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 O C is cis-acting i.e., it only affects transcription of genes it’s next to O + Z - Y + OCOC Z + Y - chromosome F’ episome O C has bp changes that prevent lac Repressor from binding to it. Lac O C = 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 I S encodes super-repressor chromosome F’ episome Evidence for allosteric regulation of lac repressor protein I S 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 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