DNA Response Elements

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DNA Response Elements

• Activate a group of genes at the same time
• development and functioning of specialized cells
• genes usually scattered in the genome
• Response elements turn on and off genes in
  response to environmental or developmental signal
• place the RE next to genes that need to be
  regulated can controlled together
• important in embryonic development and tissue
  responding to environmental and physiological
  conditions

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Hormone Response Elements

• Respond to steroid hormones
• Nuclear receptor proteins mediate the actions
  of steroid hormones
• progesterone, estrogen, testosterone and
  glucocorticoids
• Chemical messenger binds to receptor protein and
  enters the cell, transmits signals to usually
  activate transcription with an occasional
  inactivation
• Steroid hormone receptor acts as a TF binding to
  DNA control sequence Hormone Response Element

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• Steroid response elements have similar sequences
  and are upstream of promoter
• steroid hormone receptor is a zinc finger type TF
  with a hormone binding site, DNA binding site and
  a transcription activator site

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Cortisol Activationof Gene

• Glucocorticoid receptor (GR) is in cytosol and is
  bound to hsp so cannot enter the nucleus
• When cortisol is present, binds to GR and hsp is
  removed and now can enter nucleus and bind to
  response element
• Forms a GR-S dimer and activates transcription

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Common Theme

• Inverted repeat 2 copies in opposite directions
• Most other steroid receptors are already in the
  nucleus without the hormone but require it to
  release inhibition
• Occasionally can inhibit by binding to gene
• GC doesnt form dimers but depresses initiation
  oby recruiting enzyme to remove acetyl groups
  chromosomes condense and no room for
  transcription machinery

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Protein Phosphorylation

• Involves cAMP to stimulate protein kinase A
• CREB binds to cAMP response element and is
  phosphorylated by PKA, recruits transcriptional
  activator CREB binding proteins (CBP)
• CBP catalyzes histone acetylation to recruit RNA
  pol and transcription

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Signal Transducers and Activators of
Transcription (STATs)

• Interferons are the signaling molecules for STATs
  
• IFN make neighboring cells to resistant to virus
  infection
• IFN binds the receptor activates Janus Activated
  Kinase (JAK) which adds PO4 to STATs which
  dimerize and move to nucleus and bind DNA to
  increase transcription
• JAK-STAT pathway has considerable specificity
  activates a unique set of genes
• STATs are also phosphorylated by other signaling
  molecules

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Other Important Phosphorylations

• SMADs after TGF? binding
• MAP Kinase controlling cell growth and division

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Heat Shock Response Elements

• Increase in temperature activates genes
• Heat shock or stress-response genes regulates
  genes at different chomosomal sites
• Gene products help to minimize damage due to
  thermal denaturation
• Hsp70 is a molecular chaperone that helps refold
  proteins
• Heat-shock response element is a binding site
  upstream of other common promoter sequences
• heat shock TF binds to the HSRE it is inactive at
  permissive temperature but as the temperature
  increases causes conformation change that allow
  binding and further activated by phosphorylation

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Basic Principle

• Genes in different chromosomal locations are
  activated by the same signal if the same response
  element is near each of them

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Homeotic Gene

• Genes responsible for body plan
• Mutations in the homeotic gene causes changes in
  body plan
• bithorax gene complex 2 sets of wings
• antennapedia gene complex legs instead of
  antenna
• Homeotic genes encode a family of TF that
  activate/inhibit transcription of developmentally
  important genes

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Homeobox

• Near the 3 end of homeotic genes
• Homeobax encodes the homeodomain that binds to
  DNA
• highly conserved during evolution
• helix-turn-helix motif

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Post-Transcriptional Control

• All are regulatory points after transcription
• Fine tune gene expression without drastic
  transcriptional changes to transient changes in
  environment

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Control of RNA Processing and Transport

• 1º transcript to mRNA
• 5 cap, 3 polyA tail, chemical modifications
  (CH3), exon selection, intron removal and RNA
  editing
• RNA splicing is important can make different
  mRNA from 1º transcript that allows for more than
  one type of protein product
• Splicing is controlled by proteins that bind to
  the splice site and protect it from splicing

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Alternative Splicing

• IgM can either be secreted or membrane bound
  depending on the splicing that occurs at the 3
  end
• Use alternative splice site if it is to
  membrane bound, then need the exons that contain
  the hydrophobic membrane spanning region

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RNA Export

• Splicing and processing of the 1º transcript is
  important for export
• 5 capping and 3 polyA defects have a decrease
  in export of those messages to the cytoplasm
• If introns are removed from a gene, then the
  message is not transported to the cytoplasm but
  if you put at least one intron back into the gene
  there is transport to the cytoplasm of the mRNA
• mRNA genes require a nuclear export signal

