Gene regulation

Gene regulationGene regulation

Necessity of Gene RegulationNecessity of Gene Regulation

Unicellular organismsUnicellular organismso Depending on the needs of the body some Depending on the needs of the body some

genes have to be transcribed whereas the genes have to be transcribed whereas the rest have to be switched offrest have to be switched off

o Helps to adapt to the changes in environmentHelps to adapt to the changes in environment

Multicellular organismsMulticellular organisms Helps in differentiation of cellsHelps in differentiation of cells Performance of various functions in the bodyPerformance of various functions in the body

Gene Regulation Mechanisms in Gene Regulation Mechanisms in Prokaryotes and EukaryotesProkaryotes and Eukaryotes

In prokaryotes the primary control point is In prokaryotes the primary control point is the process of transcription initiation the process of transcription initiation

In eukaryotes expression of gene into In eukaryotes expression of gene into proteins can be controlled at various proteins can be controlled at various locations.locations.

Gene Gene RegulationRegulation in Prokaryotes in Prokaryotes

Operon hypothesis- Proposed by two French Operon hypothesis- Proposed by two French microbiologists, Francois Jacob and Jacques microbiologists, Francois Jacob and Jacques Monod in 1961 for which they won the nobel Monod in 1961 for which they won the nobel prize in 1965. prize in 1965.

Operon -In prokaryotes the linked genes are Operon -In prokaryotes the linked genes are clustered into units known as operons. The clustered into units known as operons. The genes in a single operon affect the same genes in a single operon affect the same biochemical pathway that is either they are biochemical pathway that is either they are expressed or repressed under similar condition.expressed or repressed under similar condition.

Polycistronic RNA – In prokaryotes a single Polycistronic RNA – In prokaryotes a single operon gets transcribed into polycistronic mRNA operon gets transcribed into polycistronic mRNA which can be translated into multiple proteins. which can be translated into multiple proteins.

Control of Transcriptional Control of Transcriptional InitiationInitiation

Promoter sequencesPromoter sequences –Help the enzyme RNA –Help the enzyme RNA polymerase to recognize the transcriptional initiation polymerase to recognize the transcriptional initiation sites sites o -35 position is TTGACA -35 position is TTGACA o -10 position is TATAAT-10 position is TATAAT

Accessory or regulatory proteinsAccessory or regulatory proteins –Control the ability –Control the ability to recognize the transcriptional initiation sites to recognize the transcriptional initiation sites o Activators Activators o Repressors Repressors

Operators - Operators - The interaction of the regulatory proteins The interaction of the regulatory proteins with the operators modulates the accessibility of with the operators modulates the accessibility of promoter regions of prokaryotic DNA. promoter regions of prokaryotic DNA.

Transcriptional Regulation in E.coliTranscriptional Regulation in E.coli

In E.coli the following two kinds of operons In E.coli the following two kinds of operons exist and in both the cases the gene exist and in both the cases the gene regulation is carried out by repressor proteins. regulation is carried out by repressor proteins. o Catabolite-regulated operonsCatabolite-regulated operons -They are the -They are the

operons which produce gene products necessary operons which produce gene products necessary for the utilization of energy. Example for the utilization of energy. Example lac lac operon.operon.

o Attenuated operonsAttenuated operons – These operons produce – These operons produce gene products necessary for the synthesis of gene products necessary for the synthesis of small biomolecules such as amino acids. These small biomolecules such as amino acids. These operons are typically attenuated by the sequences operons are typically attenuated by the sequences within the transcribed RNA. Example within the transcribed RNA. Example trp trp operon operon

The The laclac operon operon

Components of Components of laclac operon operon Regulatory geneRegulatory gene

o The regulatory gene is the The regulatory gene is the ii gene that codes for gene that codes for the repressor protein of the the repressor protein of the laclac operon. operon.

o This This ii gene is expressed all the time hence it is gene is expressed all the time hence it is also known as a constitutive gene.also known as a constitutive gene.

o The lac repressor protein has two functional The lac repressor protein has two functional domains or regions one that binds the operator domains or regions one that binds the operator sequence and the other that binds the lactose sequence and the other that binds the lactose sugar. sugar.

