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Prokaryotic Genetics III - Gene regulation. 1. Lac System Jacob and Monod, 1950's, biochemists. Not cleaved. Non-metabolic inducer. Non-inducer substrates. X-gal. ONPG. Ampicillin enrichment of lac - cells. Lac - mutants - genes mapped close together.
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Prokaryotic Genetics III - Gene regulation 1. Lac System Jacob and Monod, 1950's, biochemists
Not cleaved Non-metabolic inducer Non-inducer substrates X-gal ONPG
Lac- mutants - genes mapped close together • Lac- mutants were isolated in Z and Y, prevent utilization • lacZ - -galactosidase breaks lactose into galactose and glucose • lacY - permease is required to transport lactose into the cell One mutant allowed expression in the absence of inducer constitutive mutant, lacI First example of a mutant that affects production not activity
PaJaMo Experiment - Pardee, Jacob & Monond Hfr I+ Z+ T6sSsx F- I-Z-T6rSr
Analysis of lacI mutants LacI is trans acting - a diffusable factor
Analysis of lacOc mutants lacO is cis acting - a non-diffusable element
Analysis of lacP mutants lacP is cis acting - a non-diffusable element
LacI binds DNA as a tetramer to repress transcription Why did Jacob & Monod not find O2 and O3?
Genetic analysis of the LacI binding sites X-gal White White White Blue Blue
Distinction between factors (proteins) and elements (DNA sites) i) Regulatory factors act in trans ii) Regulatory elements act in cis
Cross feeding analysis of Trp mutants allows to analyze the biochemistry of Trp biosynthesis pathway TrpE TrpD TrpB precursor Trp
TrpE TrpD TrpC1 TrpC2 TrpA TrpB
Attenuation of trp operator expression attenuator Deletions in the attenuator increase basal synthesis of Trp enzymes
Sequences Coding for Short Peptides are Often Found in Leaders of Transcripts from Biosynthesis Operons
RNA Pol Terminator Sequences in the trp operon leader region can form different stem-loop structures
What would the phenotype be of a deletion from 118-132? What would the phenotype be of the Trp codon mutations? Stalled ribosomes block the formation of the transcription terminating stem-loop structure +Trp -Trp Can this form of regulation happen in eukaryotes?
Resistant to further infection Not resistant to infection by other phage Life cycle of a Temperate Bacteriophage
Mating by lysogen induces lytic growth in recipient cell Similar to the PaJaMa Experiment Hfr F- No repressor of the prophage in the recipient cell causes lytic growth
Screen for clear plaques How did they define the complementation groups?
WT cI, cII, cIII OL OR Turbid plaques Uninfected Cell Clear Clear Lysogen No plaques No plaques Clear Different complementation groups and phenotypes of the "clear" mutants Recessive trans Dominant - cis
Infection of WT phage into a lysogen Infecting Phage or WT cI, cII, cIII OL OR Turbid plaques Uninfected Cell Clear Clear Lysogen No plaques No plaques Clear Uninfected Lysogen
Infection of cI phage into a lysogen Infecting Phage WT cI, cII, cIII OL OR Turbid plaques Uninfected Cell Clear Clear Lysogen No plaques No plaques Clear Uninfected Lysogen Recessive trans
Infection of cI phage into a lysogen Infecting Phage WT cI, cII, cIII OL OR Turbid plaques Uninfected Cell Clear Clear Lysogen No plaques No plaques Clear Uninfected Lysogen Recessive trans Dominant - cis
15 Kb Not essential Cloning site for DNA libraries phage genome DNA replication (2) Recombination (10) Head (10), tail (12) lysis (3)
Differential gene expression in the life cycle cI Int Integrate into genome cI
Regulation of early gene expression N () and Nus (bacteria) function as anti-terminators
Regulation of late lytic gene expression 26 Kb, takes 10' Shuts off genes required for lysogeny
Regulation of late lysogenic and prophage gene expression cI shuts of all other phage genes in Prophage
Poor Media Rich Media Lytic vs lysogen decision To establish lysogen
Induction of prophage through SOS response cI Does not bind DNA
Integration and excision of phage Int/Xis ratio determines direction
