Regulation of Gene Expression

1
Regulation of Gene Expression

• Chapter 18(8th Ed)

2
The Control of Gene Expression

• An individual bacterium can cope with
  environmental fluctuations by exerting metabolic
  control
• First, cells vary the number of specific enzyme
  molecules by regulating gene expression
• Second, cells adjust the activity of enzymes
  already present (for example, by feedback
  inhibition)

3
Tryptophan Biosynthesis? Both Types of Control

• If tryptophan levels are high, some of the
  tryptophan molecules can inhibit the first enzyme
  in the pathway
• If the abundance of tryptophan continues, the
  cell can stop synthesizing additional enzymes
  in this pathway by blocking transcription of
  the genes for these enzymes.

4
Operon Model for Gene Control

• Proposed by Francois Jacob and Jacques Monod in
  1961
• An operon consists of three elements
• The genes that it controls
• A Promotor region where RNA polymerase first
  binds,
• An operator region between the promotor and the
  first gene which acts as an on-off switch.

5
Repressor Inactive? Operon is On

• By itself, an operon is on and RNA polymerase can
  bind to the promotor and transcribe the genes

Fig. 18.20a
6
Repressor Active? Operon is Off

• Repressor protein, a product of a regulatory
  gene, binds to the operator, it can prevent
  transcription of the operons genes
• Tryptophan act as a corepressor

7
trp Operon

• Repressible Operon, one that is inhibited when a
  specific small molecule binds allosterically to a
  regulatory protein
• At high concentrations of tryptophan , some
  molecules bind as a corepressor to the repressor
  protein
• This activates the repressor and turns the operon
  off.
• At low levels of tryptophan? repressors are
  inactive and the operon is transcribed.

8
Inducible Operon

• Is stimulated when a specific small molecule
  interacts with a regulatory protein
• In inducible operons, the regulatory protein is
  active (inhibitory) as synthesized, and the
  operon is off
• Allosteric binding by an inducer molecule makes
  the regulatory protein inactive, and the operon
  is on.

9
lac Operon

• Genes that code for enzymes? hydrolysis and
  metabolism for lactose
• In the absence of lactose, operon is off ? active
  repressor binds to the operator and prevents
  transcription

10

• When lactose is present in the cell allolactose,
  binds to the repressor.
• This inactivates the repressor, and the lac
  operon can be transcribed

11
Negative Control ?active repressors negative
effects on transcription

• Repressible enzymes
• Function in anabolic pathways, synthesizing end
  products
• When the end product is present in sufficient
  quantities, the cell can allocate its resources
  to other uses.

• Inducible enzymes
• Function in catabolic pathways, digesting
  nutrients to simpler molecules
• Produce the appropriate enzymes only when the
  nutrient is available, the cell avoids making
  proteins that have nothing to do.

12
Positive Gene Control ? activator interacts
directly to switch transcription on

• Even if the lac operon is turned on by the
  presence of allolactose, the degree of
  transcription depends on the concentrations of
  other substrates
• If glucose levels are low, then cyclic AMP
  (cAMP) binds to cAMP receptor protein (CRP) which
  activates transcription

13
Dual Control

• The presence / absence of lactose (allolactose)
  determines if the lac operon is on or off
• Overall energy levels in the cell determine the
  level of transcription?a volume control,
  through CRP
• CRP works on several operons that encode enzymes
  used in catabolic pathways
• If glucose is present and CRP is inactive, then
  the synthesis of enzymes that catabolize other
  compounds is slowed
• If glucose levels are low and CRP is active, then
  the genes which produce enzymes that catabolize
  whichever other fuel is present will be
  transcribed at high levels.

14
DNA Packaging

• Pp 321-322
• Study fig 16.21

15
Overview

• The typical multicellular eukaryotic genome is
  much larger than that of a prokaryote
• Cell specialization ? crucial ?limits the
  expression of many genes to specific cells
• The human genome ?estimated 25,000 genes
  includes an enormous amount of DNA that does not
  code for RNA or protein
• This DNA is elaborately organized
• DNA associated with protein (Histones) to form
  chromatin? chromatin is organized into higher
  organizational levels
• Level of packing is one way that gene expression
  is regulated
• Densely packed areas are inactivated
• Loosely packed areas are being actively
  transcribed.

