Etty Widayanti, SSi. MBiotech.Bagian Anatomi Sub Bagian BiologiFak. Kedokteran Univ. YARSI

Nov 2009

Regulation of gene expression1. 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 cellsThe 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

cell does not waste energy making enzymes it does not need.Gene expression synthesis of a gene product Constitutive

2. Regulated / inductive

1. Constitutive gene expressione.g. "housekeeping genes" like primase ssDNA binding proteinsexpression of genes at about the same level under all environmental conditions

2. Regulated gene expressionControl of the rate of protein or RNA synthesis as an adaptive response to stimuli.induction: increase in gene expressionrepression: 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 inducerB-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 BacteriaThe 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- repressorthe 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 repressorThe Operon Model

Lac Operon

Example of gene induction: Regulation of the lac operonThe lac operon is a group of genes used for catabolism of the sugar lactoseZYAlac genespromoteroperator

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:

In presence of inducer, it does not bind DNA

2. In absence of inducer, it binds strongly to lac operator DNANegative Control of the lac Operon by the lacI Repressor

Glucose indirectly inhibits lac expressionglucose lacif glucose is high cAMP is lowPositive Control of the lac Operon:CAP and Catabolite Repression Lac ZRemember 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 expressionThe "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 biosynthesisOperonsThink 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 continuesTryptophan OperonMechanism 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 levelsPOE D C B A1. repression by trp repressor (on/off)genes encoding the enzymes used for tryptophan biosynthesis2. attenuation (fine tuning by transcriptional termination)Gen trp R AporepressorTrp = 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 methods used by eukaryotesTranscription Control The most common type of genetic regulation Turning on and off of mRNA formation

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

Translational Control Regulation of the rate of Initiation

Post-Tranlational Control Regulation of the modification of an immature or inactive protein to form an active protein

DNAnew RNA transcriptmRNAmRNApolypeptide chainactive proteinA. TranscriptionB. mRNA processingC. mRNA transportD. translationE. Protein processingnucleuscytoplasm

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

Chemical modifications and chromosome duplications affect RNA polymerasesphysical access to genes.

B. mRNA processingNew 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.

C. mRNA transportRNA cannot pass through a nuclear poreunless bound to certain proteins.

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

An mRNAs 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 ControlRNA polymerase II (pol II) is a complex of some 10 different proteins. The start site is where transcription of the gene into mRNA begins. Transcription start site

Transcriptional 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 versatility. Mixing and matching of proteins into different heterodimers and homodimers means that three distinct complexes can be formed from two proteins.

Diverse in nature, but several common structures are found:- Helix-turn-helix (homeodomain) - three different planes of the helix are established and bind to the grooves of the DNA - Zinc fingers - cystine and histidine residues bind to a Zn2+ ion, looping the amion acid into a finger-like chain that will rest in the grooves of DNA - Leucine zipper - dimers result from leucine residues at every other turn of the a-helix. When the a- helical regions form a leucine zipper, the regions beyond the zipper form a Y-shaped region that grips the DNA in a scissors-like configuration

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