Regulation of Gene Expression Dr. Ishtiaq Ahmad Khan.

Regulation of Gene Expression

Dr. Ishtiaq Ahmad Khan

Today’s lecture• Gene expression

• Constitutive, inducible, repressible genes

• Specificity factors, activators, repressors

• Negative and positive gene regulation

• Lac operon

• Helix-turn-helix motifs

• Zinc-fingers

• Leucine zippers

What is gene expression?

• Biological processes, such as transcription, and in case of proteins, also translation, that yield a gene product.

• A gene is expressed when its biological product is present and active.

• Gene expression is regulated at multiple levels.

Regulation of gene expression

Plasmid

Gene (red) with an intron (green)Promoter

2. Transcription

Primary transcript

1. DNA replication

3. Posttranscriptional processing

4. Translation

mRNA degradation

Mature mRNA

5. Posttranslational processing

Protein degradationinactiveprotein

activeprotein

single copy vs. multicopy plasmids

Gene regulation (1)

Chr. I

Chr. II

Chr. III

Condition 1

“turned on”

“turned off”

Condition 2

“turned off”

“turned on”

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25 26

constitutively expressed gene

induced gene

repressedgene

inducible/ repressible genes

Gene regulation (2)

constitutively expressed gene

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25 26

Condition 3 Condition 4 upregulated gene expression

down regulated gene expression

Definitions• Constitutively expressed genes:

– Genes that are actively transcribed (and translated) under all experimental conditions, at essentially all developmental stages, or in virtually all cells.

• Inducible genes:– Genes that are transcribed and translated at

higher levels in response to an inducing factor

• Repressible genes:– Genes whose transcription and translation

decreases in response to a repressing signal

Definitions

• Housekeeping genes: – genes for enzymes of central metabolic

pathways (e.g. TCA cycle)– these genes are constitutively expressed– the level of gene expression may vary

Modulators of transcription• Modulators:

(1) specificity factors, (2) repressors, (3) activators

1. Specificity factors:Alter the specificity of RNA polymerase

Examples: -factors (TBPs

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Heat shock geneHousekeeping gene Heat shock promoter

Standard promoter

Modulators of transcription2. Repressors:

mediate negative gene regulationmay impede access of RNA polymerase to the

promoteractively block transcriptionbind to specific “operator” sequences (repressor

binding sites) Repressor binding is modulated by specific effectors

Coding sequence

Repressor

Operator

Promoter

Effector(e.g. endproduct)

Negative regulation (1)

Source: Lehninger pg. 1076

Repressor

EffectorExample: lac operon

RESULT:Transcription occurs when the gene is derepressed

Negative regulation (2)


Repressor

Effector (= co-repressor)Example: pur-repressor in E. coli; regulates transcription of genes involved in nucleotide metabolism

Modulators of transcription3. Activators:

mediate positive gene regulation

bind to specific regulatory DNA sequences (e.g. enhancers)

enhance the RNA polymerase -promoter interaction and actively stimulate transcription

common in eukaryotes

Coding sequence

Activator

promoter

RNA pol.

Positive regulation (1)


RNA polymerase

Activator

Positive regulation (2)


RNA polymerase

Activator Effector

Operons

– a promoter plus a set of adjacent genes whose gene products function together.

– usually contain 2 –6 genes, (up to 20 genes)– these genes are transcribed as a polycistronic

transcript.– relatively common in prokaryotes– rare in eukaryotes

The lactose (lac) operon

• Contains several elements– lacZ gene = -galactosidase– lacY gene = galactosidase permease– lacA gene = thiogalactoside transacetylase– lacI gene = lac repressor

– Pi = promoter for the lacI gene– P = promoter for lac-operon– O1 = main operator– O2 and O3 = secondary operator sites (pseudo-operators)

Pi P Z Y A I O3 O1 O2

The lac operon consists of three structural genes, and a promoter, a terminator,regulator, and an operator. The three structural genes are: lacZ, lacY, and lacA.

• lacZ encodes β-galactosidase (LacZ), an intracellular enzyme that cleaves the disaccharide lactose

into glucose and galactose.• lacY encodes β-galactoside permease (LacY),

a membrane-bound transport protein that pumps lactose into the cell.

• lacA encodes β-galactoside transacetylase (LacA), an enzyme that transfers an acetyl group from acetyl-CoA to β-galactosides.

• Only lacZ and lacY appear to be necessary for lactose catabolism.

Theodor Hanekamp © 2003 18

First Level• The lacI gene coding for the repressor lies nearby the lac operon

and is always expressed (constitutive).• Hinder production of β-galactosidase in the absence of lactose. • If lactose is missing from the growth medium, the repressor binds

very tightly to a short DNA sequence called the lac operator. • The repressor binding to the operator interferes with binding of RNA

Pol to the promoter, and therefore mRNA encoding LacZ and LacY is only made at very low levels.

• When cells are grown in the presence of lactose, however, a lactose metabolite called allolactose , which is a combination of glucose and galactose, binds to the repressor, causing a change in its shape.

• Thus altered, the repressor is unable to bind to the operator, allowing RNAP to transcribe the lac genes and thereby leading to higher levels of the encoded proteins.

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Second Level• The second control mechanism is a response to glucose, which

uses the Catabolite activator protein (CAP) to greatly increase production of β-galactosidase in the absence of glucose.

• Cyclic adenosine monophosphate (cAMP) is a signal molecule whose prevalence is inversely proportional to that of glucose.