16
Packaging and Organization

• A. Packaging
• B. Organization
• C. Rearrangement of the Genome

17
Packaging DNA

• Each human chromosome averages about 1.5 x 108
  nucleotide pairs
• If extended, each DNA molecule would be about 4
  cm long, thousands of times longer than the cell
  diameter
• This chromosome and 45 other human chromosomes
  fit into the nucleus
• This occurs through an elaborate, multilevel
  system of DNA packing.
• Each chromosome is
• Is combined with a large amount of protein?
  Chromatin
• Contain an enormous amount of DNA relative to
  their condensed length
• Is ordered into higher structural levels than the
  DNA-protein complex in prokaryotes

18
1st Level of Packaging

• Histone proteins are responsible for the first
  level of DNA packaging
• Their positively charged amino acids bind tightly
  to negatively charged DNA
• The histones are very similar from one eukaryote
  to another and are even present in bacteria
• Unfolded chromatin ? beads on a string, a
  nucleosome, in which DNA winds around a core of
  histone proteins

19

• The beaded string seems to remain essentially
  intact throughout the cell cycle
• Histones leave the DNA only transiently during
  DNA replication
• They stay with the DNA during transcription
• By changing shape and position, nucleosomes allow
  RNA-synthesizing polymerases to move along the
  DNA.

20
Higher Levels of Packaging

• The beaded string coils to form the 30-nm
  chromatin fiber
• This fiber forms looped domains-making a 300-nm
  fiber attached to a scaffold of nonhistone
  proteins

21
Mitotic Chromosome

• The looped domains themselves coil and fold
  forming the characteristic metaphase chromosome
• Packing steps are highly specific and precise ?
  particular genes located in the same places.

22
Interphase Chromatin

• Much less condensed than the chromatin of mitosis
• Present as 10nm- fiber with some compacted
  30nm-fiber
• Of the 30-nm fibers and looped domains remain,
  the discrete scaffold is not present
• The looped domains appear to be attached to the
  nuclear lamina and perhaps the nuclear matrix.
• The chromatin of each chromosome occupies a
  restricted area within the interphase nucleus? so
  they do not become entangled

23
Heterochromatin and Euchromatin

• Heterochromatin ? areas that remain highly
  condensed
• DNA is inaccessible to transcription
• Euchromatin? less compacted areas
• DNA is accessible and available for transcription

24
Eukaryotic Gene Expression

• 18.2 18.3 pp 356-364

25
Regulation of Gene Expression

• In prokaryotes, most of the DNA in a genome codes
  for protein (or tRNA and rRNA), with a small
  amount of noncoding DNA
• In eukaryotes, most of the DNA (about 97 in
  humans) does not code for protein or RNA
• Some noncoding regions are regulatory sequences
• Other are introns
• Even more of it consists of repetitive DNA,
  present in many copies in the genome

26
Cell Differentiation and Differential Gene
Expression

• All organisms regulate which genes are expressed
  at any given time
• During development of a multicellular organism?
  cells undergo a process of specialization in form
  and function ? cell differentiation
• Difference in cell type is due to expression of
  different genes by cells with the same genome
• In each type of differentiated cell
• A unique subset of genes is expressed

27

• Each stage ? a potential control point ,gene
  expression ? turned on or off, speeded up or
  slowed down
• A web of control connects different genes and
  their products
• These levels of control include chromatin
  packing, transcription, RNA processing,
  translation, and various alterations to the
  protein product.

28
Regulation of Chromatin Structure

• Heterochromatin genes ? usually not expressed
• Histone Modifications
• DNA Methylation
• Epigenetic Inheritance

29
Histone Modification

• Addition of an acetyl group -COCH3
• Shape is changed DNA is gripped less tightly

30
DNA Methylation

• Addition of methyl groups (-CH3) to DNA bases
  after DNA synthesis
• Inactive DNA is generally highly methylated
  compared to DNA that is actively transcribed
• For example, the inactivated mammalian X
  chromosome in females is heavily methylated
• Genes are usually more heavily methylated in
  cells where they are not expressed
• Demethylating certain inactive genes turns them
  on
• Once methylated genes stay that way thru.
  successive cell divisions
• This methylation patterns accounts for genomic
  imprinting in which methylation turns off either
  the maternal or paternal alleles of certain genes
  at the start of development.

