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INDM 3007

INDM 3007. Lecture 2. Why regulation?. Responding to changes in environment Conservation of energy by expression of genes suitable for a particular situation. Sensing. Transduction and Integration of signals. Response. Microbe. Environment. Metabolic programs e.g., Choice of nutrients

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INDM 3007

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  1. INDM 3007 Lecture 2

  2. Why regulation? • Responding to changes in environment • Conservation of energy by expression of genes suitable for a particular situation

  3. Sensing Transduction and Integration of signals Response Microbe Environment Metabolic programs e.g., Choice of nutrients homeostasis Developmental programs e.g., Stationary phase, Spore formation, Fruiting body formation Specialty programs e.g., Heat shock response, Virulence Motility pH Temperature Nutrients Oxygen Salinity Presence of hosts

  4. Gene mRNA Polypeptide Enzyme subunits/holoenzyme Regulation of gene expression Transcription Initiation Termination Stability Editing Splicing Translation Initiation (Attenuation, micRNA) Translational shifts Termination Folding Splicing (Inteins) Assembly Localization Stability Amino Acids

  5. How does RNA polymerase find a promoter? Answer lies in comparison of many promoters. Alignment leads to a consensus sequence. AAAGTTGACACACCGTTAA Promoter 1 GCGCTTGATAGCATCGTAA Promoter 2 AAAGTTGACAATTTATATT Promoter 3 GAGATTCACAACGCAATAA Promoter 4 CCTTTTGACAGGGCGGCGT Promoter 5 NNNNTTGACANNNNNNNNN Consensus Four conserved features in a bacterial (RpoD) promoter -35 region -10 region Start site Distance between them

  6. -35 Mutations affect DNA binding Recognition domain -10 Mutations affect open complex formation Unwinding Domain Note: T=A ! +1 Transcription start (not very conserved)

  7. Same sigma factor recognises variable but related promoter sequences Approximately 100-fold difference in binding affinity Approximately 100-fold difference in promoter strength. Promoter strenght is dependent On affinity of RNA polymerase for promoter Ability to clear the promoter NOT on transcription speed

  8. Operon Operon: group of genes transcribed into a single mRNA from one promoter Biochemical pathways make use of many proteins including Transport proteins Assembly proteins Regulatory proteins Enzymes Very often, BUT NOT ALWAYS, proteins of a specific metabolic function are encoded in operons

  9. mRNA Promoter lacZ lacY lacA Galactose + Glucose Lactose Lactose Lac operon

  10. How do we analalyse operon structures? Isolate mRNA from cell and separate on denaturing agarose gel Negative large fragments Positive small fragments Transfer to nylon membrane

  11. Label a gene, eg lacZ, with DIG-dUTP, using e.g., PCR Labeled LacZ DNA: probe Hybridise probe to membrane Detect probe Light CDP Alkaline phosphatase lacZ lacY lacA

  12. 2.8 kb 1.6 kb Northern blot of aceA-fadB gene cluster using aceA as probe aceA fadB Conclusions: aceA and fadB are cotranscribed: operon second smaller aceA transcript Kelly, Wall, Boland & Meijer, 2001

  13. Why two transcripts??? mRNA degradation proceeds in general from 5' to 3' via endonucleolytic cleavage followed by exonucleolytic cleavage from 3' to 5' Typical half-life of mRNA = 2 minutes

  14. aceA fadB Protects aceA from endonucleolytic degradation Answer lies in the presence of a very stable hairpin structure between aceA and fadB Simple form of regulation More aceA mRNA than fadB mRNA, despite being in one operon More isocitrate lyase is needed than 3-hydroxyacylCoA dehydrogenase

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