gene regulation sdk 2013


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  • DNA acetylation refers to the addition of an acetyl (CH3CO) group to one of the histone proteins that help hold DNA in its tightly wound configuration. When histones are altered by this change, the binding between histones and DNA is relaxed. This promotes transcription in eukaryotic cells. Read more:
  • Attachment of ubiquitin to lysine 123 (K123)
  • gene regulation sdk 2013

    1. 1. Gene RegulationExpression in Eukaryotes & ProkaryotesSDKMarch 30, 20131
    2. 2. Gene Regulation in Prokaryotesand Eukaryotes Gene is the sequence of nucleotides in DNA thatcode one mRNA molecule or one polypeptide chain In prokaryotes the primary control point is theprocess of transcription initiation In eukaryotes expression of gene into proteins canbe controlled at various locations.2
    3. 3. Check Points for Gene Expression inEukaryotes1. Synthesis of proteins is controlled right from thechromatin stage.2. Expression of gene is controlled at many stepsduring the process of transcription and translation.3
    4. 4. 1.Chromatin StructureTwo forms of chromatinEuchromatin – A lesser coiled transcriptionallyactive region which can be easily accessed by theRNA polymerases.Heterochromatin – A highly condensedtranscriptionally inactive region. The genes in thisregion cannot be accessed by the RNApolymerases for active transcription.4
    5. 5. 1.Chromatin StructureMechanisms which affect the chromatin structureand hence the expression of gene are:1.Histone modifications – These modificationsmake a region of gene either transcriptionallyactive or inactive.a)Acetylation(addition of an acetyl (CH3CO) group to one of the histone)• ↑Acetylation ----↓ Condensation of DNA ----- ↑Transcription of genes in that region5
    6. 6. 1.Chromatin Structureb. Methylation• Methylation of histone H4 on R4 (arginine residue atthe 4th position) →→ opens the chromatin structure→→ leading to transcriptional activation• Methylation of histone H3 on K4 and K79 (lysinesresidues at the 4th and 79th position) →→ opens thechromatin structure →→ leading to transcriptionalactivation• Methylation of histone H3 on K9 and K27 (lysinesresidues at the 9th and 27th position) →→ condensesthe chromatin structure →→ leading to transcriptionalinactivation6
    7. 7. 1.Chromatin Structurea) UbiquitinationUbiquitination of H2A – Transcriptional inactivationUbiquitination of H2B - Transcriptional activation2) Methylation of DNA Target sites of methylation are - The cytidine residueswhich exist as a dinucleotide, CG (written as CpG i.eCytosine bound to guanine by phosphodiester bond). ↑methylated cytidine -- ↓Transcriptional activity7
    8. 8. 2.Regulation of Transcription• The differences in the mechanisms by which thetranscription of gene is controlled in prokaryotesand eukaryotes are listed below:Prokaryotes EukaryotesThe linked genes are organizedinto clusters known as operonswhich are under the control of asingle promoter.Eukaryotic genes are notorganized into operons andeach of these genes requires itsown promoter.These genes are primarilyregulated by repressors.Regulation by repressors is veryoccasional and the primary roleof regulation is played by thetranscriptional activators knownas transcription factors.8
    9. 9. 2.Regulation of TranscriptionProkaryotes EukaryotesA promoter sequence whichcontrols an operon lies upstreamof the operon.Accessory or the regulatoryproteins control the recognition ofthe transcriptional initiation sitesby RNA polymerasesThose genes which codefor a protein have a basicstructure consisting of:Exons – Gene sequences whichencode for a polypeptideIntrons – These sequences willget removed from the mRNAbefore it gets translated.A transcription initiation sitePromoter sequences.A single operon gets transcribedinto a polycistronic mRNA whichcan be translated into multipleproteinsMonocistronic mRNAs which canproduce a single polypeptide areproduced9
    10. 10. 102.Regulation ofTranscriptionPromotersThe region necessary to initiate transcription.Consists of short nucleotide sequence thatserve as the recognition point for binding ofRNA polymerase.Located immediately adjacent to the genesthey regulate.
