Lecture 18 June 6th, 2013

Gene Expression
Regulation
Mutations

Review Session
---------------------11am-2pm WLH
Practice Final
--------------------Ledden 5-8pm
6/10/2013

From Gene to Protein

There are no
INTRONS

Central Dogma
Replication

DNA

Transcription

RNA

Translation

PROTEIN

RNA Viruses: genome is RNA Protein

Reverse

Transcriptase
(RT)

DNA

mRNA

RT

Inserts into host (e.g. human cell) genome

(INTEGRATION)

cDNA (Complementary DNA)

From Gene to Protein: Mutations

RNA*

DNA*

PROTEIN*

Mutation are changes in genetic material of a cell.

Chromosomal Mutations: (e.g. Duplications and deletions)
large-scale mutations: multiple genes are affected Multiple phenotypes
Point Mutations:

1 base pair (bp) substitution in one gene

Missense mutation

1 bp change different a.a. encoded

UGC (cys) UGG (trp)

Nonsense mutation

1 bp change STOP codon

UGC (cys) UGA (STOP)

Silent mutation

This creates a
STOP codon, so its
a missense
mutation w/ a
certain result

1 bp change codon is the same (due to wobble)

UGC (cys) UGU (cys)

From Gene to Protein: Mutations

RNA*

DNA*

PROTEIN*

Frameshift Mutations:
Insert

or

Delete a basepair

Frameshift in ORF

Frameshift in ORF

Insert or delete an a.a.

ORF:
-------------------------

From Gene to Protein: Mutations

this info WILL be
on exam

these slides
contain the bulk of
knowledge for DNA
mutation
it is imperative that
the right frame is
used to make the
right polypeptide.

(silent)

From Gene to Protein: Mutations

What about mutations that are not in the coding sequence?

Affect mRNA synthesis
- promoter
- termination
- splicing

affects ability for

DNA to translate
mRNA

Affect Translation
- mutation in RBS (Prok)
- Stability of mRNA (3UTR 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

Transcription

RNA

Regulation of Gene Expression

UTR: untranslated region

Transcriptional UNIT (DNA!)

1st base of mRNA

Promoter

P
top: master
bottom: template to
be copied

5 UTR

Terminator

Stop

Start

Coding Region

Promoter is very important

RNA polymerase binds
choses sense/template strand
Can Determine how active a gene will be!!

promoter will tell

you which strand
has the DNA

3 UTR

you could also find

the ATG / AUG
codon

Regulation of Gene Expression

Promoters DNA sequence that RNA polymerase binds
Good Promoter

RNA Pol binds tightly and lots of mRNA is made

Poor Promoter

RNA Pol binds poorly and little of mRNA is made

Encoded in the DNA of the promoter how often a gene is transcribed

Promoter sequences are constantly changing from: good to poor

HOW?

Allosteric Regulation!!
Regulation of RNA polymerase
Transcriptional Activator: Recruit RNA polymerase to a promotoer: ON!
Transcriptional Repressor: 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!
RNA Pol

ORF

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!
RNA Pol

TR

RNA Pol

TR

ORF

ORF

Binding site for repressor

TR

RNA Pol

ORF

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!
Turns GENE ON

TA

RNA Pol

ORF

Binding site for activator

automatically binds
to right region
where promoter is.

revents it from
operting if its an
oppresor,
promotes if its
beneficial.
allosteric regulation

Regulation of Gene Expression

Prokaryotic Gene Regulation
A typical scenario: An individual bacteria can respond to environmental
change by regulating gene expression

How can negative feedback

inhibition affect more than
one gene?
Prokaryotic
organisms can be
used to explain

Regulation of Gene Expression: Prokaryotic

Prokaryotic Gene Regulation
Normal food is glucose; Lactose is polymer of galactose-glucose
so the amount of lactose permease (a receptor molecule which brings
lactose inside cell) is normally low at the cell surface
lactose permease

