5 Control of Gene Expression

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  1. How are cell types determined?
    by their protein populations

    eg. neurons & cells in the liver contain the same DNA, but they are different because each cell type has a distinct pattern of gene expression
  2. Housekeeping (Constitutive) Genes
    genes expressed in all cells because they provide basic metabolic & structural functions needed for sustenance of all cells

    eg. housekeeping proteins are associated with basic metabolism, translation, transcription, replication & cell structure
  3. Histones
    • • small basic (+) proteins that DNA (-) coils around
    • • are the principal way DNA is compacted
    • • there are 2 each of histones H2A, H2B, H3 & H4 in the core group (or octomer)
    • • H1 is a linker histone between neighboring nucleosomes
  4. Nucleosome
    • the complex of octomer histones + DNA

    • are themselves further ordered into chromatin
  5. Heterochromatin
    transcriptionally inactive condensed chromatin NOT accessible to limiting amounts of DNAse I (enzyme that cleaves DNA)

    • H for HIDDEN
  6. Euchromatin
    transcriptionally active relaxed (non-condensed) chromatin accessible to limiting amounts of DNAse I

    • E for EXPRESSED
  7. Hypersensitive Site
    sections of chromatin usually in a (upstream) control region of a gene that are VERY accessible to nucleases like DNase I
  8. Locus Control Regions (LRCs)
    they regulate gene expression indirectly by regulating chromatin organization over certain chromosomal domains

    sections of DNA containing LRCs may form loops over desired genes to enhance their expression
  9. SWI-SNF
    protein complex that alters chromatin structure via nucleosome remodeling

    • complexes are thought to act globally to increase mobility of nucleosomes throughout the genome

    • some complexes may target specific genes
  10. Histone Acetylation (CH3CO-)
    • Acetylation of histones on Lysine residues → chromatin unfolding

    • • histones are very basic & Lysine is a basic AA
    • - part of DNA’s affinity to histones is that DNA is very negatively charged & histones are very positively charged
    • - Acetylating (removing the + charges) weakens the interaction between histones & DNA

    • many transcriptional activators possess histone acetyltransfrerase activity

    • many transcriptional repressors are histone deacetylases
  11. Which amino acid residue in histones is typically most susceptible to acetylation?
    Lysine (itself a +basic+ AA)
  12. Histone Acetyltransferase (HAT)
    enzyme that catalyzes acetylation of histones (specifically at lysine residues), which leads to the UNFOLDING of chromatin

    • increases the ability of transcription factors to access DNA
  13. Histone Deacetylases (HDAC)
    enzymes that REMOVE acetyl groups (O=C-CH3) from an N-acetyl Lysine amino acid on a histone

    • this allows histones to wrap the DNA more tightly (“heterochromatinizes”)
  14. Hypermethylation v. Hypomethylation
    Hypermethylation: methyl groups, particularly in promoter regions → decreased gene expression

    Hypomethylation: absence of methylation → transcriptionally active genes
  15. DNA Methyltransferases
    enzymes that methylate the 5th carbon in Cytosine bases after DNA replication has occurred to repress gene transcription
  16. DNA Methylation
    when DNA methyltransferases create 5-methylcytosine residues in a highly specific pattern

    eg. most methylated Cs are next to Gs
  17. What is CpG methylation a key mediator of?
    X- inactivation (lionization) + other epigenetic effects

    • CpG methylation provides a mechanism for producing heritable changes in gene expression that DON’T depend on DNA sequence changes
  18. Why are CpG sequences Mutation Hotspots?
    • if 5-methylcytosine is deaminated → Thymine

    • • NOT noted by repair machinery
    • (unlike when C deaminates to U → noted by mismatch repair machinery)
  19. CpG Islands
    LONG CpG rich stretches of DNA found in the promoters of genes that are actively transcribed in all types of cells (housekeeping genes)

    • 50% of human genes have a CpG island near their promoter regions

    • CpG islands almost always lack methylation, regardless of whether the associated gene is active or not
  20. Regardless of LCR, acetylation, & methylation status, what other step must take place for a gene to be transcribed?
    the chromatin might be available for transcription but if an ENHANCER isn’t (doesn’t have the correct transcriptional factors bound to it), a gene won’t be transcribed