    11. 11. 2.1: PromotersPromotersProkaryotes - There are two promoter elements orDNA sequences which are 35 and 10 base pairs inlength and seated upstream to the transcriptionalinitiation sites.The consensus sequence present at-35 position is TTGACA-10 position is TATAAT. This is also termed as Pribnow-box.Eukaryotes – There are two types of promoterswhich are:Basal promotersUpstream promoters11
    12. 12. 2.2: Promoters Basal promoter or core promoter -These promotersreside within 40bp upstream of the start site. Thesepromoters are seen in all protein coding genes.Examples are CCAAT-boxes and TATA-boxes1. TATA box The consensus sequence for TATA box isTATAT/AAT/A It resides 20 to 30 bases upstream of thetranscriptional start site This is similar in sequence to the prokaryoticPribnow-box Proteins like TFIIA, B, C interact with this TATA box12
    13. 13. 2.3: Promoters2. CCAAT-box The consensus sequence for this isGGT/CCAATCT It resides 50 to 130 bases upstream of thetranscriptional start site Protein named as C/EBP (CCAAT-box/EnhancerBinding Protein) binds this box Upstream promoters - These promoters may lie up to200bp upstream of the transcriptional initiation site. Thestructure of this promoter and the associated bindingfactors keeps varying from gene to gene13
    14. 14. PromotersPromoters for RNA polymerase IIinclude:TATA box,CAAT box,GC box,& Octamer box.Site Structure ImportanceTATA box -25 30bp upstream)from the initialpoint oftranscription8bp sequencescomposed only ofT=A pairs.Mutations in thissequence greatlyreduce transcription)Loosing the abilityto bind totranscriptionfactors(CAAT box -70 80bp upstream)from the initialpoint oftranscriptionCAAT or CCAATsequence.Mutations in thissequence greatlyreduce transcriptionGC box 110bp upstream)from the initialpoint oftranscriptionGGGCGG sequence,often present inmultiple copies.Documented bymutational analysisOctamer box -120 130bpupstream)from the initialpoint oftranscriptionATTTGCATsequence.Affects theefficiency ofpromoter ininitiatingtranscription.
    15. 15. 3. EnhancersDNA sequences interact with regulatory proteinsincrease the efficiency of initiationof transcriptionincrease its rate.
    16. 16. 3.1:Enhancers:1. Large ) up to several hundred bp long).2. Tissue- specific ( stimulate transcriptiononly in certain tissues).
    17. 17. 3.2: Enhancers EnhancersEnhancers can be located upstream, downstream orwithin the gene that is transcribedThe binding of these enhancers with enhancerbinding proteins (transcription factors) increases therate of transcription of that gene to a greater extent.Promoters are capable of initiating lower levels oftranscription.Enhancers are responsible for the cell or tissuespecific transcription.Each enhancer has its own transcription factor that itbinds to.17
    18. 18. 3.3: Enhancers1. The proteins that bind to enhancers affect the activity ofproteins that bind to promoters.2. Enhancers may allow RNA polymerase to bind to DNA andmove along the chromosome till it reaches a promoter site.3. May respond to molecules outside the cell ( e.g : steroidhormones).4. May respond to molecules inside the cell ( e.g : duringdevelopment thus the gene participates in celldifferentiation).
    19. 19. 3.4:How enhancers can control transcriptionalthough they are located away from thetranscription site.Enhancers bind to transcription factors by atLeast 20 different proteinsForm a complexchange the configuration of the chromatinfolding, bending or looping of DNA.
    20. 20. 3.5:Action of an enhancer – An enhancer binding protein has two bindingsitesBinds DNABinds the transcription factors that are bound to thepromoter20
    21. 21. 21
    22. 22. DNA looping will bring the distal enhancersclose to the promoter site to formactivated transcription complexes,then the transcription is activated,increasing the overall rate of RNA synthesis.3.6:Enhancers:
    23. 23. 4.Transcription factors“ Are the proteins that are essential forinitiation of the transcription, but they arenot part of RNA polymerase molecule thatcarry out the transcription process”.
