Chapter 11 Flashcards
Genetic Switches (bacterial transcriptional regulation)
Depends on 2 types of protein-dna interactions
1) determines when it begins - promoter
2) determines if promoter-driven transcriptin happens - activators and reperssors (repressors bind to operators)
Positive regulation
Activator protein binds to target dna site REQUIRED for transcription.
Negative Regulation
Repressor protein must be inhibited for transcription to begin
Function of activators and repressors
DNA bound activator protein tethers rna polymerase to promoter
Repressor either blocks promoter or impedes movement of RNA polymerase along DNA chain.
2 states activators and repressors must exist in
1) binding its targets
2) non-binding its targets
- Interaction of two sites in 3d structure of protein
- DNA binding domain
- allosteric site: sensor (sets domain in functional or non functional state)
- interacts with allosteric effectors
Example of allosteric effectors in lactose metabolism
- isomer of lactose = allolactose = effector
- sugar binds to regulatory protein
- inhibits expression of genes needed for lactose metabolism
- changes shape and structure of domain of regulatory protein
Lac Regulatory Circuit - structural genes
2 enzymes
- 1) permease - transport lactose into cell
-2) B galactosidase - modify lactose into allolactose (cleave lactose to yield glucose and galactose)
Structures encoded by 2 adjacent sequences
1=Y
2=Z
3=A
third adjacent encodes another enzyme (not needed for metabolism)
transcribed into mRNA, all or none enzymes transcribed
Coordinately controlled genes
Genes whose transcription is controlled by common means (simultaneously activated or repressed in parallel)
How do coordinately controlled genes occur
If genes encoding proteins make up a single transcription unit
Regulatory components of lac system
- Gene encoding reg protein
- 2 binding sites on DNA (one for reg proetin one for rna pol)
- i gene (4th gene) encodes lac repressor protein
- lac promoter site (P) - rna pol binding site - initate transcription of ZYA
- lac operator site (O) - repressor binds (located between promoter and z gene)
Induction of lac system
POZYA create an operon
Operon
Segment of DNA that encodes…
- a multigenic mRNA
-an adjacent common promoter
-regulatory region
Multigenic mRNA
Codes for multiple genes
Lac I
encodes lac repressor
- not in operon
- needed for proper regulation
Lac repressor
- Has binding site to recognize operator DNA sequence
- has allosteric site to bind allolactose
- bind tightly to O side only
- near genes its controlling
- prevents transcription by RNA pol already bound to promoter site
Allosteric transition
Occurs when allolactose binds to repressor
- prespressor changes shape and alters dna binidng site
- repressor loses affinity for operator and falls off
- rna pol can now transcribe
Requirement for control system
Presence of lactose reqiured to stimulates synthesis of genes needed for its processing
Induction
When repressor is removed via alosteric effector
Inducers
Allosteric effectors that remove a repressor protein
Summary of Lac Switch
- Absence of inducer -> repressor binds and prevents transcription of lac operon
- Presence of inducer -> effector binds to allosteric site, removes affinity of repressor, repressor falls off, transcription occurs
Requirements needed to study gene regulation
- biochemical assay - measure amount of mRNA/protein
- reliable conditions
- Genetic mutations
Need way of describing WT regulation, and mutations to change the process to further observe
Enzyme induction
appearance of specific enzyme only in presence of its substrates
Determining the function of the Lac enzymes
- Analyze mutants
- found each enzyme encoded by different gene
- therefore, coordinately controlled genes
- mapping showed very close on chromosome
Genetic evidence for operator and repressor
Examine physiological consequences of mutations
- (used synthetic inducers - prevent break down by enzyme - accurate measures of enzyme induction)
- Used IPTG
- Found multiple mutations could alter expression
- Needed diploid organisms to test on
- bacteria are haploid
- inserted F factors with lac region of genome into bacteria - made partually diploid bacteria
- created strains heterozygois for lac mutations
- could distinguish mutations in regulatory protein from those in dna site (operator vs I gene)
Mutations - Inactivation of Z and Y (Z- and Y-)
- Recessive
2 classes of reg mutations
- Oc and I-
- constitutive mutations : caused operon genes to be expressed even without inducer
Oc: opertor cant bind repressor (Switch always on)
- only affected genes on same chromosome as mutation
- cis-acting
- Suggests opertor is segemt influencing only its linked genes
Comparable genetic tests of I- mutations
I- mutations constitutive
Inducible phenotype of I+ dominnat over constitutive phenotype I-
- Showed that amound of WT encoded by one copy is enough to regulate both operators
I+ trans acting : can regulate all operon genes - doesn;t have to be on same dna molecule
Can diffuse through a cell
Genetic evidence of allostery
I_S mutation : superrepressor (continues repressing even when theres an inducer)
Dominant over I+
I_S alters allosteric site: can’t bind inducer
Therefore, operon cant turn on
Genetic analysis of Lac promoter
Promoter (P) between I and O.
Cis-acting
Characterization of repressor and operator
Monitores binidng of labelled IPTG to repressor
- Repressor binds to DNA with operator
- Comes off in presence of IPTG
- Repressor can protect specific bases of operator
- took operon dna, bound repressor, treated with enzyme, recovered short strands shielded by enzyme activity
- short strands constitutes operator sequence
- base sequence determined
-showed that operator locus is speciifc sequence 17-25 nucs just before Z gene
- showed specificity of repressor-operator recognition - can be dsirupted by single base subsititution
Catabolite repression:positive regulation
Glucose can not be present in cell’s environment for lac operon to be activated
More energy efficient to break down glucose for energy than lactose
REPRESSION OF TRANSCRIPTION OF OPERON IN PRESENCE OF GLUCOSE
Glucose = catabolite of lactose (breakdown product of lactose)
Choosing which sugar
- B galactosidase not made until all glucose has been metabolized
- cell conserves energy
1) exclude lactose from cell
2) regulate operon expression via catabolites
Glucose breakdown product odulates level of cAMP (cyclic AMP - mono not tri phosphate)
Glucose inhibits conversion of ATP to cAMP
- so cAMP levels low in cell
Glucose decreases
- ATP -> cAMP
-high cAMP activates lac operon
cAMP and lac activation
Mutants provide answer
- make cAMP, can’t activate lac enzymes (lacking another protein - CAP - catabolice activator protein)
- CAP bind sto cap binding site in operon
- INteracts with rna pol - increases enzyme affinity for lac promoter - binds RNA pol to promoter region
- CAP needs cAMP as effector to bind to binding site
- Glucose available = low cAMP = no binding of CAP = no initiation of transcription
Structure of traget dna sites
- Binding site sequences different for different proteins
- Have rotational two-fold symmetry: flip upside down, sequence remains the same
Binding of CAP complex to operon
DNA bent when CAP is bound
Can help RNA pol bind to site
