Genetics- the control of gene expression 1

Central dogma: DNA – transcription- RNA- translation- protein (Francis crick)

Important- structure of cell on WHAT protein & QUANTITY

- Differences in gene expression depend on differences in transcription & translation

Multicellular organism- contains cells with same DNA

- Different cells/tissue due to which genes in genome are expressed and at which time

GENE EXPRESSION
- Controlled at many points in pathway from DNA  Protein
- Transcriptional control

Enzyme polymerase- synthesises RNA from DNA template

In eukaryotes:
RNA polymerase I= 5.8S, 18S, 28S rRNA genes
RNA polymerase II- all protein-coding genes, plus snoRNA genes, miRNA genes, siRNA genes and
most snRNA genes.
RNA polymerase III- tRNA genes, 5S rRNA genes, some snRNA genes and genes for other small RNAs

Example: the mushroom Amanita phallodies has a death cap containing α- amanitin = stops protein
production ‫ ؞‬toxic and fatal

Promoters- specific sequence of nucleotides that bind to promoter- which then initiates
transcription (which later on in the stream). Polymerase will slow down when it gets to these regions
& once bound well -transcription begins
3’ to 5’ end read and synthesises the other way round.

Initiation of transcription:
RNA polymerase interacts with transcription factors (proteins) when binds to promoter (short
sequence of nucleotides that promoter binds to)
How does promoter get here? It reads DNA from 3’ to 5’. It whizzes along the DNA strand and
stops when it gets to promoter region. Once it is bound well it can start the initiation of
transcription.
Additional control sequences- determine
WHEN gene transcribed
Upstream is toward the 5' end of the RNA molecule and downstream is toward the 3' end.
When considering double-stranded DNA, upstream is toward the 5' end of the coding strand
for the gene in question and downstream is toward the 3' end. Due to the anti-parallel
nature of DNA, this means the 3' end of the template strand is upstream of the gene and the
5' end is downstream.

Cis-acting regulatory regions- DNA sequences recognised by proteins – regions of non-coding DNA
which regulate the transcription of neighbouring genes.
- Typically regulate gene transcription by binding to transcription factors
- Single transcription factor may bind to many CREs= control expression of many genes=
pleiotropy
- “cis”- on the side i.e. on the same molecule of DNA as the gene (s) to be transcribed.

All regions of class II genes contain two kinds of essential DNA sequences:
ENHANCERS & PROMOTERS

What are class ii genes? Genes transcribed by RNA polymerase

Enhancers/ UAS (upstream activating sequences) – regulatory sites that can be distant from
promoter

Promoter- very close to protein-coding region and includes the initiation site- where transcription
begins and a TATA box.

TATA box- site of preinitiation complex formation- first step in transcription initiation in eukaryotes

Trans-acting proteins- (transcription factors) bind to the promoter and enhancer to control
transcription from the gene

Enhancers/UAS- a short (50-1500bp) region of DNA that can be bound by proteins (activators) to
increase the likelihood that transcription of a particular gene will occur. Cis-acting.

Activators and repressors bind to the enhancers

Repressor- DNA/RNA binding protein that inhibits expression of one or more genes by binding to
operator or associated silencers.

Basal factors- including RNA polymerase II bind to the promoter

Assembly of preinitiation complex for eukaryotic transcription

1. Eukaryotic RNA polymerase ii can only bind to promoters with the

help of transcription factors
2. TATA- box binding protein (TBP) binds to the TATA box at TATA-
box binding protein subunit.
3. Conformational change induced by TBP binding to TATA box-
additional transcription factors & RNA polymerase II bind to
promoter region.
4.
5. TBP recruits TFIID complex- facilitated by TFIIA binding to
upstream part of TFIID complex
6. TFIIB then binds to TFIID-TFIIA-DNA complex- to the promoter
region (names of transcription factor relates to polymerase that it
works with e.g. TF ii- ii polymerase )
7. RNA polymerase ii and further transcription factors bind to the
promoter to give the BASAL TRANSCRIPTION COMPLEX

Generally- TATA box found at RNA polymerase II promoter regions- although some in vitro studies-
demonstrated that RNA polymerase III can recognise TATA sequences.

RNA polymerase II- recruited to this multiprotein complex- help of TFIIF and then TFIIH

Activator proteins bound to enhancers interact with the basal transcription complex
- Via mediator protein- multiprotein complex- functions as transcriptional co-activator in all
eukaryotes
- Mediator complexes function= transmit signals from transcription factors to the polymerase
- Could be through looping round of DNA- proteins can interact at that promoter site which
might be some distance away ( in linear terms of DNA)

-
 - Consensus sequence- calculated order of most common (frequent) e.g. nucleotides, amino
acids within a SEQUENCE.

