6/27/01

Lecture #3 - lac operon

rho identification via genetics

mutations in rho gene can suppress some types of polar mutations

A, B, C = genes in an operon

most mutations are nonpolar; e.g. A^- B⁺C ⁺ and mutation in A doesn't affect function of B and C

polar mutations &endash; mutation in A does cause B and C to be non-functional, A^- B^-C^-

rho⁺ A^- B^-C^- mutation only in A: nonsense mutation &endash; converts a.a. into stop codon, results in truncated protein

rho^- A^- B⁺C ⁺ if rho mutated, function of B and C restored

How do mutations in rho suppress polarity?

(Fig. 9.30)

transcription and translation are coupled in bacteria, occur simultaneously
in wild type &endash; rho bind to C rich G poor sequence, needs to gain access to RNAP but ribosome is blocking its path to RNAP so prevents rho from functioning in transcription termination
nonsense mutant in A &endash; ribosome comes to stop codon on RNA, is released, doesn't block rho; rho moves up to RNAP and results in transcription termination. B and C are mutant because the RNA for these genes is not transcribe.
Mutation in rho in combination with nonsense mutant in A. If rho isn't present, then premature transcription termination does not take place, B and C are transcribed and function normally.

Control of transcription

1. promoter strength

2. activator/ repressor &endash; most important

3. alternative sigma factor

Regulatory proteins:

activators &endash; turn on genes / operons
repressors &endash; turn off genes / operons
operator &endash; sequence that's recognized by regulatory protein
positive regulation &endash; operator situated next to promoter, relatively weak promoter
negative regulation &endash; operator overlaps promoter

Positive regulation

activator proteins
binds to operator, turn genes on
in absence of activator, genes off
if mutate activator, then gene turns off

Negative regulation

repressor protein
turn genes off
in absence of repressor, genes on
if mutate repressor, then genes turn off

Inducible vs. repressible

what happens in response to a signal?
inducible &endash; genes turn on in response to a signal - inducer is signal that causes genes to turn on
repressible &endash; genes turn off in response to a signal - corepressor is signal that causes genes to turn off

Figure 10.20

1. negative inducible &endash; lac operon

2. positive inducible &endash; catabolite repression (CAP)

3. negative repressible &endash; trp

4. positive repressible

lac operon - Figure 10.3

lac I gene &endash; encode lac repressor protein, has its own promoter
lac operon &endash; common unit of regulation
a. promoter

b. operator

c. 3 structural genes

lac Z &endash; encode ß galactosidase, converts lactose into galactose and glucose

lac Y &endash; encode permease that transports lactose into E. coli

lac A &endash; encode transacetylase, detoxification?

Figure 10.6

lac repressor &endash; functions as tetramer, not as monomer
negative regulation &endash; in absence of inducer (= lactose) repressor binds to lac operator and prevents transcription of lac Z, Y and A
in presence of inducer, lac repressor has binding site for inducer; when inducer binds, conformational change in repressor, repressor falls off operator so RNAP can bind and transcription occurs (some oversimplification, since also has positive control)

Figure 10.4

RNAP binds to about &endash;50 to +20
lac operator = downstream, overlaps transcription start site

How does lac repressor prevent transcription?

blocks open complex formation
can have RNAP and lac repressor bound to DNA at same time but don't get transcription because can't from open complex

lac repressor activity

1. bind to DNA

2. bind inducer (lactose)

3. undergoes conformational change

4. bind to itself &endash; it's a tetramer

Genetics of lac

E. coli &endash; haploid organisms so use tricks to make it diploid
put second cop of gene in plasmid, F factor or phages (viruses)
why make a diploid? &endash; can test for dominant/ recessive
called merodiploid/ partial diploid analysis

lac mutations

1. uninducible - operon can't be turned on so always off

2. constitutive - always on, can't be turned off

Trans-acting mutations

e.g., lacI mutations
capable of functioning when unlinked to genes that is being controlled
encode diffusable factor &endash; proteins, RNAs
test for trans-acting mutation from merodiploid
- I⁺ O⁺ Z^- --> lacZ - mutation in structural gene - no ß-gal activity
- I^- O⁺ Z⁺ --> constitutive repressors can't bind to operator
- I⁺ O⁺ Z^- / I^- O⁺ Z⁺ --> becomes inducible again &endash; functions normally, has restored function because lac repressor functions at other O⁺ so it's trans-acting mutation

Cis-acting mutations

e. g., operator mutation

do not function when unlinked

do not encode diffusable factors

most often mutation in sequences

test for cis-acting mutation from merodiploid

I⁺ O⁺ Z^- --> lacZ - mutation in structural gene - no ß-gal activity

I⁺ O^- Z⁺ --> constitutive &endash; operator not functional so repressor can't bind

I⁺ O⁺ Z^- / I⁺ O^- Z⁺ --> merodiploid behaves as a constitutive - O⁺ on top can't rescue O^-, there's no product that can diffuse over and help so operator unable to bind repressor, therefore, has O^cconstitutive phenotype