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Negative control

Default state is on,

Inducible
An inducer and /or
activator
De-represses a
gene, turns a gene
on or, promotes
transcription

Repressible
A repressor slows
down or stops the
expression of a
gene, but a corepressor must bind
to it first

Lac operon control



The inducer (lactose) binds to
the repressor releasing it from
the operator and preventing
repressor binding, allowing
expression -on

Tryptophan global regulation



A repressor is produced by the
trp R site, but it cannot bind to
the tryptophan operator unless
tryptophan first binds to it (i.e.
on), together tryptophan /
repressor down regulates the
expression of tryptophan

Positive control
Default state is off

RNA polymerase cannot bind to a
promoter without : unwinding
DNA from the nucleosome and an
inducer binding near the promoter
site. TBT binding to the TATA box
initiates holoenzyme assembly,
binding of other activators or
repressors.



Glucose or its catabolite represses
CAMP expression. Without CAMP,
the CAP does not bind to the 5
end of the promoter. Without CAPCAMP binding there is little
transcription by the lac operon -off.

Production regulation within eukaryotes may


be complicated, There could be control of
DNA transcription, control of splicing and
translating proteins and control through the
feedback with various metabolic pathways or
cellular processes.


Transcriptional control may be
effected by upstream enhancers on the same
strand of DNA (cis-effects). Alternatively,
hormones originating in other cells can bind to
specific receptors in a cell membrane, a signal
is moved to the nucleus and the signal is
transported into the nucleus(trans acting). In
the nucleus, a protein signal may activate
transcription by acting locally on DNA,
stimulating the expression of one or more
genes.

There can also be regulation at the splicing
level, regulation of transport out of the
nucleus, regulation of the level of translation
(number of ribosomes, degradation etc.).

(1) Remove methyl DNA tags and unwind nucleosomes


(2) TranscriptionBindingProtein at the TATA box - attracts other GTFs (TBP is part of
1, of several GeneralTranscriptionFactors)
+ RNA polymerase II core, forming the pre-initiation complex
(3) Interaction of (upstream) activator (-200 bp), cis-enhancer sequences(200 ++)
(4) Transcription Initiation
(5) Dissociation of GTP and Elongation

TBP

Nucleosome wound, promoter methylated



3

(1) Chromatin
remodeling
(changing of the
nucleosome
position),
(2) epigenetic marking
(methylation,
acetylation)and
(3) X chromosome
inactivation

Positive or negative ?

RNA polymerase cannot


bind to a promoter
without an activator
binding near the
promoter site.

DYNAMIC CHROMATIN



Sect 12.3

5

Figure 4-55 Molecular Biology of the Cell ( Garland Science 2008)

4 level of packing for


metaphase chromosomes

Unwound for
expression

2

2

3

3

4

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Figure 4-72 Molecular Biology of the Cell ( Garland Science 2008)

The structure of chromatin

H1 linker histone ; H2A, H2B, H3 H4 core histones



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DNA methylation (op promoters) inhibits transcription by : blocking binding


of GTF necessary for transcription or; they have a role in positioning
nucleosomes. Regardless, they are located on CG palindromes and they
inhibit transcription.

Pp 429 Lysine and arginine in the histone tails can be covalently and reversibly
modified (150 ways) by the attachment of methyl and acetyl groups 2nd level
of information (histone code epigenetic inheritance and control).

DNA methylation is more stable than histone


modifications such as acetylation or methylation (and
better understood). DNA methylation is often associated
with long-term gene inactivation and inherited
modifications (pp 431), whereas histone code
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modifications may be shorter term.


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Replication- old disassembled and
mixed with new nucleosomes
( epigenetic marks inherited ?),
parental strand is methylated

Histones in active genes are hyperacetylated, +



Inactive genes are underacetylated (hypoacelated -).



How might acetylation affect chromatin remodeling & gene expression ?

a) Acetylation + : predispose a nucleosome to move. remodel

b) Acetylation + : alter the packing interaction between adjacent
nucleosomes opening up a strand for transcription. remodel



c) Acetylation+ : with other histone modifications (methylation) influences
the binding of other regulatory proteins to DNA. expression

Heterochromatin, Euchromatin

Methylated DNA +

Acetylated histone -

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Methylated DNA -

Acetylated histone +


Enhanceosomes
recruit chromatin
remodelers to allow
transcription to begin

CBP coactivator binding protein


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(1) (a) Remove methyl DNA tags and b) unwind nucleosomes (+ acetylate histone tails)
(2) TranscriptionBindingProtein at the TATA box - attracts other GTFs (TBP is part
of 1, of several GeneralTranscriptionFactors)
+ RNA polymerase II core, forming the pre-initiation complex
(3) Interaction of (upstream) activator (-200 bp), cis-enhancer sequences(200 ++)
(4) Transcription Initiation
(5) Dissociation of GTP and Elongation

TBP

Nucleosome wound, promoter methylated



3

Separable, Dual Binding


and Activation Roles in
Upstream Activation
Sequences

Activators: Gal 4 dimer interacts directly


with DNA and recruits TFIID and
indirectly via a mediator protein to
recruit RNA polymeraseII

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Model for eukaryotic promoters

In eukaryotic
gene regulation
of expression
there are both
activators,
enhancers,
silencers and
repressors that
each affect the
rate of
transcription

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Eukaryotic Expression control evolved differently in different lineages (T


Phillips and L. Hoopes Nature Education 1:1)



Complexity of transcriptional control can be illustrated by comparing
the number and locations of cis-control elements in higher and lower
eukaryotes.



