Concepts of Genetics Eighth Edition Klug, Cummings, Spencer

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Concepts of GeneticsEighth EditionKlug,
Cummings, Spencer

• Chapter 17
• Regulation of Gene Expression in Eukaryotes

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Gene regulation (eukaryotes)

• 17.1 Eukaryotic gene regulation differs from
  regulation in prokaryotes
• Gene regulation more complex in eukaryotes
• Larger amount of DNA.
• Larger number of chromosomes.
• Spatial separation of transcription (nucleus) and
  translation (cytoplasm).
• Transcription of genes processed before transport
  to cytoplasm.
• mRNA processing.
• mRNA more stable.
• Cellular differentiation in eukaryotes.

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Gene regulation (eukaryotes)

• Regulation of eukaryotic gene expression can
  occur at many levels (Figure 17.1)
• Transcriptional control
• Post-transcriptional control
• Transport to cytoplasm
• Stability of mRNA
• Translational control
• Post-translational modification of protein
  product
• 17.2 Chromosome organization in nucleus
  influences gene expression
• Chromosomes occupy discrete territory in nucleus
  ? with gene-poor chromosomes located peripherally
  gene-rich chromosomes located more internally
  (Figure 17.2).

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Gene regulation (eukaryotes)

• Channels between chromosomes called
  interchromosomal compartments.
• Chromosome structure continuously rearranged so
  that transcriptionally active genes are cycled to
  edges of chromosome territories.
• Initiation of gene expression requires two steps
  ? remodeling activation of chromatin, making
  promoter sites accessible to transcription
  machinery ? recruitment of coactivators that
  assemble proteins necessary for transcription.

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Gene regulation (eukaryotes)

• 17.3 Transcription initiation is major form of
  gene regulation
• Eukaryotic genes have three types of
  cis-regulatory sequences that control
  transcription promoters, silencers and enhancers
  (Figure 17.3).
• 17.3.1 Promoters have modular organization
• Promoters ? nucleotide sequences that serve as
  recognition sites for transcriptional machinery.
• Promoters contain several elements ? TATA, CAAT
  GC boxes ? RNA polymerase II binds to TATA box ?
  core promoter (Figure 17.4).

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Gene regulation (eukaryotes)

• CAAT and GC elements bind transcription factors ?
  function like enhancers (Figure 17.5).
• 17.3.2 Enhancers control rate of transcription
• Enhancers contain several short DNA sequences ?
  often include binding sites for positive and
  negative regulatory factors (Figure 17.6).
• In yeast ? regulatory sequences similar to
  enhancers ? called upstream activator sequences
  (UAS) ? function upstream at variable distances
  and in either orientation.
• Differ from enhancers ? they cannot function
  downstream of transcription start point.

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Gene regulation (eukaryotes)

• 17.4 Transcription in eukaryotes requires several
  steps
• Eukaryotic chromosomal DNA combined with histones
  non-histone proteins to form chromatin.
• Changes in chromatin organization ? chromatin
  remodelling ? essential for binding of
  polymerases, transcription, DNA replication,
  repair recombination.
• Nucleosomes in chromatin can inhibit
  transcription (Figure 17.7).

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Gene regulation (eukaryotes)

• Chromatin remodelling involves change in
  interaction between DNA histones in
  nucleosomes.
• Remodelling carried out by protein complexes that
  have ATP-ase activity ? SWI/SNF complex (Figure
  17.8).
• These complexes can be targeted to specific DNA
  sites by transcription factors, by acetylation of
  histones or by binding to methylated DNA.

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Gene regulation (eukaryotes)

• Nucleosome remodelling complexes may alter
  nucleosome structure in several ways (Figure
  17.9)
• altering contacts between DNA histones ?
  nucleosome slide farther down DNA molecule.
• alternating path of DNA around nucleosome ?
  pulling DNA off nucleosome.
• altering structure of nucleosome core itself ?
  producing nucleosome dimer.
• Second mechanism of chromatin alteration is
  histone modification ? catalyzed by histone
  acetyltransferase enzymes (HAT) ? lessens
  attraction between histones DNA (Figure 17.10).

