Biochemistry T.Y.BSc . (Semester-V) - PowerPoint Presentation

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BiochemistryT.Y.BSc. (Semester-V)Molecular Biology (BSCOC 507 A)Unit 1Regulation of gene expression

Prepared byHarshada ParalikarAssistant ProfessorNeotech College of Applied Science and Research,SGGU, Godhra

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Principle of gene regulationThe metabolism in prokaryotes is controlled by the regulation of gene expressionThe regulation of transcription depends on the activity of RNA polymerase as for the expression of any gene ( that is formation of protein product by translation) RNA polymerase has to 1

st transcribe the gene.This process is directed by an operon which comprises of structural genes, an operator gene and a promoter gene.

DNA

RNA

Protein

Transcription

Translation

Central Dogma

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What are regulated genes and operon?Genes whose activity is controlled in response to the needs of the cell or an organism are called regulated genes.An organism also has number of genes whose products are essential to the normal functioning of a growing and dividing cell, such genes which are always active in growing cells are called

constitutive genes.Likewise an enzyme that is present throughout or continuously in the cell is called a constitutional enzyme. Genes which encode protein that work together in a cell are organized into operon. An operon is a cluster of genes the expressions of which are regulated together by operator-repressor protein interactions plus operator region itself and the

promoter

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When the gene expression is turned on by adding a substance in the medium, the genes are said to be inducible genesThe regulatory substance that brings about gene induction is called as an inducer/ effector molecule.The E.coli

lac operon is an inducible operon.There are two controls for lac operon – 1. Negative control2. Positive controlThe order of the controlling elements and the genes in the lac operon is Promoter-operator-lazZ-

lacY-lacA and the regulatory genes lacI is located close to the structural genes just upstream of the promoter. The lacI gene has its own constitutive promoter and terminator

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Negative controlWhen lactose is absent in the medium:

Source:

Peter J Russell, igenetics

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Functional state of lac operon in wild type E.coli growing in absence of the lactose-The lac repressor protein encoded by lac I gene is made constitutively but it is affected by the presence of inducerHence when lactose is absent, the repressor gene (lacI+) is transcribed constitutively, the translation of its mRNA produces 360 amino acid polypeptide, 4 of which associate and form a tetramer( functional lac repressor protein)

The lac repressor binds to the operator.The DNA sequence covered by the repressor overlap the DNA sequence recognized by RNA polymeraseWhen repressor is bound to the operator, RNA polymerase cannot bind to the promoter and transcription of the structural genes cannot occur. So these genes are not expressed.

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Basal transcription:A low level of gene transcription that produce a few molecule of the enzymes takes place even in the absence of inducer Low concentration of each of the structural gene takes place for e.g. an average of 3 molecules of β galactosidase is producedThis occurs because repressors do not just bind and stay, they bind and dissociate

In the split second, after first repressor unbinds and before another binds, an RNA polymerase could initiate transcription of the operon even in absence of an inducer

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When Lactose is present as a sole carbon source:

Source: Peter J Russell, igenetics

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When lactose is provided as sole source of carbon, the enzymes required to metabolize lactose are rapidly synthesizeLactose is a disaccharide of monosaccharide unit – glucose & galactoseWhen lactose is present 3 proteins are synthesized1) β galactosidase (encoded by lac Z) This enzyme breaks down lactose into

glucose & galactoseCatalyses isomerization of lactose to allolactose2)Lactose permease: (encoded by lac Y)Protein found in E.coli cytoplasmic membraneTransports lactose into the cell

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3)Transacetylase: In the presence of lactose, but absence of glucose amount of each enzyme increases 1000 fold as genes are actively transcribed (co-ordinate induction)Allolactose is the inducer molecule responsible for the production of 3 enzymesWhen lactose is present as a sole carbon source some lactose is converted by

β galactosidase into AllolactoseAllolactose bids to the lac repressor and change its shape, this is called Allosteric shift Repressor losses its affinity for the lac operator and dissociate from the site

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Free repressor proteins are also altered so that cannot bind to the operatorWhen no repressor is bound to the operator, RNA polymerase initiates synthesis of a single polycistronic mRNA molecule for the lac Z+, lac Y

+, lac A+ genesThe polycistronic mRNA for the lac operon is translated by a string of ribosomes to produce the 3 proteins

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Positive control:

