Gene Expression Regulation Mutations - Class...
Transcript of Gene Expression Regulation Mutations - Class...
Gene ExpressionRegulationMutations
Lecture 18 – June 2nd , 2016
From Gene to Protein
DNA RNA PROTEIN
Central Dogma
Replication
Transcription Translation
RNA Viruses: genome is RNA Protein
DNA
ReverseTranscriptase
(RT)
mRNART
cDNA (Complementary DNA)
Inserts into host (e.g. human cell) genome(INTEGRATION)
From Gene to Protein: Mutations
DNA* RNA* PROTEIN*
Mutation are changes in genetic material of a cell.
Chromosomal Mutations:large-scale mutations: multiple genes are affected
Point Mutations:
Missense mutation
Nonsense mutation
Silent mutation
Multiple phenotypes
1 base pair (bp) substitution in one gene
1 bp change different a.a. encoded
1 bp change STOP codon
1 bp change codon is the same (due to wobble)
UGC (cys) UGG (trp)
UGC (cys) UGA (STOP)
UGC (cys) UGU (cys)
(e.g. Duplications and deletions)
From Gene to Protein: Mutations
DNA* RNA* PROTEIN*
Frameshift Mutations:
Insert or Delete a basepair
1 1
2 2
3 3
Frameshift in ORF
Frameshift in ORF
Insert or delete an a.a.
From Gene to Protein: Mutations
(silent)
From Gene to Protein: Mutations
What about mutations that are not in the coding sequence?
Affect mRNA synthesis- promoter- termination- splicing
Affect Translation- mutation in RBS (Prok)- Stability of mRNA (3’UTR polyAAA tail)
Regulation of Gene Expression
Every cell in the body has the same DNA
So how can cells be so different from one another??(cell shape, function, etc)
Gene Regulation!!! DNA RNATranscription
P TStart Stop
Coding Region
Promoter Terminator
5’ UTR3’ UTR
1st base of mRNA
Transcriptional UNIT (DNA!)
Regulation of Gene Expression
Promoter is very importantRNA polymerase bindschoses sense/template strandCan Determine how active a gene will be!!
Regulation of Gene Expression
Promoters – DNA sequence that RNA polymerase binds
“Good” Promoter
“Poor” Promoter
Encoded in the DNA of the promoter how often a gene is transcribed
Promoter sequences are constantly changing from:
Allosteric Regulation!!
Regulation of RNA polymeraseTranscriptional Activator:
Transcriptional Repressor:
RNA Pol binds tightly and lots of mRNA is made
RNA Pol binds poorly and little of mRNA is made
HOW?good to poor
Recruit RNA polymerase to a promotoer: ON!
Block RNA polymerase: OFF!
Regulation of Gene Expression
Transcriptional Activators and Repressors
Repressor (TR)can bind at the promoter or a nearby sequence and block RNA pol : turns gene OFF!
P ORF
RNA Pol
Regulation of Gene Expression
Transcriptional Activators and Repressors
Repressor (TR)can bind at the promoter or a nearby sequence and block RNA pol : turns gene OFF!
P ORF
RNA Pol
TR
P ORF
RNA Pol TR
Binding site for repressor
X
X
RNA PolTR
P ORFX
Regulation of Gene Expression
Transcriptional Activators and Repressors
Activator (TA)Binds to specific sequence, next to promoter, recruits RNA pol to that promoter – turns gene ON!
P ORF
RNA Pol
Binding site for activator
TA
Regulation of Gene Expression
Prokaryotic Gene RegulationA typical scenario: An individual bacteria can respond to environmental
change by regulating gene expression
How can negative feedback inhibition affect more than
one gene?
Regulation of Gene Expression: Prokaryotic
Prokaryotic Gene Regulation
Normal food is glucose;
so the amount of lactose permease (a receptor molecule which brings lactose inside cell) is normally low at the cell surface
Lactose is polymer of galactose-glucose
Lactose ( ) is low Lactose ( ) is high
Allosteric Regulation! Bacteria will induce multiple genes which take advantage of lactose as a food source
Prokaryotic Gene Regulation: OPERONS
The Lac OPERON:
lacZ:
lacY:
lacA:
Encodes beta-galactosidase (cleaves galactose-glucose into separate molecules)
Encodes lactose permease (transports lactose inside cell)
Encodes lactose transacetylase (helps cell use lactose)
Operator:Controls RNA Pol access to gene!!
