Topic 10: Regulation of Gene Expression

  • Differences in Prokaryotes (summary of last topic)
    • DNA - histone like protein
    • Circular DNA with single origin of replication (some Archaea has more than 1), Plasmids have rolling circle replication, 1 replication bubble, 2 replication fork (multiple replication bubble/replication fork in eukaryoties)
    • Speed: prokaryotes: 2000 bp vs 100 bp
    • No nucleus, nucleoid
    • DNA polymerase - IV and V; sos reponse
    • mRNA: smaller, unstable, degrade quickly
    • operons, no introns (some in archaea) = no splicing
    • ribosome is smaller 70s instead of 80s
    • mRNA - polygenic (encode for more than 1 gene, some Archaea), no splcing
    • Polysomes: simultaneous transcription/translation - because no splicing unlike eukaryotes, thus faster
    • Protein folding occurs after complete translation
    • RF (release factor) - termination: 3 in prokaryotes (RF 1,2, 3) vs 2 in eukaryotes (RF 1 and 3)
  • Transformation Experiment by Fred Griffith (1928)
    • Streptococcus pneumoniae (gram +)
    • Some strains are pathogenic, some not
      • Those pathogenic usually have capsules
    • Cooked pathogenic bacteria and then injected into mouse (alive)
    • Added un-pathogenic and cooked pathogenic → injected into mouse (died)
      • Non-pathogenic strain took up DNA fragments (exogenous DNA) from pathogenic strain into their chromosome.
      • Incorporated into their DNA
      • Expressed the gene(s) = transformation (at least 23 genes required) - does not happen easily to eukaryotes, frequent in bacteria
  • How Bacteria Control/Regulate Expression?
    • Regulation of cellular precoesses
    • Change the rate of protein syntheis (called regulation of gene expression) because it controls the transcription and translation of genes in the formation of proteins
    • Alter the activity of enzymes and other proteins (called post=-translational control) because it occurs after the proteins are produced
    • Levels of Regulation of Gene Expression
    • occurs at many levels
      • transcription intiation
      • transcription elongation
      • translation
      • post translation
    • three domains of life differe in genome structure and regulatory mechanisms used
    • Regulation of Transcription Initiation
    • induction and repression of enzyme synthesis
      • constitutive genes: housekeeping genes that are expressed continuously
      • facultative genes: expressed as need
      • inducible genes: genes that code for inducible enzymes such as β-Galactosidase
    • Control of Transcription Initiation by Regulatory Proteins
    • induction and repression occur because of the activity of regulatory proteins (on/off gene)
    • these proteins either inhibit transcription (=negative control) or promote transcripition (=positive control)
      • modulated by inducers, corepressors and inhibitors
    • Why do bacteria need to adjust/adapt gene expression/regulation?
    • Environment changes constantly
      • example: E. coli that was on meat is now on cutting board
      • example: formed biofilm at bathroom sink, then cleaned with cleaning solution
      • example: salmonella on fruits, lunch meat, ice cream
    • Need to adapt quickly for survival; cannot waste any resources
    • Catabolite Repression
    • diauxic growth
      • a biphasic growth pattern in which there is preferential use of one carbon source over another when both are available in environment
      • lag occurs after preferred substrate is exhausted followed by the resumption of growth using the second source
      • lab - fermentation tube
    • Why does bacteria switch nutrition so fast? (i.e glucose to lactose - b/c of operons
    • Operons
      • the sequence of bases coding for one or more polypeptides along with the promoter and operator or activator binding sites
      • the lac operon is an example of “positive” transcriptional control of inducible genes
      • trp operon is an example of “negative” control
      • glucose is preferred sugar source, but lac operon can produce β-galactosidase (able to catabolize lactose - split into galactose and glucose)
    • Negative/positive control
    • presence of regulatory protein (repressor = 2) at regulatory site (operator = 3) decreases mRNA synthesis
    • absence of a regulatory protein (activator protein) at a regulatory region promotes transcription (lac operon = lactose)
    • lactose operon
      • regulated by catabolite activator protein (CAP) and cyclic AMP (cAMP)
      • CAP also called cyclic AMP receptor protein (CRP)
    • cAMP (“messenger”)
    • CAP activity is modulated by cAMP
    • levels of cAMP controlled by adenyl cyclase (converts ATP to cAMP and PPi)
      • adenyl cyclase: active only when little or no glucose is present
      • in absence of glucose, CAP is active and promotes transcription of operons used for catabolism of other sugars
    • cAMP = cyclic adenosine monophosphate
    • PPi = pyrophosphate
    • CAP = catabolite activator protein (cAMP receptor protein)
    • The lac Operon (E. coli, enterics)
    • promotor sequence
    • I = repressor (constitutive gene = “housekeeping” gene) → always on - blocks RNA polymerase? or repressor protein?
