Genetics Exam 4

• Trp operon is a ________ pathway

  anabolic
• In the absence of Trp

  RNA polymerase is able to bind to the promoter and transcription proceeds. Repressor is made but can't bind
• In the presence of Trp

  Trp binds to the repressor and undergo an allosteric change and together binds to the operator. transcription is blocked
• TrpR


  produces the repressor
• Attenuationonly in pro b/c requires co-transcription


  additional form of regulation by premature termination of transcription
• operon order(operons only in pro)


  TrpR-P-O-leader sequence- RNA leader -Leader peptide(14 codons=2 Trp
• RNA leader has 2 conformations


  1=2 and 3=4 loop structure or 2-3 loop
• RNA leader conformation 1

  termination of transcription so the rest of the operon is not transcribed (rho-independent
• RNA leader confirmation 2

  no terminator, transcription continues
• RNA leader senses existing Trp levels

  high levels of Trp= 1-2 and 3-4 , ribosomes move past Trp codon

  low Trp=2-3 ribosome stalls at Trp codon and there is no termination
• During low Trp there are multiple ribosomes


  they are chasing RNA polymerase to transcribe the end sequence and stalling on the Trp codon so the structure can't form
• What will be the level of expression of Trp operon genes on double mutants lacking both Trp repressor and the leader and attenuator sequence of the Trp operon

  100% with Trp present and 100% with no Trp. If the leader sequence is deleted you can't form the terminator sequence.
• imagine that you replaced the Rho-independent terminator in the attenuator of the Trp operon with a Rho-dependent terminator how will the modified operon behave in the presence/absence of Trp

  Structural genes of the Trp operon will not be translated in the absence or presence of Trp
• A mutant with more Trp will ensure more stalling it will be to the right of the WT


  in the Trp biosynthesis and Level of Trp graph
• Rho-independent terminator


  negative feedback
• Sigma factor


  ensure that RNA polymerase binds to the DNA at the promoter
• Alternative sigma factors


  sigma70 - promoter of housekeeping genes

  sigma 32- promoter of heat shock genes

  When there is heat shock stop producing sigma 70 and produce sigma 32
• mitochondria don't have sigma factors


  b/c they don't have bacterial RNA polymerase
• What components of the Trp operon is Cis-acting

  operator, leader and attenuator
• Gene Regulation by DNA binding proteins

  prensent in both prokaryotes and Euks, trans-acting regulatory proteins binds to a cis-acting regulatory sequence on DNA which affects the activity of the RNA polymerase by turning it on or off
• Eukaryotes respond to more stimuli

  metabolic, temperature, allosteric, subcellular localization, phosphorylation and protein stability
• Eukaryotes have a lot of transcription factors


  activators and repressors
• Regulation of TF activity

  subcellular Localization : Steroid hormone receptor, steroid doesn't bind in cytoplasm only nuclear.

  Phosphorylation and stability -Wnt: beta-catenin degraded and vice versa
• Cis-regulatory module (enhancers)


  may be far from gene and hard to study because we don't know where they are.
• Distant Enhancer 


  they are far but connected b/c DNA can loop. loop driven by TF- poly 11 interactions, other proteins connect distant DNA
• General Transcription factors


  recognize promoter -TF11D bind to tata box to separate DNA strands

  Recruit Poly 11

  limited sequence-specificity
• Regulatory Transcription factors


  binds to enhancers

  highly sequence specific (combination of transcription factors is unique for each gene

  regulate transcription
• Impact of non-coding RNA

  non-coding RNA can initiate/inhibit transcription of mRNA
• Insulators


  prevent enhancer from binding to promoter
• Nucleosoems interfere with transcription initiation 


  if nucleosomes is present there is no transcription and the cell has to remove nucleosomes for transcription to occur
• Post-transitional histone modifications


  mark nucleosomes, recognized by other proteins, TF requit co-repressors and co-activators which create chromatin readers, writer and erasers
• Chromatin remodeling


  DNA sequencing and active remodeling affect nucleosome positions
• Nucleosome assembly


  DNA to random deposition of nucleosomes and ISWI CHD assemble to around 50 base pairs between each other.
• Chromotine access


  SWI/SNF ATP-dependent chromatin remodeling complex remoces nucleosome to create a free region where gene promoter can form pre-initiation complex
• DNA methylation


  add CH3 group in the major groove so proteins can bind to DNA, repression of specific genes
• Yeast GAL system


  Galactose present: GAL 7, 10, 1 and 2 expressed

  each gene regulated by GAL4 UAS (enhancer)

  4 genes regulated in coordinated manner because they have similar enhancer sequences
• GAL 80(-/-) mutants


  constitutive expression = repression
• GAL 4(-/-) mutants


  uninducible expression = activator
• GAL 3(-/-) mutants


  uninducible expression =activator
• GAL 4= activator and GAL 80= corepressor


  corepressor blocks activation domain but GAL 4 is always bound to DNA promoter, galactose interacts with GAL3 and stops GAL 80 from binding to GAL 4.

  Gal 4 removes nucleosomes from gene promoter to allow initiation factors and reqruits general transcription factors to initiate transcription