Lecture 4: Regulating Transcription Initiation Flashcards
How can we measure the lac operon?
- There is a 1000X increase in synthesis in response to activation.
- IPTG is a synthetic inducer.
- ONPG is a chromogenic substrate. It changes from colourless to yellow. It can be measured at 420nm.
What is the structure of LacI and how does it change in response to inducers? Draw a diagram
LacI is a homotetramer. LacI binds in the absence of an inducer and stops σ70-RNAP initiation transcription.
• Each tetramer has two dimers bound to DNA. The tetramer binds two different operator sequences.
• There are four different domains of each monomer.
• The N-terminus is a helix-turn-helix motif involved in DNA binding.
• A linker connects the DNA-binding domain with the core domain (sometimes called the hinge helix).
• The core domain is involved in the binding of allolactose.
• The C-terminus is involved in tetramer formation.
• When the inducer binds, the inducer binding pocket closes, the hinge helices are disrupted and the HTH domains are freed. This leads to reduced DNA binding.
What are operator sequences and how are they involved? How have experiments shown this?
The operator sequence is downstream of the start site of transcription.
• It is actually in the part that is transcribed, however it is close enough to interact with RNAP.
• Sequence analysis shows that there are multiple operator sites.
• O2 is 401 bp downstream of O1 (in lacZ).
• O3 is 82 bp upstream of O¬1.
• Mutating these sites causes a decrease in depression, showing that they are useful.
• Disruption of O2 or O¬3 causes a 2-fold decrease in repression. Disruption of both causes a 50-fold decrease.
• Mutations in LacI that prevent tetramer formation also produce a 50-fold repression decrease.
• It is thought therefore that DNA looping is involved.
• LacI tetramer binds O1 and O¬2 simultaneously to form a barrier to transcription of 401 bp containing 3 negative supercoils.
What did Monod and Jacob discover with lac operon mutations?
Monod and Jacob then wanted to know if inducibility was due to negative or positive regulation.
• They did a complementation test with lacI wt and mutant (mutant leads to uncontrollable genes).
• When WT lacI was added, repression was restored.
• The inducible phenotype is dominant over the constitutive phenotype.
How is the lac operon structured and what are the functions of its individual parts?
There are 4 genes in the lac operon.
1) LacI: a repressor which binds to DNA. LacI is trans-dominant, it can act on other genes.
2) LacZ: β-galactosidase. An enzyme which breaks lactose down into galactose and glucose.
3) LacY: β-galactosidase permease: Transports galactosides into the cell.
4) LacA: thiogalactoside acetyltransferase. Function unclear. May detoxify non-metabolizable pyranosides by acetylating them and preventing re-entry into the cell.
The operon also contains the promoter and the operator. The operator is where the lacI protein binds. A lacO constitutive mutant will not act on other genes. It is cis-dominant.
