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Topic 8-2

Topic 8-2. 2. Repression of TranscriptionCells also possess negative regulatory elementsMechanisms:Binding to promoter elementsBlocking assembly of the preinitiation complexInhibiting binding or functioning of transcriptional activatorsModifying DNA and its interaction with nucleosomesSome tr

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Topic 8-2

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    1. Topic 8-2 1

    2. Topic 8-2 2 Repression of Transcription Cells also possess negative regulatory elements Mechanisms: Binding to promoter elements Blocking assembly of the preinitiation complex Inhibiting binding or functioning of transcriptional activators Modifying DNA and its interaction with nucleosomes Some transcription factors activate some genes and repress others

    3. Topic 8-2 3 Repression of Transcription - Mechanisms: Binding to promoter elements Blocking assembly of the preinitiation complex

    4. Topic 8-2 4 Repression of Transcription - Mechanisms: Inhibiting binding or functioning of transcriptional activators

    5. Topic 8-2 5 Repression of Transcription DNA Methylation Methyl groups may be attached to cytosine (C5 position) Methyltransferases Methyl groups provide a tag In mammals always part of a symmetrical sequence Concentrated in CG-rich domains Often in promoter regions Methylation of promoter DNA highly correlated with gene repression

    6. Topic 8-2 6 Repression of Transcription DNA Methylation Maintains a gene in inactive state rather than initiating gene repression – Example: Inactivation of genes of one X chromosome in female mammals occurs prior to a wave of methylation Shifts throughout life in DNA-methylation levels Early Zygote – most methylation tags removed Implantation – a new wave of methylation occurs Important example – Genomic Imprinting

    7. Topic 8-2 7 Repression of Transcription DNA Methylation – Genomic Imprinting Certain genes are active or inactive during early development Depending on whether they are paternal or maternal genes Eg – IGF-2 is only active in the gene from the male parent The gene is imprinted according to parental origin Mammalian genome has > 100 imprinted genes in clusters Imprinted due to selective methylation of one of the alleles

    8. Topic 8-2 8 Repression of Transcription DNA Methylation – Genomic Imprinting In the early embryo the waves of demethylation and new methylation do not affect the methylation of imprinted genes Thus the same alleles are affected in the zygote through to the adult stage in the individual

    9. Topic 8-2 9 Repression of Transcription Chromatin structure and transcription DNA is not naked – but wrapped around histone complexes to form nucleosomes How are transcription factors and RNA polymerases able to interact with DNA tightly associated with histones? Apparently nucleosome structure does inhibit initiation of transcription Initiation of transcription requires assembly of large complexes and nucleosomes block assembly at the core promoter

    10. Topic 8-2 10 Repression of Transcription Chromatin structure – role of acetylation Genes which are actively transcribed are bound by histones which are acetylated Each of the histones has a flexible N-terminal tail Extends outside the core particle and the DNA helix Acetyl groups are added to lysine residues by enzymes Histone acetyl transferases (HATs) Acetylation has two functions Neutralize the positive charge on the lysine residues Destabilize interactions between histone tails and structural proteins

    11. Topic 8-2 11 Repression of Transcription Chromatin structure – role of acetylation Some coactivators have HAT activity Links histone acetylation, chromatin structure and gene activation HAT activity of coactivator acetylates core histones bound to promoter DNA causing release of nucleosome core particles or loosening of histone-DNA interaction Subsequent binding of transcription factors and RNA polymerase Once transcription is initiated – RNA polymerase is able to transcribe DNA packaged into nucleosomes Acetylation is dynamic – enzymes also remove acetyl groups

    12. Topic 8-2 12 Repression of Transcription Chromatin structure – role of deacetylation Removal of acetyl groups Histone deacetylases (HDACs) HDACs associated with transcriptional repression HDACs are subunits of larger complexes – corepressors HDACs guided to regions of DNA by methylation patterns Example: Inactive X chromosome of female Largely deacetylated histones Active X chromosome has a normal level of histone acetylation

    13. Topic 8-2 13 Repression of Transcription Chromatin structure – Acetylation / Deacetylation

    14. Topic 8-2 14 Repression of Transcription Chromatin structure – Acetylation / Deacetylation

    15. Topic 8-2 15 Processing-Level Control Recall that the formation of multigene families is a mechanism that generates protein diversity Protein diversity also generated via alternate splicing Regulates gene expression at the level of RNA processing A mechanism by which a single gene can encode two or more related proteins Most genes (and their primary transcripts) contain multiple introns and exons Often – more than one pathway for processing of primary transcript

    16. Topic 8-2 16 Processing-Level Control Transcripts from approx 35% of human genes may be subjected to alternate splicing Simplest case – a specific segment either spliced out or retained – Example: Fibronectin: Synthesized by fibroblasts – two additional peptides compared to that synthesized by liver Extra peptides encoded by pre-mRNA retained in fibroblast

    17. Topic 8-2 17 Translational-Level Control Wide variety of mechanisms – affecting mRNA previously transported from the nucleus Subjects include: Localization of mRNA in the cell mRNA translation Half-life of mRNA Mediated via interactions between mRNA and cytosolic proteins

    18. Topic 8-2 18 Translational-Level Control mRNA noncoding segments – untranslated regions (UTRs) 5’ – UTR – from methylguanosine cap to AUG initiation codon 3’ – UTR – from termination codon to end of poly(A) tail UTRs contain nucleotide sequences which mediate translational-level control

    19. Topic 8-2 19 Translational-Level Control Cytoplasmic localization of mRNAs – Example ferritin Translation regulated by iron regulatory protein (IRP) Activity of IRP dependent on cellular iron concentration At low iron concentration – IRP binds the 5’ UTR Bound IRP interferes physically with the binding of a ribosome to the 5’ end of the mRNA At high iron concentration the IRP changes conformation and looses affinity for the 5’ UTR

    20. Topic 8-2 20 Translational-Level Control Control of mRNA stability Half-life of mRNA is variable – 10 minutes to 24 hours Specific mRNAs are recognized in the cytoplasm and treated differentially mRNAs lacking the poly(A) tail are rapidly degraded Poly(A) tail is not naked mRNA but bound by the poly(A) binding protein (PABP) Each PABP bound to about 30 adenosine residues

    21. Topic 8-2 21 Translational-Level Control Control of mRNA stability PABP protects poly(A) tail from general nuclease activity But – increases its sensitivity to poly(A) ribonuclease mRNA in cytoplasm is gradually reduced in length by poly(A) ribonuclease When the tail is reduced to approx 30 residues mRNA is rapidly degraded Degradation occurs from the 5’ end Suggests two ends held in close proximity

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