• It binds to the CAP, which in turn allows the CAP to bind to the CAP binding site (a 16 bp DNA sequence upstream of the promoter on the left in the diagram below),

• which assists the RNAP in binding to the DNA. In the absence of glucose, the cAMP concentration is high and binding of CAP-cAMP to the DNA significantly increases the production of β-galactosidase

• enabling the cell to hydrolyse (digest) lactose and release galactose and glucose.


Regulation of the lac operon

Pi P Z Y A I Q3 Q1 Q2

Inducer molecules: Allolactose: - natural inducer, degradableIPTG (Isopropylthiogalactoside)- synthetic inducer, not metabolized,

lacI repressor

Pi P Z Y A I Q3 Q1 Q2

LacZ LacY LacA

Selected DNA binding motifs1. Helix-turn-helix

• Homeodomain

2. Zinc Fingers• Cys4 zinc finger• Cys2 His2 zinc finger (e.g. TFIIIA)

3. Basic domains• Leucine zippers factors (bZIP)• Basic helix-loop-helix (bHLH)

4. Beta-scaffold factors with minor groove contacts

• HMG (High mobility group) proteins

Helix-turn-helix motifs

GENEGalR A T I K D V A R L A G V S V A T V S R V I N-cro F G Q T K T A K D L G V Y Q S A I N K A I HP22-cro G T Q R A V A K A L G I S D A A V S Q W K E

Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

alpha - helix turn alpha - helix

Structure:• about 20 amino acids long• 2 short alpha helicies ( 7 – 9 amino acids long)• DNA recognition helix (binds specific DNA sequence)• Recognition helix and 2nd helix form ~ 90° angle• very short turn ( NOT a beta-turn) • Often glycine at start of the turn (helix breaker)

How does the lac repressor bind DNA?


DNA

DNA recognition helix

LacI repressor (helix-turn-helix domain)

turn

Second alpha helix

Zinc-Finger Motifs

C

C

H

HZn

C

C

H

HZn

C

C

H

HZn

Several subtypes (Cys4, Cys2-His2 …)• Example: Cys2 His2 type• Zinc does not interact with DNA• Usually multiple zinc-fingers in a row • At least some also bind RNA• Consensus sequence:

[Y,F]-X-C-X2-4-C-XXX-F-XXXXX-L-XX-H-X3-5-H

Basic domainsLeucine zippers (bZip):• Basic region of the protein binds to DNA• Mainly act as dimers or other sometimes as other multimers• Special alpha-helices allow formation of coiled-coil

structures.• Hydrophobic residues (Leu) align on one side of the helix• Example: Jun and Fos

7 7 7 7


abcdefg

Leucine zippers

DNA

Leucines


Transcription attenuation

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Some Genes Are Regulatedby Genetic Recombination

30Example of Salmonella typhimurium

Regulation of Eukaryotic Gene Expression

Gene Regulation at DNA Level

Chromatin Remodeling

1. Changes of DNA Topo structure

Formation of ssDNA

DNase I hypersensitive site

DNA Methylation

2. DNA Methylation

CpG islands

----- are genomic regions that contain a high

frequency of CG dinucleotides.

----- CpG islands particularly occur at or near

the transcription start site of housekeeping genes.

Active transcriptionUnmethylated CpG island

TF RNA pol

Repressed transcription

Methylated CpG island

TF RNA pol

CH3 CH3 CH3

3. Histone modification

methylation

acetylation

TFTF

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Functions of Histone methylation in transcription

Most well-studied histone modifications are involved in control of transcription.Actively transcribed genesTwo histone modifications are particularly associated with active transcription:•Trimethylation of H3 lysine 4 (H3K4Me3) at the promotor of active genes •Trimethylation of H3 lysine 36 (H3K36Me3) in the body of active genesRepressed genesThree histone modifications are particularly associated with repressed genes:•Trimethylation of H3 lysine 27 (H3K27Me3)•Di and tri-methylation of H3 lysine 9 (H3K9Me2/3)•Trimethylation of H4 lysine 20 (H4K20Me3)

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Acetylated histones and nucleosomes represent a type of epigenetic tag within chromatin. Acetylation removes the positive charge on the histones, thereby decreasing the interaction of the N termini of histones with the negatively charged phosphate groups of DNA. As a consequence, the condensed chromatin is transformed into a more relaxed structure that is associated with greater levels of gene transcription.

Functions of Histone methylation in transcription

7.3 Transcriptional Regulation

1. Cis-acting element

(1) What is cis-acting element?

Concept

Cis-acting elements - DNA sequences close

to a gene that are required for gene expression

2. What is trans-acting factor?

Concept

trans-acting factors - usually they are

proteins, that bind to the cis-acting elements to

control gene expression.

These trans-acting factors can control gene

expression in several ways:

may be expressed in a specific tissue

may be expressed at specific time in development

may be required for protein modification

may be activated by ligand binding

Domains of trans-acting factors

DNA binding domain DBD

transcription activating domain

Post-Transcriptional Regulation

1. Gene Regulation of mRNA Processing

exon shuffling

alternative gene splicing

2. Gene Regulation of mRNA Editing

3. mRNA Longevity

4. mRNA Transport Control

5. RNA Interference (RNAi)

miRNA

siRNA

7.5 Translational and Post-translational Regulation

1. Translation Control

Blocking mRNA Attachment to Ribosomes

2. Regulation of Protein Processing

Protein Modification

3. Regulation of Protein Stability

Regulation of Gene Expression Dr. Ishtiaq Ahmad Khan.

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