Source: Peter J Russell, igenetics

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Glucose is a preferred source of carbon as it can directly be used for energy by the glycolytic pathwayIf lactose is present, and glucose is absent, a protein-catabolite activator protein(CAP)binds with the CAP-cAMP complexCAP protein is a dimer of 2 identical polypeptidesThe CAP-cAMP complex binds to the CAP site which is upstream of the site at which RNA polymerase binds to the promoter and transcription is initiated

When glucose is in the medium along with lactose,glucose is used preferentially because catabolite repression occursIn catabolite repression lac operon is expressed at low level

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Glucose causes amount of cAMP in the cell to be reducedTherefore insufficient CAP-cAMP complex is present to recruit RNA polymerase to the lac promoter and transcription is loweredRNA polymerase cannot bind to the promoter without the help of CAP-cAMP complex

Adenylate cyclase enzyme makes cAMP catabolite repression acts on this enzymeAn enzyme called IIIGlc in its phosphorylated form activates adenylate cyclaseTherefore when glucose is transported across the cell membrane, IIIGlc

is dephosphorylated

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Adenylate cyclase is inactivated and no new cAMP is producedFurther level of cAMP in cell decreases due to its breakdown by phoshodiesterase

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Repressible operonsThe genes encoding the enzymes for the aminoacids biosynthesis pathway are turned off when an aminoacid is present in the growth medium in case of repressible operon.

Trp operon is a repressible operon as the gene activity is repressed when the chemical (amino acid) is addedGenerally, anabolic operons like the trp operons are repressible operonsCatabolic operons –lac operon are inducible operons

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Gene organization of tryptophan biosynthesis

Source: Peter J Russell, igenetics

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Source: Peter J Russell,

igenetics

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5 structural genes(A-E) occur in trp operon Promoter and operator are upstream of trpE Between promoter-operator and trpE – trpL

(leader region) is presentAttenuator site(att) is situated within trpL, close to trpE Regulation of trp operon2 regulatory mechanism of

trp operon:Repressor-operator interactionAttenuation

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Repressor operator interactionIn presence of tryptophan(in abundance)trp R is the regulatory gene for the trp operon

It is located at some distance from operonIt codes for the aporepressor proteinAporepressor is inactive and cannot alone bind to operatorWhen trptophan is abundant in the cell,tryptophan interacts with

aporepressorHence aporepressor becomes active

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Active aporepressor binds to the operator and prevents initiation of transcription of trp operon protein coding genes by RNA polymerase Under conditions of tryptophan starvationtrp

genes are expressed to the maximum extentWhen trp starvation occurs, the structural genes are expressed

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Following is the pathway of trp synthesis:

Source: Peter J Russell, igenetics

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In presence of low concentration of tryptophanAttenuation mechanism is observed in case of presence of low concentration of tryptophanThe UGG UGG are the 2 adjacent codons for tryptophan which are important in attenuation mechanism

This sequence of codons is present on the leader regionThere are 4 regions of the leader peptide mRNA that can fold and form 20 structures by complementary base pairingPairing of 1 &2 gives pause signal,3-4 termination of transcription of transcription signal,2-3 anti termination signal for transcription to continue

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Pause of the RNA polymerase due to 1,2 pairing results in coupling of transcription and translation. So both the processes can take place simultaneouslyRegulation of transcription termination at the attenuator depends on the position of the ribosome on the leader transcriptIf tryptophan is not available,amount of trp-tRNA (or charged

tRNA) will decrease,so very few tryptophan molecules are available for aminoacylation of the tRNATherefore RNA which is translating the leader sequence will stall at the tandem trp UGG

UGG codons in region 1. As ribosome covers region 1, 1-2 pairing will not occur but 2-3 can pair as 2-3 are paired,3-4 cannot pair and 2-3 signals for anti-termination.Therefore RNA polymerase will transcribe the structural genes.

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In presence of enough tryptophanRibosome can translate the trp codon and it continues to stop codon. It covers the region 2 therefore 2-3 cannot pair, 3-4 can pair and signal for transcription terminationThus signal for attenuation is the concentration of

trp-tRNA which determines how far the ribosome moves on the leader transcript

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Regulation of gene expression in Eukaryotes

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Gene expression in eukaryotesRegulated by regulatory systems at different levels Following are the levels of at which eukaryotic genes are regulated:Control of transcription initiation-Chromatin remodelling

Activation of transcription by activators and co-activatorsBlocking transcription with repressorsCombinatorial gene regulation.Steroid HormonesGene silencing and genomic imprinting and transcription control

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mRNA transport control, mRNA translational control, m-RNA degradation control & Protein degradation controlRNA interference

