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Prokaryotic Gene Regulation Heyer 1 Prokaryotic Gene Regulation Regulation of Bacterial Gene Expression (a) Regulation of enzyme (b) Regulation of enzyme activity production Precursor Feedback inhibition Enzyme 1 Gene 1 Enzyme 2 Gene 2 Regulation of gene expression Enzyme 3 Gene 3 – Enzyme 4 Gene 4 – Enzyme 5 Gene 5 Tryptophan Figure 18.20a, b Metabolic Regulation 1. Adjust the activity of metabolic enzymes already present Operons: functional gene clusters 2. Regulate the genes encoding the metabolic enzymes • In bacteria, genes are often clustered into operons, (a) Regulation of enzyme (b) Regulation of enzyme activity production composed of Precursor Feedback –A promoter inhibition Enzyme 1 Gene 1 • Site for RNA-polymerase to bind and initiate transcription –An operator, the “on-off” switch Enzyme 2 Gene 2 Regulation of gene • Region of DNA within the promoter or between the expression promoter and the first gene Enzyme 3 Gene 3 –The genes for metabolic enzymes – Enzyme 4 Gene 4 • Usually a set of enzymes catalyzing different steps in a common metabolic pathway – Enzyme 5 Gene 5 • All the genes in the operon set are transcribed onto a single, common mRNA Tryptophan Figure 18.20a, b Regulation of Bacterial Operons: functional gene clusters Gene Expression • Conserve energy — Metabolism is precisely regulated – Make only proteins needed at a specific time • Non-regulated gene expression – RNA-poly binds freely to promoter – Constitutive genes— Enzymes always needed (e.g., glycolysis) • Negative gene regulation – Repressor protein binds operator → Block RNA polymerase → Inhibits gene expression → Decreases synthesis of enzymes • Positive gene regulation – Activator protein binds separate binding site near promoter → Enhance RNA polymerase activity • Regulon: multiple operons regulated by the same regulator. – >40 regulons identified in E. coli Heyer 1 Prokaryotic Gene Regulation Negative gene regulation Negative gene regulation Regulating the regulator: The operon default is usually •In a repressible operon turned “on” –A corepressor molecule binds allosterically to the repressor protein → repressor protein assumes active configuration Repressor protein binds operator → binds to the operator → Block RNA polymerase → shuts off transcription → Inhibits gene expression → Decreases synthesis of enzymes • In an inducible operon –A inducer molecule binds allosterically to the repressor protein → repressor protein assumes inactive configuration → cannot bind to the operator → turns on transcription (a) Regulation of (b) Regulation of enzyme activity enzyme production Feedback Precursor inhibition Repressible Operon Enzyme 1 Gene A trp operon Enzyme 2 Gene B Regulation of gene expression • Tryptophan operon Enzyme 3 Gene C – • Usually occurs in anabolism Enzyme 4 Gene D • Consists of 5 structural genes – Enzyme 5 Gene E • Repressor is inactive so tryptophan is synthesized Tryptophan • Amino acid in media – Binds to repressor activating it Heyer 2 Prokaryotic Gene Regulation trp operon trp operon DNA trp operon No RNA made Promoter Promoter Genes of operon DNA trpR trpE trpD trpC trpB trpA Operator mRNA Regulatory RNA Start codon Stop codon gene 3′ polymerasemRNA 5′ mRNA 5′ EDC BA Active Protein Inactive Protein repressor repressor Polypeptides that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on. RNA polymerase attaches to the Tryptophan DNA at the promoter and transcribes the operon’s genes. (corepressor) (b) Tryptophan present, repressor active, operon off. As tryptophan Figure 18.21a accumulates, it inhibits its own production by activating the repressor protein. Figure 18.21b Inducible Operon lac Operon • Turn on the transcription of gene • Inducible operon: enzymes to metabolize lactose • Inducer- induces transcription • Regulatory sites – Promoter- RNA polymerase • Inducible enzymes – Operator- repressor binds – Synthesized only when substrate is present • i genes code for repressor-regulatory protein – Lactose metabolism in E. coli – Outside operon – Always turned on (constitutive gene) – Binds to operator • Structural genes – lac