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 σ 70 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 Leucine zippers (figure 8.10)

8. Transcriptional Regulation Let’s be positive

9. Positive regulators - activators Activator binds to activator site or enhancer site upstream 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-ara promotor Maltose and the mal operon – (figure 8.15) cAMP + cAMP Receptor 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 NR I -P activator of the ntr regulon May involve Integration Host 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 lac operon 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

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