31
Epigenetic Inheritance

• Traits transmitted by mechanisms not directly
  involving nucleotide sequences
• Chromatin modifications that do not involve
  change in DNA
• May be passed on to future generations

32
Regulation

• Initial control? Chromatin Modification
• Gene is optimally modified? initiation of
  transcription is the most important stage of gene
  regulation

33
Organization of a typical Eukaryotic Genome
34
Regulation of Transcription Initiation

• Transcription Factors
• Enhancers
• Activators

35

• Transcription Factor binds to TATA box
• Additional factors join
• RNA pol II binds? Transcription Initiation Complex

36
Enhancers-distal control elements
37
Activators
1. Activator proteins bind to distal control
elements grouped as an enhancer in the DNA. This
enhancer has three binding sites.
2. A DNA-bending protein brings the bound
activators closer to the promoter. Other
transcription factors, mediator proteins, and
RNA polymerase are nearby
3. The activators bind to certain general
transcription factors and mediator proteins,
helping them form an active transcription
initiation complex on the promoter
38
Cell Type Specific Transcription
39
Coordinately Controlled Genes

• In prokaryotes--gt clustered into an operon with a
  single promoter and other control elements
  upstream
• The genes of the operon are transcribed into a
  single mRNA and translated together
• In Eukaryotes ? only rarely are genes organized
  this way
• Genes coding for the enzymes of a metabolic
  pathway may be scattered over different
  chromosomes
• Even if genes are on the same chromosome, each
  gene has its own promoter
• Coordinate gene expression in eukaryotes probably
  depends on the association of a specific control
  element or collection of control elements with
  every gene of a dispersed group.
• A common group of transcription factors bind to
  them, promoting simultaneous gene transcription.
• Steroid hormones enter a cell and bind to a
  specific receptor protein in the cytoplasm or
  nucleus.
• After allosteric activation of these proteins,
  they functions as transcription activators.
• Other signal molecules can control gene
  expression indirectly by triggering signal
  transduction pathways that lead to transcription
  activators.

40
Post Transcriptional Control

• RNA Processing
• mRNA Degradation
• Initiation of Translation
• Protein Processing and Degradation

41
RNA Processing

• Alternative Splicing
• Humans? more than 90,000 proteins
• Each gene generates about 3 alternately spliced
  mRNAs

42
mRNA Degradation? Micro RNAs(miRNA)
43
RNA interference (RNAi)

• Injection of double stranded RNA? turns off a
  gene
• Due to small interfering RNAs?siRNA

44
Initiation of Translation

• Can be blocked by regulatory proteins that bind
  to specific sequences or structures of the mRNA(
  prevents attachment to ribosmes)
• Stored RNA ? lacks poly-A tails? can be later
  added
• Alternatively, translation of all the mRNAs in a
  cell may be regulated simultaneously

45
Protein Processing and Degradation

• Insulin Cleavage
• Phosphorylation
• Glycosylation tagging
• Protein Degradation? Proteasomes

46
Protein Degradation

• Proteasomes recognize? protein marked for
  destruction

47
Differential Gene Expression

• 18.4

48
Differential Gene Expression

• Fertilized egg? multicellular org. w/ diff cell
  types and functions
• Cells? tissues? organs? organ systems? organism
• How does this happen??
• Genes are regulated? development

49
How do we get from egg to . . .

• Cell Division
• Cell differentiation
• Morphogenesis

50
Looking back . . .

• Differential Gene Expression
• Cell-type specific transcription
• How do different sets of activators come to be
  present in different cells????