Lactose (

) is low

Lactose (

) is high

bacteria

Allosteric Regulation! Bacteria will induce multiple genes which take advantage
of lactose as a food source

Regulation of Gene Expression: Prokaryotic

Prokaryotic Gene Regulation: OPERONS
A single promoter regulates multiple genes

physical location
on gene has
significance

Has a operator (DNA binding site) which regulates access of RNA pol to gene
ALL genes are clustered together so that a single mRNA is made
Within the single mRNA are start and stop codons for each protein to be made

A cluster of genes under control of one promoter; utilized when

The Lac OPERON: lactose is a food source

will test how loc

operon will work
only has access in
high lactose

makes one long

mRNA

Regulation of Gene Expression: Prokaryotic

Prokaryotic Gene Regulation: OPERONS
The Lac OPERON: Inducible Operon
lacZ: Encodes beta-galactosidase (cleaves galactose-glucose into separate molecules)
lacY: Encodes lactose permease (transports lactose inside cell)
lacA: Encodes lactose transacetylase (helps cell use lactose)

Operator:
Controls RNA Pol access to gene!!

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!

Lactose
Binds repressor

Transcription happens!

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: Eukaryotes

Organization of the Eukaryotic Genome
Compared to simple Prokaryotes
Much Larger Genome
Cell specialization (due to cell differentiation?)
Multicellular
Has > 25,000 Genes
Lots of DNA that does not code for RNA or protein

KEY! Multiple levels of Gene Expression Regulation!

Regulation of Gene Expression: Eukaryotes

Eukaryotic Gene Regulation
No Operons
3 polymerases

RNA pol I, II, and III

RNA Pols cant bind DNA alone

Require Transcription factors!!
DNA Promoter sequences dont act alone
Proximal Control Sequence
Enhancer Sequences
Silencer Sequences
Chromatin regulation: access to gene by RNA polymerase

Regulation of Gene Expression: Eukaryotes

Transcription factors
Bind to the minor and major grooves
of the DNA Double helix

Transcription factor DNA

binding motifs (examples)

DNA Sequence Specific!!

Recruit RNA pol turn genes ON!
Zinc helps stabilize a finger like protrusion that
binds DNA at the major groove

Helix-turn-Helix

Zinc finger

Regulation of Gene Expression: Eukaryotes

DNA Regulatory Elements (sequences!)
Proximal Control Elements
right next to promoter sequence
transcription factors bind here
allow for multiple genes in one pathway to be turned on HOW?
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

Regulation of Gene Expression: Eukaryotes

DNA Regulatory Elements (sequences!)
Enhancers Transcription activators
DNA sequences sometimes 1000s of by away
Recruit transcription factors turn genes ON!
Bend DNA to bring activators near transcription factors and RNA pol

Regulation of Gene Expression: Eukaryotes

Enhancers
(transcription activators)

bind to specific
sequences

Regulation of Gene Expression: Eukaryotes

DNA Regulatory Elements (sequences!)
Enhancers Transcription activators
DNA sequences sometimes 1000s of by away
Recruit transcription factors turn genes ON!
Bend DNA to bring activators near transcription factors and RNA pol
Silencers Transcription repressors
1000s bp away

inhibit transcription
turn genes OFF!

Regulation of Gene Expression: Eukaryotes

Chromatin
DNA-Protein (Histones) complex DNA packing
Nucleosome basic unit of DNA packing
DNA wrapped around histones supercoiling
2 M

DNA double Helix

Histones
10 M
Linker DNA
(string)

Beads on a string (TEM)

Nucleosome

Note: Chromosome (in metaphase of cell

cycle) can be 1-2 microns in diameter!!

Regulation of Gene Expression: Eukaryotes

How Can DNA Packing affect Gene Expression?
Physically hide genes Affects accessibility!
Block access to RNA Polymerase
Transcription factors must break DNA-Histone interactions and
recruit RNA Pol.