Molecular Biology and Biochemistry

694:408 / 115:512

Spring 2007, Lectures 13-14

Regulation of prokaryotic transcription

Watson et al., (2004) Mol. Biol. Of the Gene, Chapter 16

Garrett and Grisham, Biochemistry (2005), Chapter 29 (pg. 942-974)Lodish et al., (2000) Mol. Cell Biol. Chapter 10 (pg. 342); Chapter 12 (pg. 485-491)

Lewin (2000), Genes VII, Chapter 9; Chapter 10

Strong promoters contain close matches to the consensus site

A/T rich

Up element

αCTD

α NTDβ

σ

β'

-35 -10UP

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

β

70

β'

-35 -10

α β

σ32

β'

-35 -10

α

Heat Shock GenesMost Genes

Different factors in Bacillus subtilis are used at different stages of growth (vegetative vs. sporulation)

Sigma Source & Use -35 region -10 region

s43 vegetative: general genes TTGACA TATAAT

s28 vegetative: flagellar genes CTAAA CCGATAT

s37 used in sporulation AGGNTTT GGNATTGNT

s32 used in sporulation AAATC TANTGTTNTA

s29 synthesized in sporulation TTNAAA CATATT

gp28SPO1 middle expression AGGAGA TTTNTTT

gp33-34 SPO1 late expression CGTTAGA GATATT

Different factors in Bacillus subtilis are used at different stages of growth (vegetative vs. sporulation)

Sigma Source & Use -35 region -10 region

s43 vegetative: general genes TTGACA TATAAT

s28 vegetative: flagellar genes CTAAA CCGATAT

s37 used in sporulation AGGNTTT GGNATTGNT

s32 used in sporulation AAATC TANTGTTNTA

s29 synthesized in sporulation TTNAAA CATATT

gp28SPO1 middle expression AGGAGA TTTNTTT

gp33-34 SPO1 late expression CGTTAGA GATATT

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Bacteriophage - "eaters of bacteria"

Phage Early gene 28

Phage Mid. genes 33 34

Early

Middle

Transcription of phage SPO1 genes

RNAP70

RNAP28

RNAP28

Phage Late genes

LateRNAP

33 34

RNAP70 28

33 34RNAP33 34

RNAP28

Genetic regulation

lac system of E. coli

“What’s true for E. coli is true for an elephant.”

J. Monod

β-Gal is produced only when lactose is present

β-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 β-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 β-Gal under gratuitous conditions

p = (amount of β-Gal)/(total cell protein)

lac system: transcription regulation

Regulation of Transcription

1. Transcription initiation/RNA synthesis

2. mRNA Turnover RNAP

1

2mRNA

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

21

The lac locus of E. coli

β-Galgalactos

ide 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+ StrS TsXS x F- lacI- lacZ- StrR TsxR

Set a cross in the absence of inducer:

After some time, kill the donor with Str and T6

Monitor β-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

DNA

lacIRepressor

Inducer

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)

R e g u l a t o r

XR e g u l a t o r

R e g u l a t o r

X

R e g u l a t o rR e g u l a t o r

XR e g u l a t o r

X

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 O2 and O3?

X-gal

White

Blue

White

Blue

White

Genetic analysis of the LacI binding sites

O3 O1 O2

P lacZRepression

1300

1.0

1.0

1.0

O3 O1 O2

O3 O1 O2

O3 O1 O2

440O3 O1 O2

700O3 O1 O2

1.9O3 O1 O2

18O3 O1 O2

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

glucose energy

glycerolpgi

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 defectivein 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

cya crp

cAMP level highwhen 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 RNAP LacI

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

Lac

AroH

Gal

Repressor Operator Sites CAP Operator Sites

Lac

Trp

AroH

TrpR

Gal

β

β'

-35 -10

D

Act.

λPRM- cI

αCTD αNTD

α CTDαNTD β

σ

β'

-35 -10

CAct.

lac-CAP

αCTD

α NTDβ

σ

β'

-35 -10

B

UPrrnB

β

σ

β'

-35 -10

AαNTDαCTD

lacUV5

E

α CTD αNTD β

σ

β'

-35 -10

Act.Act.

CAP & cI

Different mechanisms of transcriptional activation

A) Strong promoters

B) Promoters with UP elements

C) Activation through interactions with the αCTD

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

RNAP

DNA

OpenComplex

ClosedComplex

KB ki

-35 -1015-17 bp

RNAP

-35 -1019 bp

merT

AverageProm.

Mechanism of activation by MerR

MerR

merT-35 -10

Hg++

MerR

17 bp

RNAP

Enzyme repression: the trp operator

The synthesis of Trp structural genes is controlled by unlinkedTrpR 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

precursor Trp

TrpE TrpD TrpB

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