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  21. Activators
    a class of transcription factors that are required for the assembly of basal transcription factors

    • bind to Enhancers

    • • communicate with the basal factors Coactivators
    • eg. streroid hormones, activators that activate heat shock protein (HSP) transcription during times of stress
  22. Steroid Hormone as Transcription Activators
    • • steroid hormones (estrogen, testosterone, progesterone) are soluble in lipid membranes & can diffuse into cells
    • • once inside cells they bind to specific intracellular steroid receptors
    • • when bound to hormone, these receptors are site-specific DNA binding molecules
    • • response (enhancer) elements are the DNA binding sites that hormone-steroid receptor complexes bind to
  23. Coactivators
    allow communication between activators & basal transcription factors

    • coactivator complex binds to the TATA Binding Protein (TBP)
  24. What is the result of activator-coactivator interactions?
    activator-coactivator interactions allow basal transcription factors to position RNA pol II at the beginning of the protein-coding region of a gene & set the polymerase in motion
  25. Repressors
    block the initiation of transcription by interfering with the function of activators

    • they bind to silencers
  26. DNA Microarray
    useful in determining expression levels of genes

    • used to determine differences in the mRNA population between 2 cell types (levels & patterns of gene expression)
  27. Steps of a DNA Microarray
    • 1. isolate mRNA from 2 cell types
    • 2. convert mRNA into cDNA
    • 3. cDNA from a given cell type is colored with a particular fluorescent probe
    • 4. cDNAs are pooled & hybridized to the DNA on the microarrays
    • 5. the amount of hybridization to all of the “spots” on the microarray is measured
    • 6. if a particular mRNA is present at abnormal levels in one cell type, it is easily detected (the spot will not be a blend of two colors (red + green = yellow) but will be one of the primary colors
  28. RNA-Seq
    addresses the same question as the microarray (gene expression, determines the composition & quantity of RNA in a cell) but relies on next-gen sequencing & is replacing microarrays

    • take all RNA from a cell → fragment it → convert to cDNA → sequence

    - if there’s mRNA over-expression in one cell, you’ll find that the “read” for a gene is high in one cell versus the other
  29. What is the best approach to therapy if certain diseases result from excessive production of particular mRNA?
  30. Antisense Therapy
    a fragment of nucleic acid complimentary to an mRNA of interest is introduced into a cell

    this new nucleic acid binding to a complementary fragment of mRNA blocks ribosomes from translating the mRNA

    (the antisense nucleic acid fragment is protected from intracellular degradation because it has sulfur in place of oxygen atoms that make up the phosphate links between nucleotides of the DNA backbone)
  31. Why doesn’t antisense therapy work?
    • 1. toxicity of introduced nucleic acids
    • 2. difficult to keep nucleic acids inside the cells once they are introduced
    • 3. difficult to identify genes that are causative of the diseases we intend to treat
  32. RNA Interference (RNAi)
    a technique in which double-stranded small-interfering RNA (siRNA) homologous to a gene of interest are introduced inside a cell with the goal of silencing that gene
  33. How do siRNAs silence gene expression?
    siRNAs are separated into single strands which bind to the RISC complex, facilitating degradation of complementary mRNAs before they can be translated by ribosomes.
  34. Dicer
    an enzyme that cleaves double-stranded RNAs into smaller double-stranded RNA fragments called siRNAs
  35. RISC Complex
    a protein complex that binds to single strands of siRNAs

    • the siRNAs serve as a template that recognize complementary mRNAs

    • once recognized, the RNase subunit of the RISC complex cleaves the complementary mRNAs so that they CANNOT be translated
  36. What was the RISC complex probably originally used for in cells before it's siRNA capabilities came in handy?
    it was probably present to degrade any double stranded VIRAL (harmful) RNA
Card Set:
5 Control of Gene Expression
2016-08-28 15:11:54
MedFoundationsI Biochemistry Exam1
Biochemistry Exam 1
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