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Gene regulation (eukaryotes)

• Specific transcription factors target HATs to
  genes.
• Histone deacetylases (HDACs) reverse this
  remodelling.
• Insulator elements bind specific proteins ? act
  as barriers prevent spreading of remodelling to
  neighbouring genes.
• 17.5 Assembly of basal transcription complex
  occurs at promoter
• Transcriptional control involves interaction
  between DNA sequences adjacent to promoter
  regions DNA-binding proteins.

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Gene regulation (eukaryotes)

• Prokaryotic genes all transcribed by single RNA
  polymerase but eukaryotes have three RNA
  polymerases that recognize different types of
  promoters to transcribe certain sets of genes ?
  classified as type I (ribosomal RNAs), type II
  (mRNAs snRNAs) and type III (tRNAs, 5S rRNA
  other small cellular RNAs).
• Basal (general) transcription factors act in
  trans to control initiation of transcription ?
  required for binding of RNA polymerase II to
  promoter (Figure 17.11).

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Gene regulation (eukaryotes)

• TFIID ? first general transcription factor to
  bind promoter ? binds to TATA box through TATA
  binding protein (TBP) (Figure 17.12).
• Activators ? modular proteins ? bind to enhancer
  DNA sequences and form enhanceosome, which
  interacts with transcription complex (Figure
  17.13).
• Activators have DNA-binding domain ? binds
  enhancer sequence trans-activating domain that
  activates transcription through protein-protein
  interactions with factors in transcription
  complex.

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Gene regulation (eukaryotes)

• DNA-binding domains have characteristic
  three-dimensional structural patterns ? motifs.
• Activator has helix-turn-helix (HTH) motif
  (Figure 17.14), zinc finger leucine zipper
  (bZIP).
• Typical zinc-finger protein contains clusters of
  two cysteines two histidines at repeating
  intervals (Figure 17.15).
• Another DNA-binding domain represented by basic
  leucine zipper (bZIP) ? allows protein-protein
  dimerization ? contains four leucine residues
  spaced 7 amino acids apart flanked by basic
  amino acids.

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Gene regulation (eukaryotes)

• This region forms helix with leucine residues
  protruding at every other turn ? when two such
  molecules dimerize, leucine residues zip
  together (Figure 17.16).
• Many transcription factors also contain domains
  that bind coactivators ? such as hormones or
  small metabolites that regulate their activity.
• 17.6 Gene regulation in model organism positive
  induction and catabolite repression in gal
  genes of yeast
• gal genes of yeast are inducible by presence of
  galactose, but only if concentration of glucose
  is low ? indicating that gal genes are also
  subject to catabolite repression.

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Gene regulation (eukaryotes)

• Mutation in regulator of gal genes ? GAL4 ?
  prevents activation ? indicating that
  transcription is under positive control ?
  regulator must be present to turn on gene
  transcription.
• gal genes (Figure 17.17) ? GAL1 and GAL10 ?
  controlled by central control region ? UASG ?
  contains four binding sites for Gal4 protein
  (Gal4p).
• Chromatin structure of UAS is constitutively open
  or DNase hypersensitive ? meaning that it is free
  of nucleosomes.

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Gene regulation (eukaryotes)

• Within UAS ? four binding sites for Gal4p.
• Gal4p negatively regulated by Gal80p, which
  covers Gal4p activation domain.
• Binding of phosphorylated galactose to Gal80p
  and/or Gal4p exposes activation domain of Gal4p
  (Figure 17.18).
• 17.7 DNA methylation and regulation of gene
  expression
• Methylation occurs most often in cytosine of CG
  doublets in DNA.
• Methylation state of gene can be determined by
  restriction enzyme analysis with HpaII and MspI
  (Figure 17.19).