Gal4: Activator protein example

- Activator protein= trans- regulatory – this is a transcription factor
- Found in yeast- an example for eukaryotic gene regulation
1. Yeast= single- celled eukaryote that lives in media containing sugar e.g. sucrose, galactose-
metabolises these sugars
2. To metabolise galactose- switch on metabolic pathway- including suite of proteins that are
required to break down those sugars. NOT switched on all the time.

1. Gal4 activator protein (TF) binds to UAS (enhancer). UAS is present in multiple genes that are
involved all in the same pathway- activator protein can switch on whole galactose metabolic
pathway.
2. When Galactose is absent= Gal80 inhibitor binds to Gal4- prevents transcription (no
interaction with promoter)
3. When galactose is present= Gal80 inhibitor binds with galactose. Conformational change.
Gal80 does not bind to Gal4 activator- Gal4 is free to interact with basal transcription
complex. Transcription initiated.

All genes involved in galactose metabolism are switched on and galactose is broken down.

Transcription factors bind promoter sequences and form the BTC with RNA polymerase ii

Chromatin structure plays a role in eukaryotic gene regulation

- Core histones are made from 2x histone H2A, 2x histone H2B, 2x Histone H3, 2x Histone H4
- Solenoid fiber
- Depending on the DENSITY of chromatin= accessibility on
transcription machinery- DNA tightly compacted= NO transcription
DNA made accessible to TF by multiple mechanisms:

Gene activator protein bound to histone AND then:

 Chromatin remodelling complex

1. Histone chaperone – needed to unload DNA onto histones (also unload histones from DNA)
causing:
- Remodelled nucleosomes
- Histone removal
2. Histone chaperone-
replacement with proteins
that change SHAPE of
histone complex
- Histone replacement
 Histone- modifying enzymes-
change accessibility/ability for
transcription – specific pattern
of histone modification

Core histones can be covalently modified on their N-terminal tails

(aka where their amino acids point with amine group first)
- N terminus can get synthesised first

Histones can be covalently modified at many sites on the N-terminal tail

Ubiquitylation, is the process of attaching ubiquitin, a small protein
The Post Transcriptional Modifications made to histones can impact gene expression by altering
chromatin structure or recruiting histone
modifiers.
Thus, quantitative detection of various
histone modifications would provide useful
information for a better understanding of
epigenetic regulation of cellular processes

Top one- can be modified at different sight

on proteins
Bubbles- diff type of histone modification-
diff types (methylation etc)
Addition of diff groups to amino acids K=
lysine, S= serine, R = arginine
Three amino acids:

So lysine has an acetyl group, serine/threonines have phosphate groups and lysines/arginines
have methyl groups.

Lysine is a “hot spot” for PTMs- this occurs often

The balance between these two COMPETITIVE modifications= important= establishment of
specific chromatin structures

Silent vs active domains in chromatin- involve two competitive modifications of histone H3 K9

(methylation and acetylation)

Different modifications lead to different transcriptional stage.

Lysine- adds 9 aa into proteins (methylation- gene silencing) (acetylation- gene expression)

The DNA is MORE accessible & leads to

MORE TF being able to reach DNA ‫؞‬
acetylation of histones is known to
increase the expression of genes through
transcription activation.

This diagram shows some histone H3

modifications affecting gene expression

COVALENT histone modifications- affect gene expression

- Methylation
- In vertebrates this can occur in the sequence CG
- Methylated DNA REPRESSES gene expression
 Cytosine methylation= patterns can be inherited after DNA replication
- Methylation occurs- circulating methylase – add methyl group onto cytosine
- Patterns of methylation can be passed onto cells – tissue identity
- An example of epigenetics
- Maintains patterns of gene repression in differentiated cells

- DNA methylation & histone modification- alter DNA accessibility & chromatin structure-
regulating patterns of gene expression

- Epigenetics is related to histone modifications AND DNA modification (i.e. methylation)

- Inherited- families- conserved domains that show modified histones

- Methyl- binding proteins= structural proteins- recruit variety of histone deacetylase (HDAC)
complexes & chromatin remodelling factors
- This leads to chromatin compaction & ‫ ؞‬NO transcription (transcriptional repression)
- Evidence is accumulating= epigenetic cross-talk i.e. interplay between DNA methylation &
histone acetylation- may be involved in process of gene transcription