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Levels of gene regulation

Source: Peter J Russell, igenetics

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Key differences in Eukaryotic gene expression regulationRole of chromatin structure in regulating gene expressionNecessity to add 5’cap and 3’poly (A) tail to a pre mRNA molecule and then splice the pre mRNA to remove introns and produce the mature mRNA

In case of a number of genes for alternative splicing of the pre mRNA, the possibility of producing different mRNAsRegulation of transport of mRNA from nucleus to cytoplasmOperons are rare in eukaryotes, exception C.elegans

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Control of transcription initiation Transcription is regulated by regulatory proteins(activators) binding to promoter proximal elements and to enhancer elements Such binding leads to the recruitment of proteins needed to make the chromatin accessible to the transcription machinery and then to recruit the

trancription machinery to the promoter and prepare for transciption.

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Chromatin remodellingThe eukaryotic chromosome consists of DNA complexed with histones and non-histone chromosomal proteins.The nucleosome organization of chromosome has a repressive effect on gene expression, because access to transcription machinery is physically impeded

Repression of gene activity by histonesTranscriptionally active genes in chromatin have increased sensitivity to the DNA degrading enzyme DNase I compared with transcriptionally inactive genesChromosome is less highly coiled this regionThe region of DNA around transcriptionally active genes have certain sites called hypersensitive regions which are highly sensitive to digestion by DNase I

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Activating genes by remodelling chromatinThe chromatin structure must be altered in the vicinity of the core promoter for activation of eukaryotic genes. These process is called chromatin remodellingIt involves 2 protein complexes 1) enzymes for histone acetylation are histone acetyl transferases(HATs)HATs are recruited to the chromatin when activators bind to the DNA binding sites and acetylate

lysines of the amino-terminal tails of core Histone’sAcetyl groups are –ve charged With increasing acetylation the +ve charged histone’s slowly lose affinity for –ve charged DNA & 30nm chromatin fibre changes its conformation to 10nmThe removal of acetyl group is catalysed by histone deacetylases(HDACs)

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2) Other class of chromatic modellers are ATP dependent nucleosome remodelling complexes,which are multiprotein complexes that use energy of ATP hydrolysisThis complex works in 3 ways:It may slide a nucleosome along the DNA, exposing sites for DNA binding proteins

Restructure the nucleosome in place to facilitate the association of a DNA-binding protein with a DNA sequenceTransfer the nucleosome from one DNA molecule to anotherEg. SWI/SNF-ATP dependent nucleosome remodelling complex

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Activation of transcription by activators and coactivatorsThree classes of proteinsGeneral transcription factors-required for basal transcriptionActivators-activate transcription, have two domains- DNA binding domain and transcription activation domain

eg. Helix-turn-helix,zinc finger and leucine-zipperCoactivators-large multiprotein complex that does not bind directly to DNA but activates transcription by interacting with activators and TFs. Recruitment of coactivator leads to recruitment of RNA polymerase II,then contacts TFs for initiating transcription Eg

Mediator complex

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Blocking transcription with repressorsBlock transcriptionRepressor binds to binding site near the activator and blocks the action of activator by interaction with its domainOr their binding sites may overlap and thereby prevent the binding of the activatorIf repressor binds to binding site and recruits a histone deacetylase complex (HDACs)

Combinatorial gene regulationPromoter and enhancers are important in regulating the transcription of a geneThey both bind to special regulatory protein

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Net effect of a regulatory element on transcription depends on combination of different proteins bound, their interaction with each other So if repressor has a strong effect , the gene is repressed, in this case the enhancer is called as silencer elementBy combining the regulatory proteins the transcription of different array of genes is regulated which specifies different cell types-This is called combinatorial gene regulation

Source: Peter J Russell,

igenetics

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Steroid hormone regulationSome of the genes of the eukaryotes are regulated by hormonesPolypeptide hormones act at the cell surface, activating cAMP, 20 messenger to control gene activitySteroid hormones form a specific hormone receptor complex which binds directly to the particular sequences on the cell’s genome to regulate the expression of specific genes

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Gene silencing and genomic imprintingGene silencing is phenomenon where gene is transcriptionally silent due to its location and not due to repressorProperty of heterochromatin

Telomere position effect- no protein coding genes are found at telomeres, but when active genes are moved to telomere region,the genes are silenced.Silencing is caused by deacetyated histones which bind to the silencing complexSilencing can be overcome by methylation of histone with histone methyl transferases(HMTs) which involved modifying cytosines to 5-methyl cytosine

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Genomic imprintingPhenomenon in which expression of a gene is determined by whether the gene is inherited from female or male parentEpigenetic phenomenonIn mice studies, inheritance of deletion that removed gene Igf2 showed that progeny inheriting the deletion from male parent were small but those inheriting it from female parent were of normal size.

