Etty Widayanti, SSi. Etty Widayanti, SSi. MBiotech.MBiotech.Bagian Anatomi Sub Bagian BiologiBagian Anatomi Sub Bagian Biologi

Fak. Kedokteran Univ. YARSIFak. Kedokteran Univ. YARSINov 2009

Regulation of gene expression

1. Not all genes are turned on (expressed) all the time.

2. Control of the rate of protein or RNA synthesis as an adaptive response to stimuli.

In general, they are turned ononly when needed

Why regulate gene expression?

Regulation allows cells to respond to environmental conditions by synthesizing selected gene products only when they are needed.

In Bacteria : often involves nutrient utilization pathway

In Eukaryotes: may involve the generation of specific proteins in specific types of cells

The First Model: Lactose utilization in E. coli:

B-galactosidase

lactose galactose + glucose(a disaccharide) H20 (energy)

• Cells should make the needed proteins at the right time• Do not waste energy

Gene expression synthesis of a gene product

1. Constitutive

2. Regulated / inductive

1. Constitutive gene expression

e.g. "housekeeping genes" like primase ssDNA binding proteins

expression of genes at about the same level under all environmental conditions

2. Regulated gene expression

Control of the rate of protein or RNA synthesis as an adaptive response to stimuli.

induction: increase in gene expression

repression: decrease in gene expression

Adaptation and Induction

The presence of substrate, lactose, caused the appearance of enzyme, B-galactosidase. Was this induction an "adaptation" of the enzyme to substrate just as the bacterium "adapts" to environment?

1. In absence of lactose 1-2 molecues/cellIn presence of lactose 100,000 molecules/cell

2. Synthesized nearly simultaneously and only after lac mRNA becomes detectable.

3. Lactose (and analogs) is inducer

B-galactosidase (and permease) are inducible

How does a cell "KNOW" what to make?

Monod, Jacob, Lwoff sought to explain inductionGENETICALLY

Control of Gene Expression in Bacteria

1. The lac operon (genetics)

2. Promoters and repressors

3. Other operons

1. Structural genes: lac Z, Y, A (transport & metabolism)Regulatory elements:the lac I gene- repressor

the lac O operatorthe lac P promoter

2. lac Z, Y, A in a single mRNA polycistronic

3, Promoter is adjacent to operator (lac P- - no mRNA)

4. lac I protein binds to operator represses transcription

5. Inducers, e.g. Lactose, bind to and inactivate repressor

The Operon Model

Lac Operon

Example of gene induction: Regulation of the lac operon

The lac operon is a group of genes used for catabolism of the sugar lactose

Z Y A

lac genespromoter

operator

The lac operon of E. coli

• When lactose is available, E. coli induces expression of lac operon.

• When lactose is unavailable, the catabolic enzymes are NOT needed.

The lac operon is expressed at only very low levels.

lac repressor is allosteric: it has two different conformations:

1. In presence of inducer, it does not bind DNA

2. In absence of inducer, it binds strongly to lac operator DNA

Negative Control of the lac Operon by the lacI Repressor

Glucose indirectly inhibits lac expressionglucose lacif glucose is high cAMP is low

Positive Control of the lac Operon:CAP and Catabolite Repression

Lac Z

Remember lactose galactose + glucoseIf lactose & glucose are present - no lacZ is made until glucose is depleted.How?

High cAMP is necessary for activation of lac operon cAMP is bound by CAP (catabolite activator protein) cAMP-CAP binds to distal part of promoter and facilitates transcription

• Glucose is normal energy and carbon source.

• Cell has "back-up" system to use lactose (lac).

• Even if lactose is present, it won't bother to make lacZ if glucose is present.

• Even if no glucose is present, operon isn't unduced until lactose appears.

• Lactose inhibits inhibition of lac expression

The "Logic" of Operon Control andResource Utilization

General Theme:A metabolite controls the expression of a battery ofgenes that have evolved to utilize it (CATABOLISM)

Other Examples:trp - tryptophan biosynthesisara - arabinose utilizationhis - histidine biosynthesis

Operons

Think About:Genetic logic of negative or positive controli. e. repression & activation

1. trpE is first gene in operon2. trpE mRNA has long leader (untranslated 5' region)3. region of mRNA works as attenuator:

• in presence of tryptophan, transcription is halted about 140 bases into mRNA

• in absence of tryptophan, transcription continues

Tryptophan Operon

Mechanism is complex, but logical• leader sequence contains short 11 amino acid peptide with two trp residues• when trp is abundant, trp-tRNA can be used to translate leader mRNA which terminates transcription• when trp is limiting, translation stalls and transcription is permitted

The trp operon is regulated at two levels

P O E D C B A

1. repression by trp repressor (on/off)

genes encoding the enzymes used for tryptophan biosynthesis

2. attenuation (fine tuning by transcriptional termination)

R

R

Gen trp R Aporepressor

Trp = co repressor

When the level of trp is high, trp does not have to be synthesized. Trp binds the repressor, and the repressor binds DNA and prevents RNA pol binding to the promoter.