For instance, Drosophila typically has several enhancers for a single gene
of 2 to 3 kilobases, scattered over a large (10 kilobase) region of DNA,
while,



yeast have no enhancers but instead use one UAS sequence per gene,
located upstream (activators).

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Figure 7-44 Molecular Biology of the Cell ( Garland Science 2008)

Production regulation within eukaryotes may


be complicated, There could be control of
DNA transcription, control of splicing and
translating proteins and control through the
feedback with various metabolic pathways or
cellular processes.


Transcriptional control may be
effected by upstream enhancers on the same
strand of DNA (cis-effects). Alternatively,
hormones originating in other cells can bind to
specific receptors in a cell membrane, a signal
is moved to the nucleus and the signal is
transported into the nucleus(trans acting). In
the nucleus, a protein signal may activate
transcription by acting locally on DNA,
stimulating the expression of one or more
genes.

There can also be regulation at the splicing
level, regulation of transport out of the
nucleus, regulation of the level of translation
(number of ribosomes, degradation etc.).

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Castel and Martensen Nature Reviews Genetics 14


Small RNA (<200 bp) that can regulate genes:
1) micro RNA (miRNA) hairpin derived RNA with
imperfect complementarity to targets that interfere with
translation.
2)small interfering RNA (siRNA) perfect complementarity to
their target, and they degrade transcripts.
3)PIWI interacting RNA (piRNA) which target transposon
transcripts in animals
The mechanistic details of 1-3 are converging and are more
commonly all referred to as RNAi
RNAi - temporary or reversible gene expression knockouts.
See 8.5 fig 8-21to 8-24,

siRNAs degrade mRNA from viral


genes or transposons, and may
repress gene transcription by
directing epigenetic modification of
chromatin.

Prime molecule in small
interfering (siRNA 19-40
nucleotides in the nucleus)

In the nucleus

Sense coding RNA



Antisense- complement

RNA Induced Silencing


Complex (or RITSC)

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Production regulation within eukaryotes may


be complicated, There could be control of
DNA transcription, control of splicing and
translating proteins and control through the
feedback with various metabolic pathways or
cellular processes.


Transcriptional control may be
effected by upstream enhancers on the same
strand of DNA (cis-effects). Alternatively,
hormones originating in other cells can bind to
specific receptors in a cell membrane, a signal
is moved to the nucleus and the signal is
transported into the nucleus(trans acting). In
the nucleus, a protein signal may activate
transcription by acting locally on DNA,
stimulating the expression of one or more
genes.

There can also be regulation at the splicing
level, regulation of transport out of the
nucleus, regulation of the level of translation
(number of ribosomes, degradation etc.).

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Micro Rna regulation of DNA expression.

Micro RNA (miRNA) ~19-23 nucleotide long, non-coding hairpin RNA



Function repressors in all plants and animals

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miRNAs halt translation from


targeted genes

Double stranded RNA
( complementary binding) is
necessary, miRNA are capped and
have a poly A tail otherwise they are
not exported

Prime molecule miNRA is

Dicer, an RNAIIIase, endonuclease

Unwound by Argonaute
(protein), one strand is retained
by siRISC, the other is degraded

Translational repression or
transcript degradation

Micro RNA (miRNA) ~19-23 nucleotide long, noncoding RNA Function translational repressors in all
plants and animals

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Long non-coding RNA Pauli et al.2011. Nature Reviews V12.



lncRNA > 200 bases: regulate gene expression by
transcriptional interference and chromatin modification
(epigenetic).
There is no known post transcriptional modification of lncRNA
unlike RNAi that can be used to block their effects.

Imprinting. Parental-specific, monoallelic expression of gene



clusters is based on differentially methylated imprinting control regions
(ICRs).


Problem 67 ch 2. Males
are black or orange,
females are black,
orange or calico.

A model for Xchromosome inactivation.


Long Non-coding RNA
(LncRNA) pp 444

Most genes are diploid,
regulated to co-express, but
1 X chromosome is largely
randomly inactivated
(multiple chromatin
modifications)just after cell
fate is decided in the
blastula..

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Figure 7-90 Molecular Biology of the Cell ( Garland Science 2008)

Three experiments demonstrating gene silencing

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Contrary to expected, Jorgensen et al.


observed reduced flower
pigmentation in Renilla and



Fire et al. demonstrated dsRNA
induced silencing of a gfp
transgene.



c) In a cell free- system, Renilla (RrLuc and firefly (Pp-Luc) luciferase
and homologous dsRNA and
normalized reporter activity.



d) The Dicer assay dsRNA
processed to siRNA



e) RISC assay siRNA cleaves
mRNA into smaller pieces

Liu & Paroo 2010 Ann.
Rev. Biochem.

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