operon-3 genes lac Operon * *Genes coding for enzymes to digest and utilize lactose: Z ⇒ β-galactosidase Y ⇒ permease A ⇒ transacetylase Heyer 3 Prokaryotic Gene Regulation Lactose in Medium • Binds repressor changing shape • Repressor can’t bind to Operator • RNA polymerase can bind to Promoter • Enzymes for lactose metabolism produced – Lactose transported into cell – Metabolized into glucose and galactose lac Operon lac Operon lac operon Promoter Regulatory Operator gene DNA lacl lacz lacY lacA DNA lacl lacZ RNA No 3′ polymerase mRNA 5′ RNA mRNA 5′ mRNA 5' 3′ made RNA polymerase β-Galactosidase Permease Transacetylase mRNA 5′ Protein Allo-lactose Inactive (inducer) repressor Active (b) Lactose present, repressor inactive, operon on. Allo-lactose, an isomer of Protein repressor lactose, derepresses the operon by inactivating the repressor. In this way, the (a) Lactose absent, repressor active, operon off. The lac repressor is innately enzymes for lactose utilization are induced. active, and in the absence of lactose it switches off the operon by binding to the operator. Figure 18.22a Figure 18.22b lac Positive Gene Regulation Positive regulation of the operon •InE. coli, when glucose is the preferred energy substrate. •When available glucose decreases, intracellular cAMP • Some operons are also subject to (cyclic-adenosine monophosphate) increases. positive gene regulation •cAMP binds to CAP, causing it to change into the active – Stimulatory activator protein binds separate configuration. binding site near promoter → •Active CAP enhances operon promoters for alternative Enhance RNA polymerase activity catabolic pathways, including the lac operon. → Increase gene expression & enzyme synthesis – Catabolite Activator Protein (CAP) • Activates many catabolic pathways • Including lac operon. Heyer 4 Prokaryotic Gene Regulation Positive regulation of the lac operon Positive regulation of the lac operon •The lac operon is activated by the binding of CAP • ↑glucose levels → ↓cAMP → inactive CAP Promoter Promoter DNA DNA lacl lacZ lacl lacZ CAP-binding site Operator CAP-binding site RNA Operator polymerase RNA can bind polymerase Active and transcribe can’t bind cAMP CAP Inactive Inactive lac Inactive lac CAP repressor Inactive repressor CAP (b) Lactose present, glucose present (cAMP level low): little lac mRNA (a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized. synthesized. Figure 18.23a Figure 18.23b Combined Negative & Positive Arabinose Operon Regulation of the lac Operon 1. Low lactose → repressor active → operon suppressed 2. High lactose/high glucose → repressor inactive but activator inactive → low operon activity 3. High lactose/low glucose → repressor inactive and activator active • Negative or Positive gene regulation? → high operon activity → enhanced lactose catabolism • Repressible or Inducible? Arabinose Operon Heyer 5 Prokaryotic Gene Regulation Regulation of operons in prokaryotes by Transcription Attenuation Simultaneous transcription & translation in prokaryotes RNA-polymerase DNA (gene) mRNA polypeptide ribosome Transcription Termination (prokaryotes) trp operon revisited leader trpP trpO trpL trpE trpD trpC trpB trpA promoter genes for enzymes to operator synthesize trytophan w Transcribed RNA forms GC hairpin. w Hairpin causes RNA poly to fall off, terminating transcription. Regulation of operons in prokaryotes by Regulation of operons in prokaryotes by Transcription Attenuation Transcription Attenuation 5’ UTR Section 1 Sect 2 Sect 3 Sect 4 5’ UTR Section 1 Sect 2 Sect 3 Sect 4 Start Start Sequence Start Trp Stop codon for Start Trp Sequence Stop codon for complementary to codon codons codon trpE codon codons complementary to codon trpE sects 2&4 sects 2&4 If [Tryptophan] low: If [Tryptophan] high: 1. Ribosome stalls at Trp codons 1. Ribosome continues 2. Sect 2 binds to 3 3 → blocks sect 2 2 3 3. RNA-poly continues 2. Sect 3 binds to 4 → hairpin transcribing operon 3. RNA-poly dislodged → 4. → synth Trp-enzymes 2 terminate replication → synth more tryptophan 1 4. trp genes not transcribed → 1 ↓Trp synthesis Heyer 6.
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