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

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