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Translation Rates

• Several mechanisms
• alter ribosomes or protein synthesis factors
• regulate the activity and/or stability of mRNA

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Heme-Controled Inhibitor (HCI)

• Globin synthesis is regulated by the presence of
  heme
• HCI is inhibited by heme that allows for
  translation of the globin mRNA
• When no heme, HCI phosphorylates eIF2 which
  prevents translation of globin and other messages
• no need for globin if there is no heme

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Initiation Factor Regulation

• Other kinases can also phosphorylate eIF2
• PO4 of eIF4 binds the 5 mRNA cap but rather than
  inhibiting translation it activates it
• seen in adenovirus infection
• Phosphorylation also regulates initiation and
  elongation factors as well as aminoacyl-tRNA
  synthetases
• All these are non-specific controls of translation

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Ferritin

• Iron storage protein synthesis stimulated in
  the presence of iron
• Iron-response element (IRE) in the 5
  un-translated leader sequence
• forms a hairpin loop that is required for
  expression in presence of iron

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• IRE-binding protein binds to IRE without iron
  get low level translation
• Fe present binds to the IRE binding protein and
  removes it from the hairpin loop and ribosome can
  bind and do high level translation
• Translational repressor control specific mRNA
• response to specific changes in environment

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mRNA ½ Life Regulation

• ½-life is the time to remove ½ mRNA time
  differs from mRNA to mRNA
• alters the stability of the message
• PolyA tail dictates the stabilty of mRNA
• long tail greater stability
• short tail less stability
• Other features in the 3 un-translated region
  also involved
• AU rich sequence triggers removal of polyA tail
  by degradative enzymes causes short ½ life

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Fe Control of mRNA Degradation

• Iron comes in by transferrin receptor protein
• when Fe levels drop there is an increase in
  transferrin receptor synthesis
• mRNA protected from degradation
• use IRE (similar to ferritin IRE) in the 3 end
• increase in iron in the cell causes mRNA
  degradation

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RNA Interference

• Use RNA to silence genes
• Can inhibit activity
• trigger mRNA degradation
• inhibit translation
• inhibit transcription of genes
• Response to introduction of dsRNA identified in
  plants with infection with dsRNA virus

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Specific Gene Inhibition

• Ribonuclease Dicer cleaves dsRNA into short
  fragments 21-22 bp called small interfering RNA
  (siRNA)
• siRNA combine with a group of proteins to make
  RISC (RNA-induced silencing complex)
• degrades one strand of RNA and the other strand
  will complementarily bind to target mRNA
• if match is close slicer comes in and degrades
  mRNA
• if not close match, inhibit translation but not
  degraded
• RISC can move to nucleus and siRNA can bind to
  DNA and cause methylation to histones DNA
  becomes transcriptionally inactive
• Also a useful lab tool to determine the affects
  of genes in cells and cell development

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MicroRNAs (miRNA)

• miRNA transcribed into longer RNA called primary
  miRNA
• Fold into a hairpin loop and converted to mature
  miRNA by steps
• Drosha cleave into smaller hairpins
• move to cytoplasm and Dicer will cut into small
  pieces
• bind to ribonucleoprotein complex silence
  expression
• close similarity degraded
• partial match inhibited
• Most are targeted to genes for proteins involved
  in development of the organism

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Post-Translational Control

• Modification of protein structure
• phosphorylation reversible and irreversible
• permanent change such as proteolytic cleavage
• Folding by chaperone protein
• Targeting to intra- and extra-cellular locations
• Interactions with cAMP or Ca2
• Amount of protein is a balance between synthesis
  and degradation

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Ubiquitin Targeting of Proteins

• Ubiquitin is a small protein bound to target
  proteins for proteosome
• 3 components involved
• ubiquitin-activating enzyme E1
• ubiquitin-conjugating enzyme E2
• substrate recognition protein E3
• E1 is ATP dependent transfer ubiquitin to E2,
  move to target protein lysine residue with E3

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Proteosomes

• Ubiquitin is recognized by proteosomes protease
  in the cytoplasm
• 6 proteases and ATPase and binding site for
  ubiquitin
• removes ubiquitin and then proteases chop up the
  protein into small fragments in a ATP-dependent
  fashion
• Different E3 enzymes recognize different amino
  acid sequences in amino terminus can either
  increase or decrease degradation
• Degrons internal amino acid sequences that
  allow degradation
• anaphase-promoting complex acts as an E3 for
  mitotic cyclins

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Other Methods

• Ubiquitin targeting is not the only method to
  remove proteins
• Microautophagy lysosomal membrane forms small
  vesicles that bring in proteins that are then
  degraded in the lysosome
• non-selective
• More selective process requires the recognition
  of a glutamine residue with 4 very basic, very
  acid or hydrophobic amino acids on each side