    • Z = beta = galactosidase
    • Y = permease
    • 2 mechanisms of control (positive and negative) - repressor and CAP
    • negative inducible gene
    • Regulation of the lac Operon by the lac Repressor and CAP
    • photo → the lac operon and its control elements (figure this out?)
    • CAP binding site before promotor, repressor gene is always upstream of CAP, operator after promotor
    • Low glucose & lactose available → CAP protein (cAMP) and RNA polymerase → lac genes strongly expressed (repressor protein not present)
    • High glucose & lactose unavailable → repressor protein → lac genes not expressed (no unlock of repressor so it stays because of lactose levels, no CAP because of glucose levels)
    • Low glucose & lactose unavailable → CAP protein (cAMP) and repressor protein → lac genes not expressed (repressor remains because lactose not present, CAP protein attaches because low glucose therefore no expression)
    • High glucose & lactose available → nothing present → very low (basal) level of gene expression (high glucose therefore no CAP, lactose unlocks repressor protein, causes inefficent expression - not working properly)
    • new slide: the regulator protein has a defective operator-binding site. (no binding but transcription still occurs)
    • new slide: super repressor (lactose-binding site altered; no binding to lactose → repressor always bound to operator, blocking transcripition.)
    • new slide: operator constitutive (the O^c operator sequence is defective → the repressor protein will not bind to it but transcription proceeds) - no lactose present
    • The Tryptophan (trp) Operon
    • negative repressible system
    • consists of 5 structural genes which code for enzymes needed to synthesize tryptohan
    • the operon functions only in the absence of tryptophan (“emergencency operon”)
    • has two mechanisms - Tryptophan/trp repressor and attenuation
    • Regulation of the trp Operon by Tryptophan and the trp Repressor
    • When tryptophan is present, the trp repressor binds the operator, and RNA synthesis is blocked therefore no production of tryptophan. (1st mechanism)
    • In the absence of tryptophan, the repressor dissociates from the operator, and the RNA synthesis proceeds producing tryptophan. (1st mechanism)
    • Regulation of Transcription Elongation Termination
    • trancription can also be regulated by controlling transcription termination
    • this type of regulation, called attenuation, was first demonstrated with trp operon (2nd mechanism - how to stop)
      • Tryptophan levels high (quicker) → ribosome translates sequence 1 and blocks sequence 2 before sequence 3 is transcribed; continued transcription leads to attenuation at the terminator-like structure formed by sequences 3 and 4. (try not produced)
      • Tryptophan levels low (slower) → ribosomes pauses at Trp codons in sequence 1; formation of the paired structure between sequences 2 and 3 prevents attenuation because sequence 3 is no longer availble to form the attenuator stucture with sequence 4. (try produced, but takes time to do so)
      • 1st mechanism - repressor & 2 nd mechanism - attenuation (2nd mechanism present in case 1st mechanism fails)
      • Said picture is bad on this slide b/c it looks like both will not work
  • Riboswitches: a regulatory segment of a mRNA that binds a small molecules, resulting in a change in protein production.
    • folding of leader sequence (the riboswitch) determines if transcription will continue or be terminated
    • folding pattern altered in reponse to binding of an effector molecule
    • translation regulation
  • Regulation of Translation by a Riboswitch
    • Low concentration → translation occurs
    • riboswitch attached
    • High concentration → translation blocked
    • substrate attaches to riboswitch therefore changing its shape
  • Global Regulatory Systems
    • regulatory systems that affect many genes and pathways simultaneously
    • important for bacteria since they must respond rapidly to a wide variety of chaning environmental conditons
    • Regulon (prokaryotes) - ex: biofilm
    • genes or operons controlled by a common regulatory protein
    • Modulon - multiple regulon individually controlled (lac operon) but also controlled globally
  • Regulation of Gene Expression in Archaea
    • most Archaea regulatory proteins function like bacteria activators and repressors
    • they bind DNA sites near the promoter, enhancing or blocking the binding of RNA polymerase
    • some regulatory proteins function like Eukarya regulatory transcription factors by interacting with a general transcription factor
  • Feedback Inhibition
    • also called end product inhibition
    • inhibition of one or more critical enzymes in a pathway regulates entire pathway
    • pacemake enzyme
      • catalyzes the slowest or rate-limiting reaction in the pathway
  • How to insert genes (i.e insulin)
    • Lac operon - promotor
    • Replace genes with genes of desire