Genetic Analysis of Lac Operon

Make partial diploids to do complementation tests: 1 copy of lac operon on E. coli chromosome.2nd copy of lac operon on F’ episome.

Lac OC is cis-actingi.e., it only affects transcription of genes it’s next to

O+ Z- Y+

OC

Z+ Y-

chromosome

F’ episome

OC has bp changes that prevent lac Repressor from binding to it.

Lac OC = Operator constitutive mutant allele

Y+ inducible

Z+ constitutively expressed

O+ Z- Y+

O+ Z+ Y-

chromosome

F’ episome

lac I+ gene transcribed mRNA Repressor Protein

Proteins can diffuse throughout cell

Lac I- mutants

I-: Mutant repressor protein that can’t bind to DNA

Lac I+ is trans-acting

Y+ & Z+ inducible

Lac IS encodes super-repressor

chromosome

F’ episome

Evidence for allosteric regulation of lac repressor protein

IS represses txn regardless of presence of inducer.

Both Y+ & Z+ always repressed

O+ Z- Y+

O+ Z+ Y-

Promoter Mutations

Mutations in -10 or -35 regions may reduce ability of RNA polymerase to bind and decrease ability of structural genes to be transcribed under any conditions.

RNA polymerase contacts promoter at 2 highly conserved sites (-10 & -35)

Promoter mutations are cis-acting (like Operator mutations).

+1

CAP-cAMP bound to promoter bends DNA, making it easier for RNA polymerase to bind to -10 & -35 sites.

Lac Promoter & Operator

CAP

-35

-10

Pol

Pol Repressor

Summary of lac operon regulation

Arabinose Operon

Ara I = Initiator region (both promoter & operator)Ara C codes for Activator protein (C protein)

Allosteric effector

Binds to Operator & facilitates transcription

Without arabinose, AraC binds to both ara I & a 2nd operator, ara O, prevents transcription

Regulated by catabolite repression

Eukaryotic Transcriptional Regulation

RNA Pol I makes rRNA precursors

RNA Pol II makes mRNA precursors proteins

RNA Pol III makes tRNA & 5S rRNA precursors

3 different kinds of RNA polymerases