chapter 12 Flashcards
Central Dogma of Molecular Biology
DNA -(transcription)-> RNA -(translation)->Protein
Gene expression
transcription + translation
Types of Gene Expression in Bacteria (3 types)
Constitutive transcription: Constant expression of genes (always on/expressed)
* No regulatory control
Regulated transcription: Expression only occurs under certain conditions (e.g. abundance vs scarcity of “food”). Only sometimes on
Post Transcriptional regulation: After mRNA is synthesized, its abundance can be modified to influence the amount of protein translated
Gene Expression in Bacteria (Thermostat Analogy)
It is a waste of energy if the furnace or air conditioner is always ON. Thermostats keep those systems OFF by default and only allows them to turn ON when the house gets too hot or too cold. This is how many genes are regulated
Negative vs Positive Control
Negative control of transcription involves a repressor protein binding to a regulatory DNA sequence, preventing transcription of a gene or genes (blocks RNA polymerase from having access to transcribing gene)
Positive control of transcription involves binding of an activator protein to a regulatory DNA sequence and initiating transcription of a gene or genes (directly activates transcription, regulatory molecule binds to RNA polymerase allowing it to move towards transcription site)
Negative Control: Repressors and Inducers
No inducer, just Repressor: in the absence of an inducer, repressor blocks transcription (transcription occurs when repressor is removed by inducer)
Inducer: causes an allosteric change in the repressor, causing it to release from the DNA and allow transcription
Negative Control: Repressors and Corepressors
Corepressor: with corepressor present, the repressor blocks transcription (when co-repressor removes from repressor, than repressor can move)
Just repressor, No corepressor: without corepressor, allosteric changes cause the repressor to release from the DNA and allow transcription
Allosteric Change
when interactions between proteins change their conformation and function
Positive Control: Activators and Effectors
Without effector: no transcription
With effector: transcription is activated
Positive Control: Activators and Inhibitors
With inhibitor: no transcription
Without inhibitor: transcription is activated (removal allows allosteric change to occur)
lac Operon
a System with Negative and Positive Control
Discovered in E. coli (prefer glucose, lactose is an alternative energy source. Good to keep these genes off, but have them on when secondary/emergency found source is needed)
lac operon Negative control
a repressor binds to operator sequence, preventing transcription
-Prevents synthesis of enzymes involved in lactose metabolism
-Only activates when lactose is present in environment
lac operon positive control
transcriptional elements more active when glucose is absent
Presence of lactose & absence of glucose = up-regulation of lactose-metabolizing genes (system is most active, most upregulated lactose genes)
Lactose
Glucose is the preferred source of energy for E. coli
Lactose can also be utilized as an energy source if glucose is absent from environment
First lactose needs to be transported into cell through a permease transport protein (found on cell membrane, shuffles lactose into cell)
Lactose broken down into glucose and galactose through beta-galactosidase
Glucose is now present as an energy source for various metabolic needs
Beta-galactosidase
(regulated by repressor - only expressed when lactose is present)
also converts lactose to allolactose (modified lactose form), which acts as an inducer compound that binds to the repressor protein, removing its inhibition (repressor protein) from the lac operon
The lac operon: polycistronic mRNA:
operon: is a cluster of genes undergoing coordinated
transcriptional regulation by a shared regulatory region
lacl: a regulatory gene (repressor) that is adjacent to but not part of the lac operon. Lacl repressor binds to the operator sequence and blocks transcription of the lac operon
RNA polymerase: binds to the promoter sequence
Positive Control: CAP-cAMP Complex
CAP-cAMP complex activates transcription of lac operon
Positive control: occurs at the CAP-cAMP binding region of the lac promoter
CAP = catabolite activator protein (CAP). CAP upregulates transcription by RNA polymerase. Recruit RNA polymerase to ensure high expression at promotor
cAMP = cyclic adenosine monophosphate. cAMP is synthesized from ATP by adenylate cyclase
Glucose inhibits adenylate cyclase activity, therefore:
* Low [glucose] → high [cAMP] → CAP-cAMP complex forms
* High [glucose] → low [cAMP] → CAP-cAMP complex does not form
Presence of Glucose; Absence of Lactose
Lactose unavailable (glucose available)
-Repressor bound (can’t be removed)
cAMP low (less active cAMP binding site)
-No transcription of lac operon
-Lac repressor proteins binds to the operator (lacO) sequence and inhibits transcription - no expression of LacO
-The lacl repressor is constitutively expressed and represses transcription of the lac operon
Absence of Glucose; Presence of Lactose
Lactose available (glucose unavailable)
-Repressor removed
-cAMP high (without glucose, higher lacO expression)
-Transcription of lac operon
-With repressor protein inactivated by allolactose binding, RNA polymerase carries out transcription
-Allolactose is produced from lactose which acts as an inducer to stop repression of lac operon
-RNA polymerase binds to promoter and transcription is higher than basal level
Presence of Glucose and Lactose
Lactose and glucose available
-Repressor removed
-cAMP low
-Low transcription of lac operon
(lacl removed with lactose present)
-Lac operon is transcribed at a basal level, which leads to allolactose production from lactose (when present)
Thought question: if allolactose is made from B-galactosidase, how does the cell ever have the allolactose that is needed to induce B-galactosidase?
There is always a small amount of beta-galactosidase expressed, even in the absence of allolactose (“basal” expression)
lac operon mutation:
Loss of function mutation
in lacI (repressor gene) leads to constitutive expression of lac operon. Can’t bind to operator sequence, can’t repress transcription so it’s always on. (Mutants can still regulate through positive transcription)
lac operon mutation:
prevents wild-type repressor from binding, leading to constitutive expression of lac operon (protein function, mutation is at operon = no binding)
lac operon mutation:
prevents inducer (alo lactose) from suppressing repressor, leading to no transcription of lac operon (small leaky maybe) (presence of lactose can’t repress repressor)
