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Transcriptional Regulation

Transcriptional Regulation. Getting started – Promotors, Sigma Factors, and DNA-binding proteins. Promotors. -10 and -35 consensus sequences (before transcription, not start codon) -10 TATAAT – “TATA” or Pribnow Box -35 TTGACA – “T-T-GA-CA” Altered sequence – weak promotor

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Transcriptional Regulation

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  1. Transcriptional Regulation Getting started – Promotors, Sigma Factors, and DNA-binding proteins

  2. Promotors • -10 and -35 consensus sequences (before transcription, not start codon) • -10 TATAAT – “TATA” or Pribnow Box • -35 TTGACA – “T-T-GA-CA” • Altered sequence – weak promotor • Sequence complementary to sigma factor of RNA pol

  3. Sigma factors vary • First example of global regulation – simultaneous, coordinated control of multiple genes and operons • Table 8.2 Brock 11th

  4. DNA-binding Proteins • Sigma factors σ70 • Activators • Repressors

  5. Helix-turn-Helix Motif

  6. Features of the Interaction • Repressors often act as dimers or tetramers • Each monomer has recognition domain and stabilization domain • Recognition sequence often involves inverted repeats • figure 8.8 Brock 11th

  7. Eukaryotes feature Zinc-fingers and Leucinezippers (figure 8.10)

  8. Transcriptional Regulation Let’s be positive

  9. Positive regulators - activators • Activator binds to activator site or enhancer siteupstream of promotor • Facilitate RNA pol binding to promotor • Actual touching RNA pol • “Melting”

  10. Activator binding to DNA may require small molecule - inducer • Examples • AraC protein binds L-arabinose, and then the L-arapromotor • Maltose and the maloperon – (figure 8.15) • cAMP + cAMPReceptor Protein (CRP) – will be considered in detail later

  11. Enhancer sites or activator-binding sites can occur distant from the promotor • Results in bending of DNA • May result in opening of promotor double helix • Bent DNA may be required for RNA pol-activator complex to form • Example NRI-P activator of the ntr regulon • May involve IntegrationHost Factor (IHF) protein

  12. Transcriptional Regulation Negative control of transcription: Repression and induction

  13. Importance of operator region • Protein binds operator and blocks RNA pol • LexA repressor protein blocks synthesis of DNA repair enzymes like uvrABC • When DNA is damaged, RecA protein becomes a protease that specifically degrades LexA protein

  14. Repressor frequently interacts with small molecule (effector) • Presence of small molecule prevents transcription • Frequently involved in control of amino acid synthesis (anabolic) genes • Prevents costly synthesis of unnecessary proteins

  15. Repression involves corepressor molecule binding to aporepressor protein • arginine (corepressor) binds (apo-)repressor that binds operator (fig. 8.13)

  16. One level of control of tryptophan biosynthesis • TrpR protein – 11 kD, acts as a dimer, 50 copies per cell • Binds operator when tryptophan is present • Autogenous regulation – also will block it’s own synthesis

  17. Small molecules (inducers) can bind repressor protein and prevent binding to operator • Enzymes will be synthesized only when inducer is present • Typically involves catabolic enzymes • Utilization of particular sugars

  18. lac Operon – Simple Version • Inducer binds repressor protein and reduces affinity for operator • Actual inducer is allolactose (an isomer of lactose) • Artificial inducer is isopropyl-β-D-thiogalactoside • lacoperon only transcribed if lactose is available

  19. Transcriptional Regulation Reduction of transcription after initiation: Attenuation of the trp operon

  20. Key Features of Attenuation • Leader region (trpL) occurs between promotor and first gene (trpE) • Leader region peptide requires 2 charged trp-tRNA • Inverted repeats lead to stem loop structures (including a terminator) • A second ribosome is needed (this is the secret nobody talks about)

  21. trp mRNA Synthesis at Low [tryptophan] – 10% of full expression • RNA pol slides along DNA, making transcript • Ribosome starts translating message • Ribosome sails through region 1 containing tryptophan codons • Ribosome reaches stop codon and falls off

  22. trp mRNA Synthesis at Low [tryptophan] – 10% of full expression • Consequences • Leader peptide is completed • Region 1 is free to pair with region 2 • Region 3 is free to pair with region 4 • 3:4 Stem loop is a termination stem loop and RNA pol falls off – no mRNA!

  23. Let’s take a closer look

  24. Let’s take an even closer look Shine-Delgarno

  25. trp mRNA Synthesis at Very Low [tryptophan] – full expression • RNA pol slides along DNA, making transcript • Ribosome starts translating message • Ribosome stalls at tryptophan codon

  26. trp mRNA Synthesis at Very Low [tryptophan] –full expression • Consequences • Leader peptide is not completed • Region 1 can’t pair with region 2 • Region 2 is free to pair with region 3 • 3:4 termination stem loop does not form and RNA pol continues to trpE

  27. Let’s take a closer look - again Ribosome stalls here

  28. Let’s take an even closer look Shine-Delgarno Shine-Delgarno

  29. Attenuation is a widespread control mechanism for amino acid synthesis • Threonine • Phenylalanine • Histidine • 7 straight His! • No operator needed – all attenuation control

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