Inducible Operon
Regulation of Gene Expression: Prokaryotic
Prokaryotic Gene Regulation: OPERONS
The Lac OPERON: Inducible Operon
Gene for repressor is adjacent to Lac operon!
Regulation of Gene Expression: Prokaryotic
Prokaryotic Gene Regulation: OPERONS
The Lac OPERON: Inducible Operon
When Lactose is absent: default is OFF!
No Transcription!
Regulation of Gene Expression: Prokaryotic
Prokaryotic Gene Regulation: OPERONS
The Lac OPERON: Inducible Operon
When Lactose is present: ON!
Transcription happens!LactoseBinds repressor
Regulation of Gene Expression: Prokaryotic
Prokaryotic Gene Regulation: OPERONS
The Tryptophan OPERON: Repressible Operon
When Tryptophan is absent: default is ON!
Transcription happens!
Regulation of Gene Expression: Prokaryotic
Prokaryotic Gene Regulation: OPERONS
The Tryptophan OPERON: Repressible Operon
When Tryptophan is present: OFF!
No Transcription!
Regulation of Gene Expression: Prokaryotic
Regulation of Gene Expression: Eukaryotes
Organization of the Eukaryotic Genome
Compared to simple ProkaryotesMuch Larger GenomeMulticellularHas > 25,000 GenesLots of DNA that does not code for RNA or protein
KEY! Multiple levels of Gene Expression Regulation!
Cell specialization (due to cell differentiation?)
Regulation of Gene Expression: Eukaryotes
Eukaryotic Gene Regulation
No Operons
3 polymerases
RNA Pol’s can’t bind DNA alone
Require Transcription factors!!
DNA Promoter sequences don’t act alone
Proximal Control SequenceEnhancer SequencesSilencer Sequences
Chromatin regulation: access to gene by RNA polymerase
RNA pol I, II, and III
Helix-turn-Helix Zinc finger
Transcription factor DNA binding motifs (examples)
Regulation of Gene Expression: Eukaryotes
Transcription factorsBind to the minor and major grooves of the DNA Double helix
DNA Sequence Specific!!
Recruit RNA pol – turn genes ON!
Zinc helps stabilize a finger like protrusion that binds DNA at the major groove
Regulation of Gene Expression: Eukaryotes
DNA Regulatory Elements (sequences!)
Proximal Control Elementsright next to promoter sequencetranscription factors bind hereallow for multiple genes in one pathway to be turned on
Can share similar DNA Seq. in front of promoters for different genes
Example: “SRE” sequences (stress response element)
transcription factors will bind SRE element and turn multiple genes
HOW?
Regulation of Gene Expression: Eukaryotes
DNA Regulatory Elements (sequences!)
Enhancers
DNA sequences sometimes 1000’s of by away
Recruit transcription factors
Bend DNA to bring activators near transcription factors and RNA pol
turn genes ON!
Transcription activators
Regulation of Gene Expression: Eukaryotes
Enhancers (transcription activators)
Regulation of Gene Expression: Eukaryotes
DNA Regulatory Elements (sequences!)
Enhancers
DNA sequences sometimes 1000’s of by away
Recruit transcription factors
Bend DNA to bring activators near transcription factors and RNA pol
Silencers
1000’s bp away
turn genes ON!
inhibit transcription
turn genes OFF!
Transcription repressors
Transcription activators
Regulation of Gene Expression: Eukaryotes
Chromatin
DNA-Protein (Histones) complex
Nucleosome basic unit of DNA packingDNA wrapped around histones
DNA packing
Nucleosome
Beads on a string (TEM)
Note: Chromosome (in metaphase of cell cycle) can be 1-2 microns in diameter!!
supercoiling
DNA double Helix
2 M
10 M
Linker DNA(string)
Histones
Regulation of Gene Expression: Eukaryotes
How Can DNA Packing affect Gene Expression?
Physically hide genes
Block access to RNA Polymerase
Transcription factors must break DNA-Histone interactions and recruit RNA Pol.
Affects accessibility!