Biology 331: Chapter 6

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Biology 331 Chapter 6

• Regulation of Gene Transcription

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Introduction

• Every cell contains all of the genes of that
  organism
• How are genes only turned on in the proper
  setting?
• Tissue types
• Changes in the environment
• Cells must be able to turn transcription on and
  off
• Cells must be able to recognize environmental
  conditions

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Prokaryotic transcription control and the lac
operon
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Anatomy of the lac operon

• The lac operon consists of 6 parts
• It contains three genes that deal with lactose
  metabolism (Z,Y,A)
• These genes are transcribed as one mRNA

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Promoter and operator

• Up stream of these genes is a promoter region
• The promoter is required for RNA polymerase to
  bind
• Between the promoter and the genes is an operator
  region

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The I gene

• Up stream of the promoter region is the I gene
• The I gene codes for a repressor protein
• The repressor protein binds to the operator
  region
• This blocks transcription

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Operon

• A set of adjacent genes which are transcribed as
  one unit plus related regulatory sequences

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So how does it all work?

• If there is no lactose in the environment do I
  want to produce enzymes to digest lactose?
• The I gene constantly produces a repressor which
  binds to the operator (O) region
• Since the action of RNA polymerase is blocked by
  the repressor transcription does not take place

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Lac operon
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Allosteric changes
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Add lactose to the environment

• We want to have enzymes to metabolize lactose
• Lactose binds to an allosteric site on the
  repressor causing a confirmational change
• In this case lactose is acting as the "inducer"
• The repressor no longer binds to the operator
• Transcription proceeds
• This is termed negative control since the
  repressor normally blocks production

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Lac operon
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Mutations in the system
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The I gene

• Constitutive Mutants
• Enzymes are always expressed in an uncontrolled
  fashion
• What happened?
• This mutation is recessive.....explain

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I- Mutations
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The Is mutation

• The allosteric site is altered
• The protein can no longer bind to the inducer (in
  this case lactose)
• The lactose enzymes are never produced
• Explain
• This mutation is dominant
• Also called trans (across) dominance

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Is mutation
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The O gene

• Oc mutations
• Changes the sequence of the operator region so
  the repressor won't bind
• Genes on that chromosome are always transcribed
• This is a cis (adjacent) dominant mutation
• It only affects transcription of genes on the
  same chromosome
• Explain...

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Oc mutation
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The promoter region

• Changes in the promoter region may cause RNA
  polymerase not to bind
• Blocks transcription
• Cis Dominant

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Catabolite repression of the lac operon

• A second control system on the lac system
• Some energy sources are preferred over others
• If glucose AND lactose are present E.coli prefers
  the glucose
• So we must find a way to shut down the lac system
  in the presence of glucose
• This is termed positive control since it only
  works in the presence of a substance

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How does it work?

• The presence of a glucose metabolite interferes
  with the production of cAMP from ATP
• CAP is produced by the crp gene
• CAP binds to cAMP forming a complex
• This complex binds to the promoter region and
  increases the affinity of RNA polymerase for the
  promoter
• Thus with too much glucose we get too little cAMP
  and RNA polymerase affinity is not increased

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CAP-cAMP system
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Glucose No Lactose
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Glucose and Lactose
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Lactose no Glucose
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Multiple Positive Controls

• The arabinose operon
• Enzymes are the araB, araA, and araD genes
• araI (initiator) region contains the promoter
• A product from the araC gene binds to arabinose
• This complex activates transcription

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This system also contains the same CAP-cAMP
system already discussed
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Positive control
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Alternate action of araC

• araC protein binds to both the araO and araI
  regions when arabinose is not present
• Forms a loop preventing transcription
• The same protein has opposite affects depending
  on the environment

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The Loop (negative control)
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Transcription in Eukaryotes

• Many of the problems are similar to those in
  prokaryotes
• However, often the actions of different tissues
  must be coordinated
• Development and physiological changes

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Cis acting sequences

• Has multiple regions

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The core promoter

• The RNA polymerase II binding site
• Transcription start sequence
• TATA box 30 bp up stream of initiation site

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Promoter-proximal cis acting sequences

• Helps binding of RNA polymerase II to the
  promoter
• CCAAT box and GC rich segment
• 100-200bp up stream

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Distance independent elements

• Act at some distance from the promoter
• Enhancers Increase transcription rates
• Silencers Decrease transcription rates
• Typically they are cis acting sequences affected
  by trans acting regulatory proteins
• The distance can be 50kb or more from the
  promoter!
• Can be up stream or down stream from the promoter

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Mechanism of action from a distance

• How can they function so far away?
• DNA forms loops
• Proteins bound to regulatory regions are brought
  into contact with the promoter region

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Action from a distance
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Tissue specific transcription

• Some genes only turn on in particular tissues
• Regulatory substances may only be present in
  certain tissues
• Can be a repressor or an enhancer
• The complex regulation requires a large number of
  regulatory genes
• Hormones

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Reporter genes and gene regulation

• Splice in a reporter gene
• A reporter gene produces an obvious phenotype
• Presence of the reporter substance indicates one
  or more enhancers must be near
• Moving the spliced bit localizes the enhancer
• We gain knowledge of where an enhancer works
• In time we can isolate trans acting regulatory
  compounds

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Reporters
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Regulatory mutations from chromosomal
rearrangements

• Can be due to chromosomal rearrangements
• Move the enhancer of one gene next to the
  transcription unit of another

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Bar eye mutation

• An eye regulatory sequence is placed next to a
  gene not normally expressed in the eye
• This compound leads to the death of many cells in
  the eye

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Tab (trans abdominal) mutation

• Genes normally only expressed in the abdomen are
  expressed in parts of the thorax