Regulation of Bacterial Gene Expression
Describe the lac operon if lactose and glucose are present
Lactose and glucose present - cAMP levels will be low - lac repressor is still inactive - CRP complex hasn't been active - rna polymerase has lower affinity for promoter - little mRNA will be synthesized
What is the function of lactose permease?
Lactose permease functions to transport lactose from the medium into the cytoplasm
How does b-galactosidase cleave lactose?
The hydrolytic cleavage of the lactose sugar is achieved by beta galactosidase working on that beta galactoside linkage between the the two monosaccharides - gives the two monomers from the cleavage
What is allolactose?
•Once glucose is exhausted, E.coli will switch to catabolism of lactose through inhibition of lac repressor binding. •Initially some lactose is converted to allolactose, the lac operon physiological inducer.
Describe the lac operon if glucose is absent
Glucose is absent - cAMP levels will be high - the active CRP complex because the repressor has been inactivated is able to bind at a position within the promoter - enhances the attraction of rna polymerase to the promoter region. - so abundant mRNA can be synthesized from the genes contained in the lac operon
Describe the lac operon when lactose is absent
24 nucleotide sequence - operator - downstream of promoter - RNA polymerase can't travel past - barrier to the function of RNA polymerase - none of the genes will be transcribed Repressor has a site for allolactose to bind (intermediate of lactose catabolism) - required for the induction of the lac operon - this is why we need to have low levels of expression of b galactosidase When bound to repressor allolactose causes a conformational change making it inactive - cant remain attached - lock and key style (no longer fits)
What is IPTG?
An artificial lac inducer, performing the same function as allolactose. •Addition of IPTG to medium with no glucose allows significant cloned gene expression.
Define polycistronic
Can encode more than one polypeptide
Describe the lac operon when lactose is present
So when lactose is present and it causes the repressor to be released from the operator, RNA polymerase is now free to begin transcribing those three genes in the operon. And the resultant product of that is the single molecule of messenger RNA - polycistronic - can produce the three functional proteins that are essential for the full metabolism of lactose (B galactosidase, permease and transacetylase) will all now be produced.
What is the lac operon required for?
The operon is required for the transport and metabolism of lactose - glucose is the preferred source of carbon but the lac operon allows for the effective digestion of lactose when glucose is not available through the production and activity of proteins that will contribute to the catabolism of lactose - primarily the production of beta glactasidase - there are another two proteins that feed into the process - one which is a transporter and another that codes for a trans-acetylase
What is the function of B galactosidase?
b-galactosidase catalyses cleavage of lactose into glucose and galactose
Describe the lactose operon
•An operon is a region of DNA containing genes with related functions •The operon is transcribed as a single unit, producing a single mRNA ie polycistronic. •The lac operon consists of: -5' promoter and operator transcriptional control DNA sequences, -DNA encoding the structural genes lacZ, lacY(and lacA - encodes a transacetylase). •Upstream is the lacI gene which encodes the lac repressor protein.
What does binding of the CRP-cAMP complex do?
•Binding of CRP-cAMP complex is necessary to turn on lac operon transcription. •CRP-cAMP bends the lac promoter into a shape that RNA polymerase efficiently binds to - process of transcription can take place effectively. •In this way CRP-cAMP forms part of the binding site for RNA polymerase - essential for the recognition and attachment of the RNA polymerase to the promoter region within the lac operon.
What is catabolite repression?
•Glucose controls expression of the lac operon through catabolite repression. •When glucose is abundant cytoplasmic cAMP levels are low, and once glucose is depleted cAMP levels rise. •cAMP is bound by cAMP receptor protein (CRP), this complex then attaching to the lac operon promoter region. •This allows transcription, but only in the absence of lac repressor bound to the operator ie positive regulation.
How is activity of the lac operon regulated?
•Lac repressor protein is continuously manufactured, and in the absence of lactose binds the operator region of the lac operon. •Lac repressor binding prevents transcription by blocking RNA polymerase access to the promoter site ie negative regulation.
Why is lactose only metabolised when glucose is unavailable?
•Only when E.coli needs to metabolise lactose are the necessary enzymes synthesised. •Hence, E.coli does not waste resources synthesising enzymes unnecessarily. •The enzymes are b-galactosidase and lactose permease, produced by the lacZ and lacYgenes, respectively.
What happens when e.coli grows in medium containing both glucose and lactose?
•When E.coli grows in medium containing both glucose and lactose a biphasic logarithmic growth curve is observed. •Glucose is exclusively catabolised until exhausted. •Only then is lactose used.
In bacteria how does regulation of gene expression occur?
•mainly at the level of transcription. •This relates to the short lifespan of bacterial mRNA within the cell i.e. up to 5 minutes. •Efficient mechanisms with different modes for controlling transcription have developed eglactose operon in E.coli. •When the cloned gene is inserted into the lacZsequence, transcription is controlled by the promoter of the lac operon.