EPIGENETIC WRITERS- enzymes that modify histone

- Enzymes like histone acetyltransferases (HATs), histone methyltransferases (HMTs/KMTs),
protein arginine methyltransferase (PRMTs) and kinases are responsible for adding
epigenetic marks on histones
EPIGENETIC READERS-
- Recognise and bind to epigenetic marks made by writers- determine functional outcome
- Include proteins containing bromodomains, chromodomains & Tudor
- Readers- induce chromatin compaction/ act as shield- PREVENT- binding of proteins
involved in RNA transcription.
Epigenetic modification
- Epigenetic changes are not passed on from parent to offspring
- During development of germ cells/immediately after fertilisation- germ cells erase
epigenetic modification
Control of eukaryotic transcriptional initiation:
- Activator proteins bind upstream to enhancer sequences & activate BTC

 Multiple epigenetic mechanisms control expression via CHROMATIN CONDENSATION

- Histone modifications
- In vertebrates, direct DNA methylation on cytosine helps maintain patterns of transcription
from parent to offspring cell (cell repression in somatic cells)
- A repressor- prevents DNA polymerase from binding ‫ ؞‬NO transcription
Bacterial gene expression
- NO nuclear membrane- DNA in cytoplasm
- One cytoplasmic compartment
- NO histones- site of DNA transcription = same site for translation= coupled

Bacterial protein synthesis

- Where transcription &
translation take place
simultaneously in
cytoplasm
- Coupling- means that
ribosomes bind to mRNA
& protein synthesis
(translation) begins while
mRNA is still being
synthesised ( transcribed)
- AKA same length of DNA
is being worked on so
when one thing is
transcribed it then goes to be translated

Recruiting RNA polymerase to the bacterial promoter

Initiation
Sigma subunit binds first to initiation (promoter
sequence- closer to start of protein) , and recruits the
rest of the RNA polymerase

DIFFERENT GENES= DIFFERENT SIGMA SUBUNIT

Essentially:

1. Initiation- Sigma subunit binds to promoter region of DNA – promoter region is close to start
of protein- recruits the rest of the RNA polymerase. Different genes= different sigma
subunit.
2. RNA polymerase (only one type in prokaryotes) moves along the DNA- reaches protein
coding region site. Unwraps DNA double helix through breaking of hydrogen bonds. Free
nucleotides come to close proximity of base and interact and bind. Make mRNA sequence.
3. Elongation- mRNA strand gets longer
4. Ribosomes bind with mRNA strand- translation begins- tRNA molecules & ppc forms.
Multiple ribosomes may bind. The direction of translation is TOWARD the mRNA strand.

Bacterial gene expression

1. Genes of related function- often clustered into OPERONS.

2. An operon usually has ONE promoter
3. All genes in an operon are transcribed together
4. mRNA translated to give separate proteins

Lac operon

- Lactose operon- required for the transport & metabolism of lactose in E.coli
- Lac operon allows for effective digestion of lactose when glucose NOT available
- LacZ, lacY, lacA
- Lacz= β-galactosidase- CLEAVES lactose- disaccharide – into GLUCOSE & GALACTOSE
- lacY- encodes β-galactosidase permease- protein which embeds in cytoplasmic membrane to
enable transportation of lactose INTO cell
- lacA- encodes β-galactosidase transacetylase - degrades lactose
-

Polycistronic transcripts- able to produce multiple proteins from ONE mRNA transcript. (vs
Monocistronic in eukaryotes) polycistronic transcripts- very rare in eukaryotes

General structure of a bacterial operon:

No lactose= No transcription

ABSENCE of lactose- lac repressor binds to lacO operon region. Lac-l produces protein
(repressor) that BLOCKS RNA polymerase from binding to promoter site as binds to lacO region. ‫؞‬
NO transcription.

Lactose= transcription

PRESENCE of lactose- lactose binds to repressor. Allosteric shift. Repressor cannot bind to
operator- RNA polymerase- transcribe lac genes (Z, Y, A) ‫ ؞‬transcription ‫ ؞‬higher levels of encoded
proteins

Lac operon undergoes negative inducible regulation. Gene is turned off by the regulatory factor (lac
repressor) unless some molecule (lactose) is added.

Other extra stuff:

There are two proteins- β-g & permease- membrane-bound protein- direct route for lactose outside
cell- IMPORTED into cell- greater rate than passive transfer- translation continues so more permease
so greater rate.

Once lactose concentration reduced, lactose which is bound to repressor is released. Repressor
binds to protein conducting region & gene expression is haulted.

• Functional genes are clustered in operons:

• Single transcript

• Multiple proteins