Trp Attenuation

The latest estimates are that a human cell, a eukaryotic cell,

contains approximately 35,000 genes.

How is gene expression regulated?

There are several There are several methods used by methods used by eukaryoteseukaryotes

Transcription Control Transcription Control  The most common type of genetic regulation The most common type of genetic regulation Turning on and off of mRNA formationTurning on and off of mRNA formation

Post-Transcriptional ControlPost-Transcriptional Control Regulation of the processing of a pre-mRNA into a Regulation of the processing of a pre-mRNA into a

mature mRNAmature mRNA

Translational ControlTranslational Control Regulation of the rate of InitiationRegulation of the rate of Initiation

Post-Tranlational ControlPost-Tranlational Control Regulation of the modification of an immature or inactive Regulation of the modification of an immature or inactive

protein to form an active proteinprotein to form an active protein

DNA

new RNA transcript

mRNA

mRNA

polypeptide chain

active protein

A. Transcription

B. mRNA processing

C. mRNA transport

D. translation

E. Protein processing

nucleus

cytoplasm

Binding of transcription factors tospecial sequences in DNA slows or speeds transcription.

Chemical modifications and chromosome duplications affect RNA polymerase’sphysical access to genes.

A.Transcription

New mRNA cannot leave the nucleusbefore being modified, so controls overmRNA processing affect the timing oftranscription.

Controls over alternative splicing influence the final form of the protein.

B. mRNA processing

RNA cannot pass through a nuclear pore

unless bound to certain proteins.

Transport protein binding affects wherethe transcript will be delivered in thecell.

C. mRNA transport

An mRNA’s stability influences how long it is translated.

Proteins that attach to ribosomes or initiation factors can inhibit translation.

Double-stranded RNA triggers degradation of complementary mRNA.

D. Translation

A new protein molecule may become activated or disabled by enzyme-mediated modifications, such as phosphorylation or cleavage.

Controls over these enzymes influence may other cell activities.

E. Protein processing

Transcriptional Transcriptional Control Control 

RNA polymerase IIRNA polymerase II ( (pol IIpol II) is a complex of) is a complex of s some 10 different ome 10 different proteins. proteins.

The start site is where transcription of theThe start site is where transcription of the gene into mRNA begins. gene into mRNA begins.

Transcription start siteTranscription start site

Transcriptional Transcriptional Control Control 

The basal promoter contains a sequence of 7 bases (TATAAAA) called the TATA box (this is very similar to the -10 box or Pribnow box found in prokaryotes) .

It can be bound by Transcription Factor IID (TFIID read TF2D) which is a complex of some 10 different proteins including

- TATA-binding protein (TBP), which recognizes and binds to the TATA box

- other protein factors which bind to TBP - and each other - but not to the DNA.

The basal or core promoter is found in all protein-encoding genes. This is in sharp contrast to the upstream promoter whose structure and associated binding factors differ from gene to gene (i.e. they are unique to each specific gene).

The basal promoter

PROMOTER PROKARIOT & EUKARIOT

Just how do proteins bind to DNA?

DNA : Protein and Protein : Protein interactions are important for transcription factor function.

Note modular structure of transcription factors: one part of the protein is responsible for DNA binding, another for dimer formation, another for transcriptional activation (i.e. interaction with basal transcription machinery). 

Dimer formation adds an extra element of complexity and Dimer formation adds an extra element of complexity and versatility. Mixing and matching of proteins into different versatility. Mixing and matching of proteins into different heterodimers and homodimers means that three distinct complexes heterodimers and homodimers means that three distinct complexes can be formed from two proteins. can be formed from two proteins. 

Diverse in nature, but several common structures are found:Diverse in nature, but several common structures are found:- - Helix-turn-helix (homeodomain) - three different Helix-turn-helix (homeodomain) - three different

planes of the helix are established and bind to the planes of the helix are established and bind to the grooves of the DNA grooves of the DNA

- - Zinc fingers - cystine and histidine residues bind to Zinc fingers - cystine and histidine residues bind to a Zn2+ ion, looping the amion acid into a finger-like a Zn2+ ion, looping the amion acid into a finger-like chain that will rest in the grooves of DNA chain that will rest in the grooves of DNA

- - Leucine zipper - dimers result from leucine residues Leucine zipper - dimers result from leucine residues at every other turn of the a-helix.  When the a-at every other turn of the a-helix.  When the a- helical regions form a leucine zipper, the regions helical regions form a leucine zipper, the regions beyond the zipper form a Y-shaped region that grips beyond the zipper form a Y-shaped region that grips the DNA in a scissors-like configurationthe DNA in a scissors-like configuration

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