51
How do different cells get different signals?

• Cytoplasmic Determinants? Maternal substances in
  the cell that influence early development
• Induction from neighboring cells
• Pattern Formation

52
Cytoplasmic Determinants

• After fertilization cell mitotic divisions cell
  is exposed to diff sets of CD express diff genes

53
Induction from Neighboring Cells

• These signals result in
• Different mRNAs
• Differentiation

54
Pattern Formation

• CD inductive signals? spatial organization

55
Development from Egg to Larva

• Controlled by specific genes ?Homeotic genes

56
Abnormal Pattern Formation
57
Mutation in Reg. Genes?Homeotic Genes
58
Normal and double winged Drosophila
59
Homeobox-containing genes as switches

• How a particular cell differentiates depends on
  how many of these switches are thrown

60
Homeotic Genes and Pattern Formation

• Axis Establishment? Maternal Effect genes? egg
  polarity genes
• Morphogens? establish embryo axes
• Bicoid Gene? codes for a morphogen that specifies
  the head
• Bicoid mutant? no head, 2 posterior ends
• Bicoid mRNA? confined to anterior end of the
  embryo

61
(No Transcript)
62
Eric Wieschaus/ Nusslein-Volhard1995 Nobel
63
Cancer Results From Genetic Changes

• 18.5

64
Cancer Results from Genetic Changes

• Genetic changes in the cell cycle
• mutations in genes that regulate the cell cycle
• 1911 Peyton Rous? virus causes cancer in chicken
• Tumor Virus cause cancer in humans
• Epstein Barr Virus? infectious mononucleosis
• Burkitts Lymphoma
• Papilloma virus?cancer of the cervix
• HTLV-1? adult leukaemia

65
Oncogenes and Proto-oncogenes

• Oncogenes
• Cancer causing gene
• 1st found in retroviruses
• similar to normal human genes
• Prto-oncogenes
• code for proteins that stimulate cell growth and
  division
• can become oncogenes

66
Prot-oncogenes ? Oncogenes

• A genetic change that leads to an increase in the
  gene product or the activity of the proteins
• Three main categories
• Translocation/transposition
• Gene amplification
• Point mutations

67
Genetic Changes

• Translocation/transposition
• Cancer cells? contain chromosomes that have
  broken rejoined incorrectly
• If translocation ends up near an active promoter?
  transcription increases? oncogene
• Amplification ?increases the of proto-oncogenes
  in the cell
• Point mutations
• Control element
• Proto-oncogene itself

68
Prot-oncogenes ? Oncogenes
69
Tumor Suppressor genes

• Cells have genes whose products
• Promote cell division
• Inhibit cell division? tumor suppressor gene
• Prevent uncontrolled cell growth
• Mutation in this gene? cancer

70
Tumor Suppressor Gene Products

• Repair damaged DNA? prevent accum. of cancer
  causing mutations
• Controls adhesion of cells to each other and the
  extracellular matrix
• Are components of cell signaling PWs

71
Interference with Normal Cell Signaling

• ras gene
• G-protien that leads to a kinase cascade
• Mutant form ? 30 of human cancers
• Dominant
• Positive regulator
• p53
• Guardian angel of the genome
• AKA anti-oncogene
• Recessive
• Negative regulator

72
p53 Gene

• Three ways it prevents a cell from passing on
  mutations due to DNA damage
• Activates gene p21 ? product halts cell cycle by
  binding to cyclin-dependent kinases? allows cell
  to repair DNA damage
• Activates DNA repair enzymes genes
• Activates cell suicide genes when damage is
  irreparable? apoptosis

73
Signaling that STIMULATES Cell Growth
74
Signaling that INHIBITS Cell Growth
75
Effects of Mutation

• Increased cell Division
• Cell cycle over stimulated
• Cell cycle not inhibited

76
Multistep Model of Colorectal Cancer
77
Viruses and Cancer

• 15 of human cancers are caused by viruses
• Retroviruses donate an oncogene
• Viral genome may disrupt the tumor suppressor
  gene
• Convert a proto-oncogene to an oncogene
• May produce proteins that inactivates p53 and
  other tumor suppressor genes

78
Cancer

• If occurs between 25-30? inherited predisposition
• All in tumor suppressor genes
• Oncogene mutation in fetus causes spontaneous
  abortions
• Why incidence increases with age?
• 5-7 independent mutations are required
• Mutations in both onco, tumor supp genes
• Telomerases are activated

79
Colon Cancer
80
Inherited Predisposition to Cancer

• Individuals who inherit a mutant oncogene or
  tumor-suppressor allele
• Have an increased risk of developing certain
  types of cancer

81
Inherited Cancer

• BRCA1 gene? women with this gene are at a higher
  risk for breast cancer

82
Types of DNA in the Human Genome

• Only about 15
• codes for protein