gene regulation ch 18.1-4, campbell 9 th edition
TRANSCRIPT
Gene regulation
Ch 18.1-4, Campbell 9th edition
Lac operon – inducible operon• Normally, the
repressor IS bound to the operator, so lac operon is OFF.
• In the "induced" state, the lac repressor is NOT bound to the operator site.
18.1 Gene Regulation in Prokaryotes:
Trp operon - repressibleTrp operon is normally ON
When tryptophan is present it binds to the repressor, which activates it. The activated repressor will bind to the operator, “repressing” the operon
Positive Gene Regulation• Positive activation of lac operon• Bacteria uses carbohydrates for energy – glucose vs.
lactose• How is this signaled to the bacteria?
Positive activation is through the CAP protein, which is activated by cAMP.
cAMP accumulates when glucose is scarce.
18.2 Gene Regulation in Eukaryotes
Gene is expressed when it makes a protein
Expression regulated at various levels:• Chromatin structure• Transcription factors*• Alternative splicing• Non-coding RNAs that
degrade other mRNAs
Chromatin packing
Chromatin structure
• Histone acetylation- acetyl groups are added to histones– loosens chromatin structure – promotes transcription– Deacetylation – acetyl groups removed, reduces
transcription
• Methylation – methyl groups added to certain bases in DNA – Reduces transcription in some species– In genomic imprinting, regulates expression of
maternal or paternal alleles of certain genes
Regulation of Transcription:Typical Eukaryotic Gene Organization
• Control elements, segments of noncoding DNA, are associated with eukaryotic genes. Control elements act as binding sites for transcription factors that help regulate transcription
• Control elements and the transcription factors that bind them allow for precise control of gene regulation
Enhancer(distal control
elements)
DNA
UpstreamPromoter
Proximalcontrol
elementsTranscription
start site
Exon Intron Exon ExonIntron
Poly-Asignal
sequenceTranscriptiontermination
region
DownstreamPoly-Asignal
Exon Intron Exon ExonIntron
Transcription
Cleaved3 end ofprimarytranscript
5Primary RNAtranscript(pre-mRNA)
Intron RNA
RNA processing
mRNA
Coding segment
5 Cap 5 UTRStart
codonStop
codon 3 UTR
3
Poly-Atail
PPPG AAA AAA
Eukaryotic gene & transcript
• Transcription factors – general ones are required for the RNA polymerase binding. They bind first to the DNA, and then recruit the RNA polymerase. Specific transcription factors bind with control elements for regulation
• Enhancers – groups of control elements upstream of a gene, have binding sites for specific transcription factors
• Activators - a protein that binds to an enhancer and stimulates transcription of a gene–have two domains, one that binds DNA and a
second that activates transcription• Repressors - transcription factors that inhibit
expression of a particular gene by a variety of methods
DNA
Activationdomain
DNA-bindingdomain
MyoD – a specific transcription factor that acts as an activatorMyoD is a master regulatory gene
• Gene Switches • http://www.hhmi.org/biointeractive/gene-
switch
ActivatorsDNA
EnhancerDistal controlelement
PromoterGene
TATA boxGeneraltranscriptionfactors
DNA-bendingprotein
Group of mediator proteins
RNApolymerase II
RNApolymerase II
RNA synthesisTranscriptioninitiation complex
Model for Transcription Initiation
• 1. Transcriptional activators bind to DNA & recruit chromatin remodeling complexes and histone acetyltransferases
• 2. These open up the chromatin to expose promoter & regulatory sequences
• 3. Transcriptional factors bind to enhancers• 4. DNA bending protein protein brings activators,
mediator proteins, and general transcirption factors together to form transcription initiation complex on promoter
• Regulation of Eukaryotic DNA Transcription
• http://www.hhmi.org/biointeractive/regulation-eukaryotic-dna-transcription
Coordinately controlled genes in eukaryotes
• Genes coding for enzymes of a metabolic pathway are often scattered over different chromosomes
• Coordinate gene expression depends on simultaneous expression of the genes
• Chemical signalling is often used for coordinate gene expression – i.e. hormones
Alternate Gene Splicing
• Post transcriptional regulation through RNA processing
• Different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns
Exons
DNA
Troponin T gene
PrimaryRNAtranscript
RNA splicing
ormRNA
1
1
1 1
2
2
2 2
3
3
3
4
4
4
5
5
5 5
mRNA degradation
• Lifespan of mRNA in cytoplasm affects protein synthesis
• mRNA in eukaryotes lasts longer than prokaryotic mRNA
Translation
• Initiation of translation can be blocked by proteins that bind to parts of mRNA
Protein processing & degradation
• Post-translational protein processing includes cleavage, and addition of functional groups
• Proteasomes are giant protein complexes that bind protein molecules and degrade them
18.3 Non coding mRNAs
• A large part of the genome is made up of DNA that is transcribed into non-coding mRNAs (ncRNA)
• These can affect translation and chromatin expression
• RNA• Important in many cellular machines:
• Ribosome rRNA• Spliceosome snRNA• Telomerase telomerase RNA
Interference with Translation
• MicroRNAs (miRNA) are single-stranded RNA molecules that can bind to mRNA