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Gene regulation (eukaryotes)

• In eukaryotes, several observations suggest that
  DNA methylation plays role in gene regulation
• First, inverse relationship exists between degree
  of methylation and degree of gene expression ?
  inactivated X chromosome has higher level of
  methylation than active X chromosome in mammalian
  females.
• Second, methylation patterns are tissue specific
  and heritable for all cells in that tissue.
• Incorporation of 5-azacytidine (Figure 17.20)
  causes undermethylation of sites of incorporation
  and changes in pattern of gene expression.

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Gene regulation (eukaryotes)

• 17.8 Post-transcriptional regulation of gene
  expression
• Although transcriptional control is perhaps major
  type of regulation in eukaryotes,
  post-transcriptional regulation also occurs in
  many organisms.
• Eukaryotic mRNAs modified prior to translation ?
  noncoding introns removed ? remaining exons
  spliced together ? mRNA modified by addition of
  cap at 5 end and poly-A tail at 3 end ? message
  then exported to cytoplasm.

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Gene regulation (eukaryotes)

• 17.8.1 Alternative splicing pathways for mRNA
• Alternative splicing can generate different forms
  of mRNA from pre-mRNA ? giving rise to number of
  proteins from one gene (Figure 17.21).
• Alternative splicing increases number of proteins
  made from each gene ? as result, number of
  proteins made by cell (its proteome) is not
  directly related to number of genes in genome.

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Gene regulation (eukaryotes)

• 17.8.2 Alternative splicing and cell function
• Inside cochlea of inner ear, each hair cell
  responds to different narrow range of
  frequencies (Figure 17.22).
• Response of these cells controlled by alternative
  splicing of pre-mRNA transcripts of SLO gene.

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Gene regulation (eukaryotes)

• 17.8.3 Alternative splicing amplifies number of
  proteins produced by genome
• Drosophila Dscam gene encodes protein that guides
  axon growth, ensuring that neurons are correctly
  wired together ? Dscam gene can encode 38 016
  different versions of DSCAM protein (Figure
  17.23).
• 17.8.4 RNA silencing of gene expression
• RNA silencing is called RNA interference (RNAi)
  in animals and post-transcriptional gene
  silencing (PTGS) in plants.

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Gene regulation (eukaryotes)

• RNAi uses protein called Dicer to cleave double
  stranded RNA molecules into short interfering
  RNAs (siRNA) that bind to RNA-induced silencing
  complex (RISC) for unwinding (Figure 17.24).
• Single-stranded RNAs target mRNAs with
  complementary sequences to mark them for
  degradation (Figure 17.25).
• In animals ? microRNAs (miRNAs) mediate RNA
  silencing by binding to 3 untranslated regions
  of mature mRNAs to block translation.

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Gene regulation (eukaryotes)

• In plants ? miRNAs arrest translation or initiate
  mRNA degradation.
• Short RNAs can also mediate RNA-directed DNA
  methylation (RdDM) of cytosine.
• RdDM ? highly specific process ? limited to
  region of RNA-DNA pairing.
• In RdDM ? CG dinucleotides and other C residues
  in promoter regions are methylated ? leading to
  gene silencing.

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Gene regulation (eukaryotes)

• 17.9 Alternative splicing and mRNA stability can
  regulate gene expression
• Sex lethal (Sxl), transformer (tra) doublesex
  (dsx) genes are part of hierarchy of gene
  regulation for sex determination in Drosophila
  (Figure 17.26).
• Sxl gene acts as switch that selects pathway of
  sexual development by controlling splicing of dsx
  transcript in female-specific fashion.

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Gene regulation (eukaryotes)

• Another way to control mRNA stability ? through
  translation level control ? translation of
  message controls its stability.
• Translation plays role in mRNA stability for
  tubulin genes and has been proposed as regulatory
  mechanism for other genes as well.
• This type of translational regulation is known as
  autoregulation.