L13Biol261ERegulation2013

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Negative control

Default state is on,

Positive control

Default state is off-

Inducible An inducer and /or

activator

De-represses a

gene, turns a geneon or, promotes

transcription

Lac operon control

The inducer (lactose) binds to

the repressor releasing it from

the operator and preventing

repressor binding, allowing

expression -on

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.

Repressible A repressor slows

down or stops theexpression of a

gene, but a co-

repressor must bindto it first

Tryptophan global regulation

A repressor is produced by the

trp R site, but it cannot bind tothe tryptophan operator unless

tryptophan first binds to it (i.e.

on), together tryptophan /

repressor down regulates the

expression of tryptophan

Glucose or its catabolite represses

CAMP expression. Without CAMP,

the CAP does not bind to the 5’

end of the promoter. Without CAP-

CAMP binding there is little

transcription by the lac operon -off.

C



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 moregenes.

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.).

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.

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(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

3

Nucleosome wound, promoter methylated



(1) Chromatin

remodeling

(changing of the

nucleosome position),

(2) epigenetic marking

(methylation,

acetylation)and

(3) X chromosome

inactivation

4

Positive or negative ? RNA polymerase cannot

bind to a promoter

without an activator

binding near the

promoter site.



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

DYNAMIC CHROMATIN

Sect 12.3

5




1

2

3

4

2

3

4

4 level of packing formetaphase chromosomes

Unwound forexpression

6



7



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 bindingof GTF necessary for transcription or; they have a role in positioningnucleosomes. Regardless, they are located on CG palindromes and they

inhibit transcription.



DNA methylation is more stable than histonemodifications such as acetylation or methylation (and

better understood). DNA methylation is often associatedwith long-term gene inactivation and inheritedmodifications (pp 431), whereas histone code

modifications may be shorter term.

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

8



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 adjacentnucleosomes opening up a strand for transcription. remodel

c) Acetylation+ : with other histone modifications (methylation) influences

the binding of other regulatory proteins to DNA. expression

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

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Heterochromatin, Euchromatin

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Methylated DNA + Acetylated histone -

Methylated DNA - Acetylated histone +



Enhanceosomesrecruit 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

3

Nucleosome wound, promoter methylated



Separable, Dual Bindingand Activation Roles inUpstream ActivationSequences

Activators: Gal 4 dimer interacts directlywith DNA and recruits TFIID and

indirectly via a mediator protein torecruit RNA polymeraseII

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Model for eukaryotic promoters

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In eukaryoticgene regulationof expression

there are bothactivators,enhancers,silencers andrepressors that

each affect therate oftranscription



Eukaryotic Expression control evolved differently in different lineages (TPhillips and L. Hoopes Nature Education 1:1)

“Complexity of transcriptional control can be illustrated by comparingthe number and locations of cis-control elements in higher and lowereukaryotes.

For instance, Drosophila typically has several enhancers for a single geneof 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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hormones originating in other cells can bind tospecific receptors in a cell membrane, a signal















cellular processes.

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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 withtranslation.

•

2)small interfering RNA (siRNA) perfect complementarity totheir target, and they degrade transcripts.

• 3)PIWI interacting RNA (piRNA) which target transposontranscripts in animals

•

The mechanistic details of 1-3 are converging and are morecommonly all referred to as RNAi

• RNAi - temporary or reversible gene expression knockouts.

See 8.5 fig 8-21to 8-24,



Prime molecule in smallinterfering (siRNA – 19-40nucleotides in the nucleus)

Sense –coding RNA

Antisense- complement

In the nucleus

RNA Induced SilencingComplex (or RITSC)

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siRNAs degrade mRNA from viralgenes or transposons, and mayrepress gene transcription by

directing epigenetic modification ofchromatin.






hormones originating in other cells can bind tospecific receptors in a cell membrane, a signal















cellular processes.

15



Micro Rna regulation of DNA expression.

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Micro RNA (miRNA) ~19-23 nucleotide long, non-coding hairpin RNAFunction – repressors in all plants and animals



miRNAs halt translation fromtargeted genes

Double stranded RNA

( complementary binding) isnecessary, miRNA are capped andhave a poly A tail otherwise they arenot 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 ortranscript degradation

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Micro RNA (miRNA) ~19-23 nucleotide long, non-coding RNA Function translational repressors in all

plants and animals



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• lncRNA > 200 bases: regulate gene expression bytranscriptional interference and chromatin modification

(epigenetic).

• There is no known post transcriptional modification of lncRNA – unlike RNAi that can be used to block their effects.

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

Imprinting. Parental-specific, monoallelic expression of geneclusters is based on differentially methylated imprinting control regions(ICRs).



A model for X-

chromosome inactivation.Long Non-coding RNA(LncRNA) pp 444

Most genes are diploid,regulated to co-express, but1 X chromosome is largelyrandomly inactivated(multiple chromatinmodifications)just after cell fate is decided in theblastula..

Problem 67 ch 2. Malesare black or orange,females are black,

orange or calico.

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Three experiments demonstrating gene silencing

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Contrary to expected , Jorgensen et al.observed reduced flowerpigmentation in Renilla and

Fire et al. demonstrated dsRNA –induced silencing of a gfptransgene.

c) In a cell free- system, Renilla (Rr-

Luc and firefly (Pp-Luc) luciferaseand homologous dsRNA andnormalized reporter activity.

d) The Dicer assay dsRNAprocessed to siRNA

e) RISC assay siRNA cleavesmRNA into smaller pieces

Liu & Paroo 2010 Ann.Rev. Biochem.

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