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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)

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slide1

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

slide4

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

gp28 SPO1 middle expression AGGAGA TTTNTTT

gp33-34 SPO1 late expression CGTTAGA GATATT

slide5

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

gp28 SPO1 middle expression AGGAGA TTTNTTT

gp33-34 SPO1 late expression CGTTAGA GATATT

slide7

Transcription of phage SPO1 genes

70

70

28

RNAP

RNAP

RNAP

RNAP

RNAP

RNAP

RNAP

28

28

28

34

33

34

34

33

33

Phage Early gene 28

Early

Phage Mid. genes 33 34

Middle

Phage Late genes

Late

slide8

Genetic regulation

lac system of E. coli

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

J. Monod

slide10

b-gal induction can be due to

1. Activation of preexisting enzyme (i.e., removal of repressor)

2. Synthesis of new enzyme

slide11

Lactose is both an inducer and a substrate for b-Gal

Gratuitous inducers do not act as substrates

Some substrates do not work as inducers

Action of the enzyme on the inducer is neither necessary

nor sufficient for induction

slide12

Induction kinetics of b-Gal under gratuitous conditions

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

slide14

RNAP

1

mRNA

2

Regulation of Transcription

1. Transcription initiation/RNA synthesis

2. mRNA Turnover

slide16

1

2

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

slide17

The lac locus of E. coli

galactoside permease

b-Gal

galactoside transacetylase

lacZ mutants are Lac-

lacY mutants are cryptic

lacA mutants are Lac+

lacI mutants are constitutive (first example of mutants that

affect production, not activity)

slide19

The PaJaMo experiment

Set a cross in the absence of inducer:

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

After some time, kill the donor with Str and T6

Monitor b-Gal in the presence or in the absence of inducer

slide24

lac operator

Most bacterial operator sequences are short inverted repeats;

Most transcription regulators are dimeric

slide25

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

slide26

Distinction between factors (proteins) and elements (DNA sites)

i) Regulatory factors act in trans

ii) Regulatory elements act in cis

slide28

LacI binds DNA as a tetramer to better repress transcription

Why did Jacob & Monod not find O2 and O3?

slide29

Genetic analysis of the LacI binding sites

X-gal

White

4

4

0

Blue

O

O

O

1

3

2

White

7

0

0

O

O

O

1

3

2

White

1

8

O

O

O

1

3

2

1

.

9

O

O

O

1

3

2

Blue

R

e

p

r

e

s

s

i

o

n

P

l

a

c

Z

1

3

0

0

O

O

O

1

3

2

1

.

0

O

O

O

1

3

2

1

.

0

O

O

O

1

3

2

1

.

0

O

O

O

1

3

2

slide30

Glucose effect:

no response to inducers in the presence of glucose

slide31

Catabolism

???

glucose

energy

pgi

glycerol

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)

slide32

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.

slide33

Mutants defective in catabolite regulation occur in two distinct loci

cya

crp

codes for CAP (catabolite activating protein).

CAP, when bound to cAMP, binds to lac regulatory region and activates transcription of structural genes

cAMP level high

when glucose is low

slide34

LAC Operon and catabolite repression

Positive control of the lac operon is exerted by cAMP-CAP Catabolite Activator Protein

slide35

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

slide38

Residues that interact with RNAP

CAP binding bends the DNA

slide40

Different mechanisms of transcriptional activation

A) Strong promoters

B) Promoters with UP elements

C) Activation through interactions with the aCTD

D) Activation through interactions with other components of RNAP

E) Activation through interactions with components multiple components of RNAP by multiple activators

slide42

Changes in DNA topology affect isomerization

step in formation of the open complex

slide43

Mechanism of activation by MerR

RNAP

RNAP

merT

MerR

-10

-35

19 bp

Hg++

MerR

merT

-10

-35

17 bp

Average

Prom.

-10

-35

15-17 bp

slide44

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

slide45

Crossfeeding analysis of Trp mutants allows to analyze

the biochemistry of Trp biosynthesis pathway

TrpE

TrpD

TrpB

precursor

Trp

slide46

Attenuation of trp operator expression

attenuator

Deletions in the attenuator increase basal synthesis of Trp enzymes

slide48

Attenuation occurs due to formation of alternative secondary

RNA structures in the leader sequence in the presence or absence or Trp

slide51

The l repressor idea

Zygotic induction

Immunity of lysogens to superinfection with lwt

The existence of c and vir mutants. l are immune to lc, but not lvir

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