Chapter 17

• Eukaryotic gene expression


  • Comparing to prokaryotes
  • Chromatin structure
  • Transcription initiation
  • RNA processing
  • RNA interference

  
• Cap


  5' cap on mRNA
• AAA


  Poly(A) tail on mRNA
• Eukaryotic gene regulation


  1. mRNA transport

  2. chromatin remodeling

  3. transcription

  4. RNA processing

  5. translation

  6. protein modification

  7. degradation

  
• Epigenetics


  Differences in gene expression not due to changes in DNA sequence

  
• Epigenetic variation


  • Differences in flower shape in toadflax (Linaria vulgaris) due to methylation of the Lcyc gene
• Epigenetic marks can be passed from one generation to the next
• Eukaryotic transcription initiation


  • Transcription factors bound to regulatory promoter and enhancers interact via the mediator to affect transcription
  • Assembled TFII complex attracts RNA polymerase and allows initiation
  • TBP and TFIID bind the TATA box
  • The basal transcription apparatus is the complex of RNA polymerase, TFs, and other proteins that assemble to carry out transcription

  
• TATA binding protein (TBP) of TFIID


  • Binds to the TATA box
  • Recruits additional TFII proteins
  • Attracts RNA polymerase II
  • Completes assembly and commits to transcription
  • Required for basal levels of expression for all pol II genes

  
• Core promoter


  Very similar from gene to gene

  
• Regulatory promoter


  • Upstream of the core promoter and is bound by additional transcription factors (TFs)
  • Regulatory elements bound by activator proteins

  
• Mediator complex


  Component of the basal transcription apparatus that interacts with TFs directly or via coactivators

  
• Enhancers


  Cis elements that interact with activators, chromatin remodelers, or TFs to increase expression

  
• Silencers


  Cis elements that interact with repressors, chromatin remodelers, or TFs to decrease expression
• Enhancers and silencers can be quite far (1kb or more) from the transcription start site</b>
• Unique combinations of regulatory sequences and the TFs that bind them allow for effectively infinite combinations of TFs and cell-type specific gene expression

  
• Topologically associated domains (TADs)


  Large, spatially interacting regions of chromatin that are separated physically from other TADs by insulators
• Insulators


  DNA sequences that block the effects of enhancers
• Some response elements found in eukaryotic cells

  • Heat-shock element
  • Glucocorticoid response element
  • Phorbol ester response element
  • Serum response element
• Eukaryotes use response elements, consensus sequences found in the regulatory promoter of coordinated genes

  
• One gene may have multiple response elements so it can be activated by different signals
• Multiple genes may have the same response element so that they can all be activated by the same signal
• GAL genes in yeast

  • Code for enzymes required to metabolize galactose
  • Induced by galactose
  • Each gene has an independent promoter but are all up-regulated by the same signal

  
• UASG


  Upstream activating sequence for GAL4 that contains multiple GAL4 binding sites and acts as an enhancer

  
• GAL4


  An activator that binds to UASG sequences and enhances transcription of nearby genes

  
• GAL80


  A repressor that binds to GAL4 and prevents it from activating transcription

  
• GAL3


  A protein that binds to galactose (when present) and GAL80, preventing GAL80 from binding to GAL4

  
• gal4- mutants are uninducible, gal80- mutants are constitutively active, gal4- mutants are epistatic to gal80- mutants
• Alternative splicing


  • Some pre-mRNAs can splice out introns in more than one way
  • 95% of human genes display alternative splicing
  • May change the reading frame, alter amino acids or introduce stop codons
  • Splicing may be regulated differently in different tissues or in response to external factors
  • Regulation is accomplished by trans-acting proteins binding to cis-acting RNA sequences and either blocking or promoting splice site choice

  
• Sxl


  • The master regulator of sex determination in Drosophila
  • Functional protein in XX, non-functional protein in XY
  • Activity determines the sex of the fly
• Flipping the Sxl switch

  1. PE (early promoter) expresses Sxl in XX, not XY embryos

  2. Functional Sxl protein is made in XX, not XY embryos

  3. PM (mature promoter) expresses Sxl in both XX and XY embryos

  4. If Sxl protein is absent, Sxl is spliced into an inactive, truncated form

  5. If Sxl protein is present, Sxl is spliced into active form
• Phenotypes of sex-determination mutants

  • Sxl null mutant: XX phenotype, XY phenotype, embryonic lethal
  • tra null mutant: male, male
  • dsx null mutant: sterile intersex, sterile intersex
• mRNA stability


  • mRNA is a relatively short-lived molecule
  • The amount of mRNA available for translation is a function of both the rates of transcription and the rate of degradation
  • The half-life of mRNA varies between transcripts
  • Short-lived RNA allows faster transcriptional control, long-lived RNA is more efficient for housekeeping genes
  • Bacterial genes have very short half lives (minutes) & lack control at the level of mRNA stability

  
• Factors affecting mRNA stability

  • PolyA tail and 5' cap
  • 3' UTR sequences
  • miRNAs & siRNAs
  • Binding of poly(A)-binding proteins (PABPs) to the poly(A) tail protects the tail from degradation
  • Shortened poly(A) tails lead to removal of 5' cap and degradation of mRNA
  • RNAi (siRNA & miRNA) can target mRNA for degradation

  
• siRNA and miRNA


  • Both are cleaved by Dicer into short dsRNA molecules
  • Both bind a protein to form an RNA-induced silencing complex (RISC)
  • siRNAs form perfect pairings with target mRNA and often lead to cleavage and degradation of target mRNA
  • miRNA form imperfect pairing with target mRNA and generally act by blocking translation
  • miRNA from independent genomic locus, siRNA transcribed from target locus

  
• RNAi as a genetic tool

  Pick a gene, construct dsRNA that matches the gene, inject dsRNA into experimental organism, observe phenotype

  
• Translational and posttranslational control of gene expression

  • Control of translation via abundance of required proteins can slow or speed up translation
  • Many proteins undergo posttranslational modifications that affect their ability to function

  
• Comparison of gene control in bacteria and eukaryotes

  • Cascades of gene regulation
  • DNA-binding proteins
  • Negative and positive control
  • Levels of regulation
  • Role of chromatin structure
  • Presence of operons
  • Initiation of transcription
  • Enhancers
  • Transcription and translation

  
• Eukaryotic gene regulation involves mRNA transport, chromatin remodeling, transcription, RNA processing, translation, protein modification, and degradation

  
• mRNA is a relatively short-lived molecule


  The amount of mRNA available for translation is a function of both the rates of transcription and the rate of degradation