Deletion of Igf2 gives mutant phenotype only when the deletion is inherited from male parent, because the DNA is methylated.

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Post transcriptional controlRNA processing controlAll the RNAs –mRNA,rRNA,tRNA are synthesized as larger precursor moleculesAlternative splicing produces different functional mRNAsEg

of alternative splicing in CALC geneAlternative spilicing and polyadenylation results in tissue specific products of human calcitonin gene (CALC)CALC consists of 5 exons and 4 introns and is transcribed in certain cells of the thyroid gland and in certain neurons of the brain

Alternative polyadenylation occurs with polyadenylation site next to exon 4,pA1 used in thyroid cells and polyadenylation site next to exon 5,pA2 used in the neuronal cells

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Source: Peter J Russell,

igenetics

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Alternative splicing occurs at the time of intron removalThe pre-mRNA in thyroid is spliced to remove the three introns and bring together exons 1,2,3,4 –Here exon 4 is encodedThe pre-mRNA in neuronal cells is spliced to remove the three introns and bring together exons 1,2,3, and 5;exon 4 is excised and discarded-here exon 5 is encodedThese mRNAs are translated to produce 2 different polypeptides, calcitonin in thyroid cells and CGRP(Calcitonin gene-related peptide) in neuronal cells

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mRNA transport controlRegulation of the number of transcripts that exit the nucleus to the cytoplasmHow is the transport of mature mRNA from the nucleus pore complexes?

The mRNA exits through the nuclear pore complexes, mRNA must be capped for them to exitUnspliced mRNA are not exported due to snRNPs as it cannot interact with the nuclear pore

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mRNA translation controlTranslational control of mRNA is by ribosome selectionDifferential translation can affect gene expressionStored mRNAs are associated with proteins which protect the mRNAs and inhibit their translationPoly(A) tail promotes initiation of translation Stored, inactive mRNAs have shorter poly(A) tails than active mRNAs

Translation control can be reversed by deadenylation

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mRNA degradation controlOnce in the cytoplasm, all RNA species are subjected to degradation controlRate of RNA breakdown is regulated2 pathways of mRNA degradation-

a. deadenylation dependent decay pathway- poly(A) tails are deadenylated until the tails are too short to bind PAB binding proteinThe 5’ cap is removed by decappingThen it is degraded from 5’end by a 5’ to 3’ exonuclease

b. deadenylation independent pathwaymRNAs are decapped without being deadenylated, by either 5’ to 3’ exonuclease or an endonuclease

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Protein degradation controlRegulatory mechanisms exist at the post translational levelThere are various ways to regulate the amount of protein in a cellThe degradation of proteins (proteolysis) in eukaryotes has been shown to require the protein cofactor ubiquitinThe binding of ubiquitin to a protein identifies it for degradation by proteolytic enzymesUbiquitin is released intact during degradation enabling it to be used to tag other proteins for degradation

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RNA interferenceMechanism by which a small fragment of dsRNA whose sequence matches part of a gene’s sequence, interferes with the expression of that geneRNAi has natural functions-Protection against viral infectionsRegulation of developmental processes

How does dsRNA result in silencing of the expression of a specific gene?dsRNA in the cell is cleaved by the enzyme Dicer into 21-23bp dsRNA fragments called short interfering RNA (siRNA)Dicer cuts so as to leave 3’ overhangs.

The siRNA-Dicer complex recruits other proteins to which the siRNA is transferred to form the RNA-induced silencing complex(RISC)

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Source: Peter J Russell,

igenetics

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RISC is then activated in an ATP-dependent manner which leads to unwinding of the ds siRNAActivated RISC used ss siRNA to pair with complementary mRNA and then cleaves the mRNA with an endoribonucleaseAs a result, the gene expression is silencedThe cleaved mRNA is degradedActivated RISC may block translation by ribosomes

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Or it can migrate to the nucleus, where siRNA binds complementary DNA and recruits chromatin remodelling complex which modifies the chromosome around the gene’s promoter thereby silencing the transcription of the geneActivated RISC can also cause amplification of interference signalRNA interference can be used as an alternative to gene deletion techniques such as ‘knock out’ gene function

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ReferencesPeter J Russell, igenetics A molecular approachWatson, Baker, Bell, Gann,levin,losick, Molecular Biology of gene,

5th edition