1. Chapter 11 Lecture and Animation Outline
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2. Catabolite activator protein (blue) binding to the DNA double helix (orange and white) to activate transcription

3. Chapter 11 Gene Regulation
Chapter Outline:
Overview of Gene Regulation
Regulation of Transcription in Bacteria
Regulation of Transcription in Eukaryotes: Roles of Transcription Factors
Regulation of Transcription in Eukaryotes: Changes in Chromatin Structure and DNA Methylation
Regulation of RNA Processing and Translation in Eukaryotes

4. Overview of Gene Regulation
Gene regulation refers to the ability of cells to control their level of gene expression
Most genes are regulated to ensure proteins are produced at the correct time and amount
Saves energy by producing only when needed
Constitutive genes are unregulated and have essentially constant levels of expression

5. Bacterial gene regulation
Responds to changes in the environment
ex: Escherichia coli and lactose
When lactose is available, two proteins are made:
lactose permease – transports lactose into the cell
β-galactosidase – breaks down lactose
When lactose levels drop, the proteins are not made

7. Living organisms use energy
BIOLOGY PRINCIPLE
Living organisms use energy
Gene regulation provides a way for cells to avoid wasting energy and to make proteins only when they are needed.
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8. Eukaryotic gene regulation
Necessary to produce different cell types in an organism – cell differentiation
All of the organism’s cells contain the same genome but express different proteomes due to gene regulation
Different proteins
Different amounts of the same protein

9. (a) Skeletal muscle cell (b) Neuron (c) Skin cell
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118 μm
179 μm
71 μm
(a) Skeletal muscle cell
(b) Neuron
(c) Skin cell
a: © Ed Reschke/Getty Images; b: © Triarch/Visuals Unlimited; c: © SIU BioMed/Custom Medical Stock Photo

10. Developmental gene regulation in mammals
ex: Hemoglobin in fetal vs. adult humans
Fetal human stage characterized by continued refinement of body parts and a large increase in size
Gene regulation determines which globin polypeptides are made to become functional hemoglobin
Fetal hemoglobin has a higher affinity for oxygen than adult hemoglobin
Helps fetus to harvest oxygen from maternal blood

11. 41 weeks 8 weeks Fertilization Embryo Fetus Adult Embryo Fetus Adult
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41 weeks
8 weeks
Fertilization
Embryo
Fetus
Adult
Embryo
Fetus
Adult
Hemoglobin
protein
2 ζ-globins
2 ε-globins
2 α-globins
2 β-globins
2 α-globins
2 β-globins
Oxygen
affinity
Highest
High
Moderate
Gene expression
α-globin gene
β-globin gene
γ-globin gene
ζ-globin gene
ε-globin gene
Off On On
Off
Off On
Off On
Off On
Off
Off On
Off
Off

12. Living organisms grow and develop
BIOLOGY PRINCIPLE
Living organisms grow and develop
Gene regulation is an important process that allows organisms to proceed through developmental stages.
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13. Gene regulation occurs at different points
Bacterial gene regulation
Most commonly occurs at the level of transcription
Also can control rate of translation
Can be regulated at protein or post-translation level

14. Eukaryotic gene regulation
Transcriptional regulation is common
RNA processing
Translation
Post-translation

15. Regulation of Transcription in Bacteria
Involves regulatory transcription factors
Bind to DNA in the vicinity of a promoter and affect transcription of one or more nearby genes
Repressors inhibit transcription
Negative control
Activators increase the rate of transcription
Positive control

17. Operon
Operon in bacteria is a cluster of genes under transcriptional control of one promoter
Regulatory region called operator
Transcribed into mRNA as polycistronic mRNA – encodes more than one protein
Allows coordinated regulation of a group of genes with a common function

18. lac operon In E. coli contains genes for lactose metabolism
lacP – lac promoter
Three structural genes
lacZ – β-galactosidase
lacY – lactose permease
lacA – galactosidase transacetylase

19. Near the lac promoter are two regulatory sites
lacO – operator – provides binding site for repressor protein
CAP site – activator protein binding site
lacI gene – codes for lac repressor
Considered a regulatory gene since its sole function is to regulate other gene’s expression
Has its own promoter (not part of lac operon)