Components of Components of laclac Operon Operon Structural genesStructural genes

o lac Zlac Z codes for β-galactosidase (β-gal), which codes for β-galactosidase (β-gal), which is primarily responsible for the hydrolysis of the is primarily responsible for the hydrolysis of the disaccharide, lactose into its monomeric units, disaccharide, lactose into its monomeric units, galactose and glucose.galactose and glucose.

o Lac Y Lac Y codes for permease, which transports codes for permease, which transports lactose into the cell.lactose into the cell.

o Lac A Lac A codes for transacetylase whose function codes for transacetylase whose function is not considered hereis not considered here

OperatorOperator PromoterPromoter

Structure of a Structure of a laclac operon operon

Image reference - http://www.emunix.emich.edu/~rwinning/genetics/proreg.htm

In the presence of an inducer that is In the presence of an inducer that is lactoselactose

Lactose binds repressorLactose binds repressor Repressor undergoes conformational changeRepressor undergoes conformational change Repressor does not bind operatorRepressor does not bind operator Lac Lac operon is transcribed operon is transcribed

Image reference - http://www.emunix.emich.edu/~rwinning/genetics/proreg.htm

In the absence of an inducer that is In the absence of an inducer that is lactoselactose

Repressor protein is constitutively producedRepressor protein is constitutively produced Enzymes required for the lactose metabolism Enzymes required for the lactose metabolism

are not producedare not produced

Image reference - http://www.emunix.emich.edu/~rwinning/genetics/proreg.htm

Catabolite RepressionCatabolite Repression Feed back control of Feed back control of lac lac operon through Catabolite repressionoperon through Catabolite repression

Transcription of Transcription of lac lac operon takes place with the help of another protein operon takes place with the help of another protein named catabolite activator protein (CAP for short).named catabolite activator protein (CAP for short).

When a small molecule called cyclic AMP (cAMP) binds CAP it is able When a small molecule called cyclic AMP (cAMP) binds CAP it is able to bind the promoter region of the to bind the promoter region of the laclac operon operon

In the absence of cAMP, CAP fails to bind to the promoter region and In the absence of cAMP, CAP fails to bind to the promoter region and hence no transcription takes place.hence no transcription takes place.

cAMP is produced by an enzyme called adenylcyclasecAMP is produced by an enzyme called adenylcyclase In the presence of glucose in the environment the following changes In the presence of glucose in the environment the following changes

take place-take place-o Synthesis of adenylcyclase is inhibitedSynthesis of adenylcyclase is inhibitedo cAMP production drops downcAMP production drops downo (cAMP – CAP) complex does not form(cAMP – CAP) complex does not formo CAP fails to bind to the promoter sequenceCAP fails to bind to the promoter sequenceo Transcription of Transcription of laclac operon does not take place operon does not take place

Acts like a feed back mechanismActs like a feed back mechanismo Lactose ------- Beta-galactosidase -----> Glucose ↑ + GalactosidaseLactose ------- Beta-galactosidase -----> Glucose ↑ + Galactosidase

o Transcription of Transcription of lac lac operon is inhibitedoperon is inhibited

Inducible and Repressible OperonsInducible and Repressible Operons

Inducible operonInducible operon - the effector molecule - the effector molecule interacts with the repressor protein such that it interacts with the repressor protein such that it can not bind to the operator.can not bind to the operator.o Lac operon is an inducible system as this operon is Lac operon is an inducible system as this operon is

always turned off except in the presence of an inducer always turned off except in the presence of an inducer that is lactose.that is lactose.

