Gene Expression Regulation Mutations - Class...

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Gene Expression Regulation Mutations Lecture 18 – June 2 nd , 2016

Transcript of Gene Expression Regulation Mutations - Class...

Page 1: Gene Expression Regulation Mutations - Class Websitesclasses.biology.ucsd.edu/bild1.SP16/Lect-18.pdf · UGC (cys) UGG (trp) UGC (cys) UGA (STOP) UGC (cys) UGU (cys) (e.g. Duplications

Gene ExpressionRegulationMutations

Lecture 18 – June 2nd , 2016

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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)

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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)

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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.

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From Gene to Protein: Mutations

(silent)

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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)

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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

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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!!

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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!

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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

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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

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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

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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?

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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

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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

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Prokaryotic Gene Regulation: OPERONS

The Lac OPERON: Inducible Operon

Gene for repressor is adjacent to Lac operon!

Regulation of Gene Expression: Prokaryotic

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Prokaryotic Gene Regulation: OPERONS

The Lac OPERON: Inducible Operon

When Lactose is absent: default is OFF!

No Transcription!

Regulation of Gene Expression: Prokaryotic

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Prokaryotic Gene Regulation: OPERONS

The Lac OPERON: Inducible Operon

When Lactose is present: ON!

Transcription happens!LactoseBinds repressor

Regulation of Gene Expression: Prokaryotic

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Prokaryotic Gene Regulation: OPERONS

The Tryptophan OPERON: Repressible Operon

When Tryptophan is absent: default is ON!

Transcription happens!

Regulation of Gene Expression: Prokaryotic

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Prokaryotic Gene Regulation: OPERONS

The Tryptophan OPERON: Repressible Operon

When Tryptophan is present: OFF!

No Transcription!

Regulation of Gene Expression: Prokaryotic

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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?)

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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

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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

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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?

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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

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Regulation of Gene Expression: Eukaryotes

Enhancers (transcription activators)

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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

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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

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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!