20. The lac operon is under negative control by a repressor protein
In the 1950s, Jacob and Monod studied a phenomenon called enzyme adaptation
An enzyme appears in a cell only after exposure to the substrate
Exposing E. coli to lactose increased levels of lactose enymes
They discovered the lac repressor – that binds the operator to block transcription of lac genes
When lactose is in the environment, the lac repressor binds a byproduct of it and then cannot bind DNA, allowing transcription

22. When lactose is absent
Lac repressor protein binds the lac operator site and blocks transcription
RNA polymerase can bind but not move forward

23. When lactose is present
Allolactose binds lac repressor to block DNA binding
Process is induction – the lac operon is “inducible”

24. Lac operon also under positive control by activator protein
CAP (catabolite activator protein) is an activator
Catabolite repression – glucose, a catabolite, represses lac operon
Small effector molecule, cAMP, binds to activator protein called catabolite activator protein (CAP) or cAMP receptor protein (CRP)
Operon is turned off when CAP is not bound
Glucose inhibits production of cAMP and so prevents binding of CAP to DNA

25. Gene regulation involving CAP and cAMP an example of positive control
When cAMP binds to CAP, complex binds to CAP site near lac promoter
Resulting bend in DNA enhances RNA polymerase binding which increases transcription

26. When both lactose and glucose are high, the lac operon is shut off
Glucose uptake causes cAMP levels to drop
CAP does not activate transcription
Bacterium uses one sugar at a time, glucose

27. When lactose is high and glucose is low, the lac operon is turned on
Allolactose levels rise and prevent lac repressor from binding to operator
CAP is bound to the CAP site
Bacterium uses lactose

28. When lactose is low and glucose is high or low, the lac operon is shut off
Under low lactose conditions, lac repressor prevents transcription of lac operon

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30. trp operon
In E. coli, encodes enzymes required to make amino acid tryptophan
Regulated by a repressor protein encoded by trpR gene
Binding of repressor to trp operator site inhibits transcription
When tryptophan levels are low, trp repressor cannot bind to operator site and operon genes transcribed
When tryptophan levels are high, tryptophan turns off the trp operon
Tryptophan acts as a small repressor molecule or corepressor

32. lac repressor binds to its operator in the absence of its small effector molecule
Inducible – allolactose induces transcription
Operons for catabolism are often inducible
Genes turned off unless appropriate substance available
trp repressor binds to its operator only in the presence of its small effector molecule
Repressible – tryptophan represses transcription
Operons for anabolism are often repressible
When enough of the product is present, genes are turned off to prevent overproduction

33. Some of same principles as in prokaryotes
Regulation of Transcription in Eukaryotes: Roles of Transcription Factors
Some of same principles as in prokaryotes
Activator and repressor proteins influence ability of RNA polymerase to initiate transcription
Many regulated by small effector molecules
Many important differences
Genes almost always organized individually
Regulation more intricate

34. Combinatorial control
Activators – activator proteins stimulate RNA polymerase to initiate transcription
Repressors – repressor proteins inhibit RNA polymerase from initiating transcription
Modulation – small effector molecules, protein–protein interactions, and covalent modifications can modulate activators and repressors
Chromatin – activator proteins promote loosening up of the region in the chromosome where a gene is located, making it easier for RNA polymerase to transcribe the gene
DNA Methylation – usually inhibits transcription, either by blocking an activator protein or by recruiting proteins that make DNA more compact

35. Three features of most promoters
TATA box
5’ – TATAAAA – 3’
25 base pairs upstream from transcriptional start site
Determines precise starting point for transcription
Transcriptional start site
Where transcription begins
With TATA box forms core promoter
By itself results in low level basal transcription
Regulatory or response elements
Recognized by regulatory proteins that control initiation of transcription
Enhancers and silencers

37. Preinitiation complex
RNA Pol II transcribes genes that encode proteins
Requires five different general transcription factors (GTFs) to initiate transcription
Come together at the core promoter

38. Transcriptional regulation
Activators – bind to DNA regions called enhancers
Repressors – bind to DNA regions called silencers
Regulate rate of transcription of a nearby gene
Most do not bind directly to RNA polymerase II, instead they influence the function of GTFs

40. Regulation of Transcription in Eukaryotes: Changes in Chromatin Structure and DNA Methylation
DNA is associated with proteins to form compact chromatin
Chromatin packing affects gene expression
Transcription is difficult or impossible in the closed conformation of tightly packed chromatin
Access to the DNA is allowed in the loosely packed open conformation