Repressible operonRepressible operon - the effector molecule - the effector molecule interacts with the repressor protein such that it interacts with the repressor protein such that it can bind to the operator can bind to the operator o trptrp Operon is an example of repressible system which Operon is an example of repressible system which

means that it is automatically switched on and stops means that it is automatically switched on and stops only when a repressor becomes active and binds it only when a repressor becomes active and binds it

The The trptrp Operon Operon Components of the Components of the trptrp operon are the operon are the

promoter, operator and five structural promoter, operator and five structural genes which are responsible for genes which are responsible for tryptophan biosynthesis that is tryptophan biosynthesis that is trpE, D, C, trpE, D, C, BB and and AA. . trpLtrpL gene lies between operator gene lies between operator and and trpE gene.trpE gene.

The The trptrp operon is controlled by two operon is controlled by two mechanismsmechanismso Negative control systemNegative control systemo Translation-induced transcriptional Translation-induced transcriptional

attenuation attenuation

Negative control systemNegative control system

Presence of Presence of Co-repressorCo-repressor that isthat is tryptophan tryptophan moleculemolecule

Translation-induced transcriptional Translation-induced transcriptional attenuationattenuation

Role of trpLRole of trpL GeneL which is situated GeneL which is situated

immediately at the 5' of the immediately at the 5' of the trpEtrpE gene is 160 bp in length gene is 160 bp in length and controls the expression of and controls the expression of the trp operon by the process the trp operon by the process of attenuation.of attenuation.

The geneL codes for the The geneL codes for the leader region of RNA and this leader region of RNA and this region is located at the 5' end region is located at the 5' end of RNA.of RNA.

This region of RNA is capable This region of RNA is capable of forming different stable stem of forming different stable stem loop structures.loop structures.

Characteristics of the leader region Characteristics of the leader region of the mRNAof the mRNA

The leader region coded by gene L is made up The leader region coded by gene L is made up of 1-4 domains and contains tandem tryptophan of 1-4 domains and contains tandem tryptophan codons.codons.

The domain 3 of mRNA can base pair with either The domain 3 of mRNA can base pair with either domain 2 or domain 4.domain 2 or domain 4.

Domain 4 is known as attenuator as its presence Domain 4 is known as attenuator as its presence is required to stop the transcription. is required to stop the transcription.

The domain1 of the leader region of mRNA The domain1 of the leader region of mRNA codes for a chain of 14 amino acids which have codes for a chain of 14 amino acids which have two tryptophan residues.two tryptophan residues.

Under high levels of TryptophanUnder high levels of Tryptophan

As the cellular levels of tryptophan are high the levels of As the cellular levels of tryptophan are high the levels of tryptophan tRNA are also high.tryptophan tRNA are also high.

The ribosome complex moves through the domain 1 The ribosome complex moves through the domain 1 translating it into a small peptide.translating it into a small peptide.

The ribosome complex quickly moves to the domain 2 as The ribosome complex quickly moves to the domain 2 as there is an abundance of tryptophan tRNAthere is an abundance of tryptophan tRNA

When the ribosome complex lies associated with the When the ribosome complex lies associated with the domain 2 the domain 3 and 4 are free to form a stem domain 2 the domain 3 and 4 are free to form a stem loop structure.loop structure.

This stem loop structure is an intrinsic transcription This stem loop structure is an intrinsic transcription terminator which does not allow the RNA polymerase to terminator which does not allow the RNA polymerase to move furthermove further

This stops the transcription and related translationThis stops the transcription and related translation

Under low levels of TryptophanUnder low levels of Tryptophan

As the cellular levels of tryptophan are low the levels of As the cellular levels of tryptophan are low the levels of tryptophan tRNAs are also low making the translation of tryptophan tRNAs are also low making the translation of domain 1 slow.domain 1 slow.

Hence domain 2 is free to get associated with domain 3Hence domain 2 is free to get associated with domain 3 This also forms a stem loop structure but it is not a This also forms a stem loop structure but it is not a

terminator of transcriptionterminator of transcription This allows the continued transcription of the operon.This allows the continued transcription of the operon. Thus trp E –A are translated and the enzymes necessary Thus trp E –A are translated and the enzymes necessary

for the synthesis of tryptophan are producedfor the synthesis of tryptophan are produced..

Image reference - http://www.ndsu.edu/pubweb/~mcclean/plsc431/prokaryo/prokaryo3.htm

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