Molecular Biology and Biochemistry 694:408 / 115:512 Spring 2007, Lectures Regulation of prokaryotic transcription Watson et al., (2004) Mol. Biol. Of the Gene, Chapter 16 Garrett and Grisham, Biochemistry (2005), Chapter 29 (pg ) Lodish et al., (2000) Mol. Cell Biol. Chapter 10 (pg. 342); Chapter 12 (pg ) Lewin (2000), Genes VII, Chapter 9; Chapter 10

Strong promoters contain close matches to the consensus site

Transcription from some promoters is initiated by alternative sigma (  ) factors

Different  factors in Bacillus subtilis are used at different stages of growth (vegetative vs. sporulation) Sigma Source & Use-35 region-10 region s 43 vegetative: general genes TTGACATATAAT s 28 vegetative: flagellar genesCTAAACCGATAT s 37 used in sporulationAGGNTTTGGNATTGNT s 32 used in sporulationAAATCTANTGTTNTA s 29 synthesized in sporulationTTNAAACATATT gp 28 SPO1 middle expressionAGGAGATTTNTTT gp SPO1 late expressionCGTTAGAGATATT

Different  factors in Bacillus subtilis are used at different stages of growth (vegetative vs. sporulation) Sigma Source & Use-35 region-10 region s 43 vegetative: general genes TTGACATATAAT s 28 vegetative: flagellar genesCTAAACCGATAT s 37 used in sporulationAGGNTTTGGNATTGNT s 32 used in sporulationAAATCTANTGTTNTA s 29 synthesized in sporulationTTNAAACATATT gp 28 SPO1 middle expressionAGGAGATTTNTTT gp SPO1 late expressionCGTTAGAGATATT

Bacteriophage - "eaters of bacteria"

Phage Early gene 28 Phage Mid. genes Early Middle Transcription of phage SPO1 genes RNAP  70 RNAP  28 RNAP  28 Phage Late genes Late RNAP  33  34 RNAP  70  28  33  34 RNAP  33  34 RNAP  28

Genetic regulation lac system of E. coli “What’s true for E. coli is true for an elephant.” J. Monod

 -Gal is produced only when lactose is present

 -gal induction can be due to 1. Activation of preexisting enzyme (i.e., removal of repressor) 2. Synthesis of new enzyme

Gratuitous inducers do not act as substrates Lactose is both an inducer and a substrate for  -Gal Some substrates do not work as inducers Action of the enzyme on the inducer is neither necessary nor sufficient for induction

Induction kinetics of  -Gal under gratuitous conditions p =  (amount of  -Gal)/  (total cell protein)

lac system: transcription regulation

Regulation of Transcription 1. Transcription initiation/RNA synthesis 2. mRNA Turnover RNAP 1 2 mRNA

Selection of Lac - mutants (negative selection nutritional marker) +Lac

Tricks use chromogenic substrates (X-gal) and gratuitous inducers (IPTG) to select for Lac mutants (Lac + - blue, Lac - - white) use diagnostic plates (EMB) to elect for absence of sugar fermentation 2 1

The lac locus of E. coli  -Gal galactoside permease galactoside transacetylase lacY mutants are cryptic lacI mutants are constitutive (first example of mutants that affect production, not activity) lacA mutants are Lac + lacZ mutants are Lac -

The PaJaMo experiment Hfr lacI + lacZ + Str S TsX S x F - lacI - lacZ - Str R Tsx R Set a cross in the absence of inducer: After some time, kill the donor with Str and T6 Monitor  -Gal in the presence or in the absence of inducer

The properties of lacO mutants provide genetic proof of operon model

lac operator Most bacterial operator sequences are short inverted repeats; Most transcription regulators are dimeric

The presence of inducer changes the conformation of LacI repressor so that it can no longer bind DNA

Distinction between factors (proteins) and elements (DNA sites) ii) Regulatory elements act in cis i) Regulatory factors act in trans

The LAC OPERON

LacI binds DNA as a tetramer to better repress transcription Why did Jacob & Monod not find O 2 and O 3 ?

X-gal White Blue White Blue White Genetic analysis of the LacI binding sites O 3 O 1 O 2 P lacZ Repression O 3 O 1 O 2 O 3 O 1 O 2 O 3 O 1 O O 3 O 1 O O 3 O 1 O O 3 O 1 O 2 18 O 3 O 1 O 2

Glucose effect: no response to inducers in the presence of glucose

glucose energy glycerol pgi pgi- mutants grown on glycerol induce lac genes even in the presence of glucose Interpretation: glucose effect is due a product of glucose catabolism (catabolic repression) Catabolism ???

Catabolite repression occurs for a wide range of sugars Catabolite repression mutants must therefore be defective in utilization of wide range of sugars (cells will be permanently repressed). Select on EMB agar.

Mutants defective in catabolite regulation occur in two distinct loci cyacrp cAMP level high when glucose is low codes for CAP (catabolite activating protein). CAP, when bound to cAMP, binds to lac regulatory region and activates transcription of structural genes

LAC Operon and catabolite repression Positive control of the lac operon is exerted by cAMP-CAP Catabolite Activator Protein

Cooperative binding of cAMP-CAP and RNA polymerase to the lac control region activates transcription

The lac control region contains three critical cis-acting sites CAP RNAPLacI RNAP

lac operator: the regulatory region

CAP binding bends the DNA Residues that interact with RNAP

Operator sites can be in different places with respect to the start of the promoter

Different mechanisms of transcriptional activation A) Strong promoters B) Promoters with UP elements C) Activation through interactions with the  CTD D) Activation through interactions with other components of RNAP E) Activation through interactions with components multiple components of RNAP by multiple activators

Different types of negative and positive control of transcription

Changes in DNA topology affect isomerization step in formation of the open complex

bp RNAP bp merT Average Prom. Mechanism of activation by MerR MerR merT Hg ++ MerR 17 bp RNAP

Enzyme repression: the trp operator The synthesis of Trp structural genes is controlled by unlinked TrpR repressor. TrpR binds to Trp operator in the presence of Trp (product inhibition). Both trpR and trpO mutants are derepressed

Crossfeeding analysis of Trp mutants allows to analyze the biochemistry of Trp biosynthesis pathway precursorTrp TrpETrpDTrpB

Attenuation of trp operator expression attenuator Deletions in the attenuator increase basal synthesis of Trp enzymes

the trp attenuator region

Attenuation occurs due to formation of alternative secondary RNA structures in the leader sequence in the presence or absence or Trp

The repressor idea The existence of c and vir mutants. are immune to c, but not vir Immunity of lysogens to superinfection with wt Zygotic induction