41. Some activators diminish DNA compaction near a gene
Recruit proteins to loosen DNA compaction – the chromatin remodeling complex
Histone acetyltransferases attach acetyl groups to histone proteins so they don’t bind DNA as tightly

43. Histone code
Many different amino acids in the amino terminal tails of histone proteins are subject to several types of covalent modification
Pattern of modifications (histone code) affects degree of chromatin compaction

44. Nucleosome-free regions
A nucleosome-free region (NFR) is found at the beginning and end of many genes
Less regular distribution elsewhere

45. DNA Methylation DNA methyltransferase attaches methyl groups
Common in some eukaryotes but not all
In mammals, 5% of DNA is methylated
Usually inhibits transcription
CpG islands near promoters in vertebrates and plants
Cytosine and guanine connected by phosphodiester bonds
Unmethylated CpG islands are correlated with active genes
Repressed genes contain methylated CpG islands

46. Methylation can inhibit transcription two ways:
The Methylation of CpG islands may prevent an activator from binding to an enhancer element
Converting chromatin from an open to a closed conformation
Methyl-CpG-binding proteins bind to methylated sequences and recruit proteins that condense the chromatin

48. Regulation of RNA Processing and Translation in Eukaryotes
Unlike bacteria, gene expression is commonly regulated at the level of RNA processing and translation
Added benefits include…
Produce more than one mRNA transcript from a single gene (gene encodes 2 or more polypeptides)
Faster regulation achieved by controlling steps after RNA transcript made

49. Alternative splicing of pre-mRNAs
In eukaryotes, a pre-mRNA transcript is processed before it becomes a mature mRNA
When a pre-mRNA has multiple introns and exons, splicing may occur in more than one way
Alternative splicing causes mRNAs to contain different patterns of exons.
Allows same gene to make different proteins
At different stages of development
In different cell types
In response to a change in the environmental conditions

50. Linear order of exons is maintained in both versions
Products usually have similar functions, because most of the amino acid sequence will be the same
Alternative splicing produces proteins with specialized characteristics
Advantage of alternative splicing:
Different polypeptides from a single gene
Bigger proteome with the same size genome

51. Alternative splicing tends to be more prevalent in complex eukaryotic species
Alternative splicing increases the proteome size without increasing the total number of genes
For organisms to become more complex, as in higher plants and animals, evolution has produced more complex proteomes
General trend is that less complex organisms tend to have fewer genes
Frequency of alternative splicing increases with more biological complexity

53. miRNAs and siRNAs
Both are short (~22 nts) RNA molecules that silence the expression of mRNAs
miRNA precursors are encoded by genes
They form a hairpin structure
Usually partly complement mRNA to bind and inhibit
siRNAs are derived from a virus, or created by a researcher for an experiment
Usually a perfect match to mRNA, lead to degradation
RISC complex causes mRNA degradation
Originally called RNA interference (RNAi)

54. Synthesized as pre-miRNA Cut by dicer to release miRNA
Associates with cellular proteins to become RNA-induced silencing complex (RISC)
Upon binding, either
mRNA degraded
Or, RISC inhibits translation
Either way, mRNA is silenced

55. Structure determines function
BIOLOGY PRINCIPLE
Structure determines function
Because the structure of an miRNA or siRNA is complementary to an mRNA, it is able to inhibit the function of the mRNA.

56. Iron toxicity in mammals
Another way to regulate translation involves RNA-binding proteins that directly affect translational initiation
Iron is a vital cofactor for many cellular enzymes
However, it is toxic at high levels
To prevent toxicity, mammalian cells synthesize a protein called ferritin, which forms a hollow, spherical complex that can store excess iron
mRNA that encodes ferritin is controlled by an RNA binding protein known as iron regulatory protein (IRP)

57. When iron in cytosol is low and ferritin is not needed, IRP binds to a response element within the ferritin mRNA known as the iron regulatory element (IRE)
Binding of IRP to the IRE inhibits translation of the ferritin mRNA

58. When iron is abundant in the cytosol, the iron binds directly to IRP and prevents it from binding to the IRE
Ferritin mRNA is translated to make more ferritin protein
Faster than transcriptional regulation, which would require gene activation and transcription prior to the synthesis of more ferritin protein