    24. 24. Function:Each RNA polymerase requires a number oftranscription factors which help in:1. Binding of the enzyme to DNA template.2. Initiation and maintenance oftranscription.3. Control the rate of gene expression.Transcription factors
    25. 25. Structure & Mechanism of actionThese proteins contain 2 functional domains, thatperform specific function.1. DBD: DNA binding domain: binds to DNA sequencespresent in regulatory regions (e.g : TATAbinding protein).2. AD: Transcriptional activating domain: activatetranscription via protein-protein interaction
    26. 26. Types of transcription factors:1. Basal transcription factors:The initiation of transcription by RNA polymeraseII requires the assistance of several basaltranscription factors.Each of these proteins binds to a sequencewithin the promoter to facilitate the properalignment of RNA polymerase on the templatestrand of DNA.
    27. 27. The basal TFs must interact with thepromoters in the correct sequence to initiatetranscription effectively.TFIID is the 1stbasal TF that interact with thepromoter ; it contains TATA- Binding Protein.Followed by TFII B, F, E, H & J.Types of transcription factors:
    28. 28. 2. Special TFs:Involved in regulation of heat, light, and hormone induciblegenes.They bind to:a. enhancers.b. Basal TFs.c. RNA polymerase that bind to the gene promoter.Therefore, special TFs can regulate the transcriptional activityof the gene.Types of transcription factors:
    29. 29. How is the gene transcription controlledat this point• The unique combination of the promotersites, transcription factors and enhancerschosen ultimately decides which genegets switched on and which one getsswitched off.33
    30. 30. 5.Regulation of RNAProcessing RNA processing involvesAddition of 5 capAddition of a 3 poly (A) tailRemoval of introns The RNAs which get translated to proteins aretransported out from the nucleus to cytoplasm. Depending on the final combination of exons aftersplicing different kinds of proteins are obtainedwhich can perform different functions in the cell.34
    31. 31. 5.1:Exon Shuffling• The functions of two proteins synthesized from the samemRNA are different in different cells as differentcombination of exons exist in different cells.35
    32. 32. 6.Regulation of RNA Transport• Only some RNAs function within thenucleus whereas all other RNAs which aremeant for protein synthesis have to betransported from the nucleus to thecytoplasm via nuclear pores.36
    33. 33. 6.1:Regulation of RNA Longevity• mRNAs from different genes havedifferent life spans.• The information of the life span of mRNAis found in the 3 UTR(Un-translatedReagion).• The sequence AUUUA within 3 UTR actsas a signal for early degradation.• More the number of times the sequence isrepeated  Shorter the lifespan of mRNA37
    34. 34. 38three prime untranslated region (3 UTR)6.1:Regulation of RNA Longevity
    35. 35. 6.Regulation of TranslationTranslational initiationThe expression of a gene product also dependson the ability of the ribosome to recognize thecorrect AUG codon out of the multiple methioninecodons present in the mRNA.Control of translational processIn many animals large amounts of mRNAs areproduced by the eggs but all of them do not gettranslated until the egg is fertilized.39
    36. 36. 7.Post Translational Control PointsPost translational modificationsFunctional state of protein depends onmodifications like glycosylation, acetylation, fattyacylation, disulfide bond formations.ChaperonsProtein transportTransportation to the site of action also regulategene expression.Protein stabilityThe lifespan of a protein depends on the specificamino acid sequence present within them40
    37. 37. Summary of the Class• The expression of genes is controlled at various levels ineukaryotes.• At the chromatin stage the level of condensationdetermines whether the genes will remaintranscriptionally active or not.• The unique combination of the promoter sites,transcription factors and enhancers regulates thetranscriptional rate of a gene.• After transcription the gene expression is controlled byRNA processing.• The expression of gene is also controlled at the level oftranslation and after translation.41
    38. 38. 42THANK YOU