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Molecular Biology and Biochemistry 694:408 / 115:512 Spring 2007, Lectures 13-14 PowerPoint PPT Presentation


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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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Molecular Biology and Biochemistry 694:408 / 115:512 Spring 2007, Lectures 13-14

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Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

Strong promoters contain close matches to the consensus site


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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 TTGACATATAAT

s28vegetative: flagellar genesCTAAACCGATAT

s37used in sporulationAGGNTTTGGNATTGNT

s32used in sporulationAAATCTANTGTTNTA

s29synthesized in sporulationTTNAAACATATT

gp28SPO1 middle expressionAGGAGATTTNTTT

gp33-34SPO1 late expressionCGTTAGAGATATT


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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 TTGACATATAAT

s28vegetative: flagellar genesCTAAACCGATAT

s37used in sporulationAGGNTTTGGNATTGNT

s32used in sporulationAAATCTANTGTTNTA

s29synthesized in sporulationTTNAAACATATT

gp28SPO1 middle expressionAGGAGATTTNTTT

gp33-34SPO1 late expressionCGTTAGAGATATT


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

Bacteriophage - "eaters of bacteria"


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

Genetic regulation

lac system of E. coli

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

J. Monod


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

b-Gal is produced only when lactose is present


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

b-gal induction can be due to

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

2. Synthesis of new enzyme


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

Induction kinetics of b-Gal under gratuitous conditions

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


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

lac system: transcription regulation


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

RNAP

1

mRNA

2

Regulation of Transcription

1. Transcription initiation/RNA synthesis

2. mRNA Turnover


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

Selection of Lac- mutants (negative selection nutritional marker)

+Lac


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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)


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

The properties of lacO mutants provide genetic proof of operon model


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

lac operator

Most bacterial operator sequences are short inverted repeats;

Most transcription regulators are dimeric


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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

i) Regulatory factors act in trans

ii) Regulatory elements act in cis


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

The LAC OPERON


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

LacI binds DNA as a tetramer to better repress transcription

Why did Jacob & Monod not find O2 and O3?


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

Glucose effect:

no response to inducers in the presence of glucose


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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)


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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.


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

LAC Operon and catabolite repression

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


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

The lac control region contains three critical cis-acting sites

RNAP

CAP

RNAP

LacI


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

lac operator: the regulatory region


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

Residues that interact with RNAP

CAP binding bends the DNA


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

Different types of negative and positive control of transcription


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

Changes in DNA topology affect isomerization

step in formation of the open complex


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

Crossfeeding analysis of Trp mutants allows to analyze

the biochemistry of Trp biosynthesis pathway

TrpE

TrpD

TrpB

precursor

Trp


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

Attenuation of trp operator expression

attenuator

Deletions in the attenuator increase basal synthesis of Trp enzymes


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

the trp attenuator region


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

Attenuation occurs due to formation of alternative secondary

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


Molecular biology and biochemistry 694 408 115 512 spring 2007 lectures 13 14

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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