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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

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



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


Different alternative sigma ( 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


Different alternative sigma ( 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



Transcription of phage SPO1 genes alternative sigma (

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


Genetic regulation alternative sigma (

lac system of E. coli

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

J. Monod


b alternative sigma (-Gal is produced only when lactose is present


b alternative sigma (-gal induction can be due to

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

2. Synthesis of new enzyme


Lactose is both an inducer and a substrate for alternative sigma (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


Induction kinetics of alternative sigma (b-Gal under gratuitous conditions

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


lac alternative sigma (system: transcription regulation


RNAP alternative sigma (

1

mRNA

2

Regulation of Transcription

1. Transcription initiation/RNA synthesis

2. mRNA Turnover


Selection of Lac alternative sigma (- mutants (negative selection nutritional marker)

+Lac


1 alternative sigma (

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


The alternative sigma (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)


The PaJaMo experiment alternative sigma (

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


The properties of alternative sigma (lacO mutants provide genetic proof of operon model


lac alternative sigma ( 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)

i) Regulatory factors act in trans

ii) Regulatory elements act in cis


The sites)LAC OPERON


LacI binds DNA as a tetramer to better repress transcription sites)

Why did Jacob & Monod not find O2 and O3?


Genetic analysis of the LacI binding sites 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


Glucose effect: sites)

no response to inducers in the presence of glucose


Catabolism sites)

???

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)


Catabolite repression occurs for a wide range of sugars sites)

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

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


LAC distinct loci 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 distinct locilac control region activates transcription


The distinct locilac control region contains three critical cis-acting sites

RNAP

CAP

RNAP

LacI


lac distinct locioperator: the regulatory region


Residues that interact with RNAP distinct loci

CAP binding bends the DNA



Different mechanisms of transcriptional activation the start of the promoter

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



Changes in DNA topology affect isomerization transcription

step in formation of the open complex


Mechanism of activation by MerR transcription

RNAP

RNAP

merT

MerR

-10

-35

19 bp

Hg++

MerR

merT

-10

-35

17 bp

Average

Prom.

-10

-35

15-17 bp


Enzyme repression: the transcriptiontrp 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 transcription

the biochemistry of Trp biosynthesis pathway

TrpE

TrpD

TrpB

precursor

Trp


Attenuation of transcriptiontrp operator expression

attenuator

Deletions in the attenuator increase basal synthesis of Trp enzymes


the transcriptiontrp attenuator region


Attenuation occurs due to formation of alternative secondary

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


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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