• Small Interfering RNA (siRNA) act similarly to miRNAs, but have a longer, double stranded precursor
• They can degrade mRNA or block its translation• This is called RNA interference - RNAi
(a) Primary miRNA transcript
HairpinmiRNA
miRNA
Hydrogenbond
Dicer
miRNA-proteincomplex
mRNA degraded Translation blocked(b) Generation and function of miRNAs
5 3
microRNAs
• A novel class of ncRNA gene• Products are ~22 nt RNAs• Precursors are 70-100 nt hairpins• Gene regulation by pairing to mRNA• Unknown before 2001• Forms RISC – RNA inducing silencing complex
Small Interfering RNAs - siRNA
• RNA interference (RNAi) – when double stranded RNA injected into the cell, it turned off expression of gene with same sequence as the RNA
• siRNAs are the cause of this RNAi– Similar to miRNA, but formation is different– Many siRNAs are formed from a longer, double
stranded RNA molecule– Some siRNAs can bind back to chromatin and cause
changes in the chromatin
siRNA
Chromatin & ncRNA
• In some yeasts, siRNAs can play role in heterochromatin forming, and block parts of chromosome
• Small ncRNAs can induce heterochromatin, which blocks parts of chromosome, blocking transposons
• RNAi (~5 min)]• http://www.youtube.com/watch?v=cK-
OGB1_ELE
18.4 Differential gene expression leads to different cell types in multicellular organism
• One fertilized egg can give rise to many different cell types
• Differential gene expression results from genes being regulated differently in each cell type
• Materials in the egg can set up gene regulation that is carried out as cells divide
Cell development
Zygote cell – totipotent – has potential to develop into a complete organism
Cell determination – cell has committed to a final fate, it is unable to change at this point
Cell differentiation – cell produces tissue-specific proteins, cell has clear cut identity
Morphogenesis – organization of cells into tissues & organs
Cytoplasmic Determinants
• Based on uneven distribution of cytoplasmic determinants in egg
• The cytoplasm has RNA & proteins that were encoded by the mother’s DNA
• When cell divides, the two cells have different amounts of the determinants, which can determine the cell’s fate
(a) Cytoplasmic determinants in the egg
Unfertilized egg
Sperm
Fertilization
Zygote(fertilized egg)
Mitoticcell division
Two-celledembryo
Nucleus
Molecules of twodifferent cytoplasmicdeterminants
Environment around the cell, especially signals from nearby embryonic cells influence development of cells
The changes in gene expression lead into observable cellular changes
In the process called induction, signal molecules from embryonic cells cause transcriptional changes in nearby target cells
Interactions between cells induce differentiation of specialized cell types
Induction signals and cell differentiation
Signal Induction - types• Signals from one group of
cells influence another group of cells
Diffusion: signal diffuses from distance to receptor– i.e. hormone, or other signal molecule - receptor can transmit signal through second messengers in signal transduction pathway
Signal Induction - types
Direct contact – neighboring cells
Gap junction – cytoplasm of 2 cells is connected
Muscle cell determination
Pattern formation• What controls the body plan of an
organism? How do organs get in the right place?
• 2 general models: –Morphogen gradient– Sequential induction
Sequential induction• Differentiation due to production & release of
a series of chemical signals
Morphogen gradientA diffusible chemical signal, or morphogen, is produced. The concentration is higher closer to the source, and lower farther away from the source.The fate of the cell depends on its exposure to the different threshold levels.
Drosophila- model organism
• Lewis studied development by looking at mutants with bizarre developmental defects, and through this discovered homeotic genes
• Homeotic genes specify the identity of body segments
• Mutations in these genes lead to structures in the wrong place
• In a fruit fly, for example, Hox genes lay out the various main body segments—the head, thorax, and abdomen. Here we see a representation of a fruit fly embryo viewed from the side, with its anterior end to the left and with various Hox genes shown in different colors. Each Hox gene, such as the blue Ultrabithorax or Ubx gene, is expressed in different areas, or domains, along the anterior-to-posterior axis.
Drosophila development
• Cytoplasmic determinants in egg establish axes of drosophila body
• Bicoid mRNA from mother is translated into the Bicoid protein in the Drosophila zygote
• Bicoid is transcription factor that turns on genes in different levels
a. Bicoid concentration & 4 genes affectedb. concentration gradient of Bicoid in zygote– more at rightc. concentration gradient in embryo after several divisionsd. hunchback protein – green, kruppel protein - orange
• Eric Wieschaus – Bicoid gradient (3:28)
• http://www.youtube.com/watch?v=pAoK-KOUTZM
• Bicoid animation (2:15)• http://www.youtube.com/watch?v=
uaedzlrnBGY
• Bonnie Bassler – Quorum sensing & gene expression
• http://media.hhmi.org/hl/09Lect2.html?start=32:28&end=39:40
• http://www.hhmi.org/biointeractive/molecular-cascade-bacterial-quorum-sensing
