INDM 3007

1. INDM 3007 Lecture 5

2. Rpol This is typical for transcriptional activators Repressor binding sites often overlap the promoter Exception: activators of sigma-54 RNA polymerase These bind far upstream, more common for eukaryotic promoters lysR proteins bind adjacent to the CbbR promoter

3. Conclusion from previous: two LysR dimers bind side by side to the same side of the DNA helix Proper orientation between proteins bound to DNA is extremely important Facilititates protein protein contacts Essential for transcriptional activation cooperative binding

4. One protein-DNA Complex No protein-DNA interaction IR1 IR2 IR3 GCCACTTCAGATTTCCTGAATGCCTACTTCATATCATTTAAATTTACCTGAAATCGGCGCGGGGGCA Conclusion: strong binding to binding site 1 binding to binding site 2 requires CbbR at binding site 1

5. GCCACTTCAGATTTCCTGAATGCCTACTTCATATCATTTAAATTTACCTGAAATCGGCGCGGGGGCAGCCACTTCAGATTTCCTGAATGCCTACTTCATATCATTTAAATTTACCTGAAATCGGCGCGGGGGCA 5 0 GC CT 6 0 GC CT 10 66 GC CT CT 12 0 GC 14 0 GC CT cbbR Cbb operon Promoter Activity (%) Spacing (nt)

6. Effect of spacing Cbb operon Spacing 0, 10 bp RNA polymerase CbbR Spacing 5, 6, 12,14 bp

7. Conclusion binding between CbbR molecules is cooperative Cooperative binding: binding of a protein to binding site 1 helps binding of a second protein to binding site 2 Common for LysR type proteins, and many other regulatory proteins Dependent on protein protein interactions

8. Chemical signal Binding to regulator Activation of transcription Transcriptional regulators are switches: turn RNA polymerase ON and OFF Signal about physiological state of the cell What is the chemical signal for CbbR???

9. 200 uM No addition NADPH NADP+ NADH NAD+ Complex 1 Complex 2 Free Fragment LysR type proteins bind to DNA in the absence of an effector molecule BUT binding is increased 3 fold in the presence of an effector Allowed identification of effector molecule for CbbR Conclusion: NADPH is the effector

10. RNA pol RNA pol Binding of the effector to the regulator activates the activator How does this affect transcription? Many transcriptional regulators affect DNA architecture, e.g., bending LysR type regulators induce a bend in the DNA, relaxed after binding of the effector

11. Isolation of suppressor mutants, promoter now active Promoter inactive Promoter fused to lacZ as reporter gene Relaxation of DNA bending may serve to produce productive contacts between RNA polymerase alpha subunit and the LysR-type activator How do we know??? LysR mutants have been constructed which prevent activation of transcription

12. Alpha subunits Suppressor mutations for OxyR and CysB (LysR type proteins) Alpha subunit gene Suppressor mutants are analysed: mutations map to alpha subunit Relaxation of DNA bend may serve to produce productive contacts between activator and RNA polymerase

13. Signal recognition domain Hinge region DNA binding domain and RNA polymerase contact domain LysR type proteins are examples of simple transcriptional regulators One protein binds to DNA Recognises the ‘signal’ Transduces the signal to RNA polymerase Other examples are: CRP, FNR and lacI

14. LysR type proteins • Are often a dimer of identical subunits • Two dimers bind side by side adjacent to promoter • Binding of the dimers is cooperative • recognise a low molecular weight compound, ie NADPH • DNA binding occurs in absence of effector but is increased upon binding of effector • Introduce a bend in the DNA • Bending is decreased upon binding of the effector • Contact the alpha subunit of RNA polymerase • Contacts could affect RNA polymerase binding or open complex formation