Lecture 13 Transcription 3 Catabolic Operons Flashcards
LacI
Encodes repressor protein. LacI acts in trans - it can affect the expression of a gene even if not adjacent
LacO
Operator region
The site at which the repressor protein binds. The operator acts in cis - it only affects the expression of genes adjacent to itself
Repressor
Blocks RNA polymerase’s access to promoter by stearic hinderance.
Catabolic repression lactose/glucose
Glucose will be used first by bacteria as it’s easier/requires less energy to digest.
Only once all glucose is used will the lac operon be activated (induced) and beta galactosidase activity begins.
Catabolism of glucose represses lac operon aka catabolic repression and this works for other sugar operons too
Glucose overrides lactose operon system
Cyclic AMP discovered in 1960 is a signalling molecule
Adenyl cyclase enzyme takes ATP removes 2 phosphate groups to make cyclic adenosine monophosphate (CAMP) linking 3’ and 5’ C on sugar of ATP linking them by the one remaining phosphate.
Growth of the bacteria on glucose inhibits adenyl cyclase so CAMP levels are low
In absence of glucose adenyl cyclase works fine and lots of CAMP produced
CAMP is an inverse indicator of glucose presence
cAMP function deducted by mutants
E. Coli cya mutants have defects in the enzyme adenyl cyclase and can’t make cAMP
Cya mutants are unable to induce any of the ‘sugar operons’ lac,gal,ara,mal or xyl even when glucose is absent
Addition of cAMP to cya mutant strains immediately restores operon inducibility
So cAMP must be required for inducibility
Phosphoenolpyruvate (PEP): glucose phosphotransferase system (PTS)
Lactose enters bacteria through lac permease channel, because sugars are water soluble chemicals they must always come through a protein channel
IN PRESENCE OF GLUCOSE
Glucose enters via protein llC and is phosphorylated to glucose 6 phosphate so cell can use it. Protein llC is connected to llB llA and HPR that sit on top of Adenylate cyclase.
Protein llA-Glc becomes dephosphorylated upon glucose transport. The dephosphorylated form inhibits adenyl cyclase so cAMP levels drop.
IN ABSENCE OF GLUCOSE
The phosphorylated form of llA-glc it stimulates adenylate cyclase allowing it to form cAMP
So cAMP levels opposite to glucose levels
How does cAMP aid transcription
CAMP allows CAP - catabolite gene activator to bind to CAP site.
When glucose levels are high cAMP levels are low and CAP protein is detached so transcription cannot occur
When glucose low cAMP is high. CAMP attaches to CAP protein causes a confirmational change activating the protein allowing it to move to and bind to promotor- activating transcription
CAP binds to DNA in the promotor
CAP binding site is in the promoter and consists of palindromic sequences. Bending DNA and opening the coils.
Most bacterial promotors
-35:TTGACA
-10: TATAAT
Lac promotor
-35 TTGACA
-10 TATGTT
Changing one A to G forms a stronger bond making it slightly too difficult to open with just DNA pol so CAP protein bending needed to open it up
Lac operon summary
+glucose + lactose
Operon off CAP not bound
+glucose -lactose
Operon off because lac repressor bound and CAP not bound
-glucose -lactose
Operon off because lac repressor bound (CAP bound)
-glucose +lactose
Operon on
PEP: glucose PTS
Dephosphorylated llA-GLC also represses lac permease
Phosphorylation of glucose, dephosphorylates llAGlc and it represses lac permease. Less lactose import, less allolactose, more functional lacI repressor, less lac operon transcription
Biosynthetic operons
Expression of enzymes that together synthesise small molecules e.g. amino acids, nucleotides and vitamins
If they are not in the growth media the bacterium has to make them - operon switches ON
If present in growth media they don’t need to be synthesised - operon switches OFF
Charles Yanofksy - Trp operon
Tryptophan operon :
Promotor/operator/leader region and genes Trp E,D,C,B,A
( In anti alphabetical order)
Separate gene encodes Trp repressor
Trp repressor
(aporepressor - incomplete)
+
Tryptophan
=
Holo-repressor
Holo-repressor binds to the operator stopping transcription
In lower Tryp levels less repression - equilibrium maintained
Trp operon
Biosynthetic operons should be off when molecules they synthesise are available
Trp itself is corepressor for Trp apo-repressor (co+apo>holo)
Under high Trp more repressor is bound to operator- Less transcription of operon
Trp controls it’s own production
Pos and neg control
Two types of regulation
Pos controlled - activators bond to operator to allow transcription no transcription in absence of activator
Neg controlled- repressors bond to operator to prevent transcription, transcription allowed in absence of repressor
