-A tail), spliced, and then transported from the nucleus to the cytoplasm to initiate translation
Each of these events represent potential control points
Example: When the short poly-A tail of an mRNA is elongated, mRNA becomes activated and is translated

61. Pre-mRNA Processing
Alternative splicing patterns allow the same gene to produce one type of protein in one tissue and a different type of protein in another tissue
Typically, such a gene includes at least one segment that can be either an intron or an exon

62. Alternative Splicing

63. Functional mRNA in tissue A Functional mRNA in tissue B
Potential splice sites
Exon
or intron
Exon
Intron
Exon
pre-mRNA
Alternative splicing
Exon
Exon
Exon
Exon
Exon
Figure 14.14: Regulation of mRNA processing: alternative splicing.
In some cases, a pre-mRNA molecule is processed in more than one way to yield two or more mature mRNAs, each of which encodes a related, but different, protein. In this generalized example, the gene contains a segment that can be an exon in tissue A (left) but an intron in tissue B (right).
Functional mRNA in tissue A
Functional mRNA in tissue B
Fig , p. 319

64. Stability of mRNA Molecules
Controlling the lifespan of an mRNA molecule regulates the number of protein molecules translated from it
In some cases, mRNA stability is under hormonal control

65. Posttranslational Chemical Modifications
Another way to control gene expression is by regulating the activity of the gene product
In proteolytic processing, proteins are synthesized as inactive precursors, which are converted to an active form by removal of a portion of the polypeptide chain
Chemical modification (adding or removing functional groups) reversibly alters the activity of an enzyme
- Enzymes that add phosphate groups are called
kinases; those that remove them are phosphatases

66. Posttranslational Chemical Modifications (cont.)
Posttranslational control of gene expression can also involve protein degradation
Proteins that are selectively targeted for degradation in a proteasome are covalently bonded to ubiquitin
Proteasomes are large macromolecular structures that recognize ubiquitin tags
Proteases (protein-degrading enzymes) associated with proteasomes hydrolyze peptide bonds

67. Protein Degradation
As protein is degraded, ubiquitin molecules are released intact and reused

68. Gene Regulation in Eukaryotic Cells

69. Figure 14.7: Gene regulation in eukaryotic cells.
Cytosol
Nucleus
Regulation of chromatin
DNA must be unpacked; heterochroma- tin cannot be transcribed.
Methylated DNA is inaccessible to transcription machinery; demethylated DNA can be transcribed.
Regulation of transcription
Selective transcription: promoter and enhancer elements in DNA interact with protein transcription factors to activate or repress transcription.
Regulation of mRNA processing Control mechanisms, such as rate
of intron/exon splicing, regulate mRNA processing.
Alternative splicing of exons produces different proteins from same mRNA.
Regulation of mRNA transport Controlling access to, or efficiency of, transport through nuclear pores regulates mRNA transport from nucleus to cytosol.
Nuclear pore
Figure 14.7: Gene regulation in eukaryotic cells.
Regulation of translation Translational controls determine how often and how long specific mRNA is translated.
Degradation of mRNA Translational controls determine degree to which mRNA is protected from destruction; proteins that bind to mRNA in cytosol affect stability.
Modifications to protein Chemical modifications, such as phosphorylation, affect activity of protein after it is produced.
Degradation of protein
Selective degradation targets specific proteins for destruction by proteasomes.
Fig. 14-7, p. 315

70. KEY CONCEPTS 14.3
Gene regulation in eukaryotes occurs at the levels of transcription, posttranscription, translation, and posttranslation