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.
Dec 18, 2015
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