Chapter 11

Question 1

Genetic Switches (bacterial transcriptional regulation)

Answer 1

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)

Question 2

Positive regulation

Answer 2

Activator protein binds to target dna site REQUIRED for transcription.

Question 3

Negative Regulation

Answer 3

Repressor protein must be inhibited for transcription to begin

Question 4

Function of activators and repressors

Answer 4

DNA bound activator protein tethers rna polymerase to promoter

Repressor either blocks promoter or impedes movement of RNA polymerase along DNA chain.

Question 5

2 states activators and repressors must exist in

Answer 5

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

Question 6

Example of allosteric effectors in lactose metabolism

Answer 6

• 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

Question 7

Lac Regulatory Circuit - structural genes

Answer 7

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

Question 8

Coordinately controlled genes

Answer 8

Genes whose transcription is controlled by common means (simultaneously activated or repressed in parallel)

Question 9

How do coordinately controlled genes occur

Answer 9

If genes encoding proteins make up a single transcription unit

Question 10

Regulatory components of lac system

Answer 10

• Gene encoding reg protein
• 2 binding sites on DNA (one for reg proetin one for rna pol)

1. i gene (4th gene) encodes lac repressor protein
2. lac promoter site (P) - rna pol binding site - initate transcription of ZYA
3. lac operator site (O) - repressor binds (located between promoter and z gene)

Question 11

Induction of lac system

Answer 11

POZYA create an operon

Question 12

Operon

Answer 12

Segment of DNA that encodes…
- a multigenic mRNA
-an adjacent common promoter
-regulatory region

Question 13

Multigenic mRNA

Answer 13

Codes for multiple genes

Question 14

Lac I

Answer 14

encodes lac repressor
- not in operon
- needed for proper regulation

Question 15

Lac repressor

Answer 15

• 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

Question 16

Allosteric transition

Answer 16

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

Question 17

Requirement for control system

Answer 17

Presence of lactose reqiured to stimulates synthesis of genes needed for its processing

Question 18

Induction

Answer 18

When repressor is removed via alosteric effector

Question 19

Inducers

Answer 19

Allosteric effectors that remove a repressor protein

Question 20

Summary of Lac Switch

Answer 20

• 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

Question 21

Requirements needed to study gene regulation

Answer 21

1. biochemical assay - measure amount of mRNA/protein
2. reliable conditions
3. Genetic mutations

Need way of describing WT regulation, and mutations to change the process to further observe

Question 22

Enzyme induction

Answer 22

appearance of specific enzyme only in presence of its substrates

Question 23

Determining the function of the Lac enzymes

Answer 23

• Analyze mutants
• found each enzyme encoded by different gene
• therefore, coordinately controlled genes
• mapping showed very close on chromosome

Question 24

Genetic evidence for operator and repressor

Answer 24

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)

Question 25

Mutations - Inactivation of Z and Y (Z- and Y-)

Answer 25

- 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

Question 26

Comparable genetic tests of I- mutations

Answer 26

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

Question 27

Genetic evidence of allostery

Answer 27

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

Question 28

Genetic analysis of Lac promoter

Answer 28

Promoter (P) between I and O. Cis-acting

Question 29

Characterization of repressor and operator

Answer 29

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

Question 30

Catabolite repression:positive regulation

Answer 30

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)

Question 31

Choosing which sugar

Answer 31

- 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

Question 32

cAMP and lac activation

Answer 32

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

Question 33

Structure of traget dna sites

Answer 33

- Binding site sequences different for different proteins - Have rotational two-fold symmetry: flip upside down, sequence remains the same

Question 34

Binding of CAP complex to operon

Answer 34

DNA bent when CAP is bound Can help RNA pol bind to site