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SIGMA FACTOR

&
LAMBDA PHAGE CYCLE

Bhaskar Ganguly
Ph.D. , M.V.Sc., B.V.Sc. & A.H.

The RNApol holoenzyme


Definition of holoenzyme
Complete, working version
of an enzyme
cf. apoenzyme - missing
specific
cofactors
that
allow it to perform its job

Examples of cofactors
common prosthetic groups (haem) or metal ions
(magnesium)
Dissociable protein subunits sigma () factor.

The RNApol holoenzyme


All multi-subunit RNA polymerases have 5 core
subunits.
Bacterial RNApol have additional subunit
Has function in binding to promoter
In bacteria, RNApol binds a promoter via
In eukaryotes, RNApol binds via TF complex

Bacterial RNApol is regulated purely by


(initiation phase), but eukaryotic RNApol is
regulated both by the TFs and by various gene
regulatory proteins.
Although promoters are similar, the bacterial
promoter tends to be highly conserved.

RNAP binds specific promoter


sequences
Sigma factors recognize consensus
-10 and -35 sequences

General Properties of
factor

RNA polymerase holoenzyme binds directly to DNA via its


subunit
Promoter consensus sequence (below) is highly conserved in
Sequence
bacteria alignment
of E. coli promoters
reveal
a
predominance
of
certain residues at
positions -35 and
10 relative to start
point
of
transcription (+1).
Most common is the
70 subunit the
generic
sigma
subunit

RNA polymerase promoters

TTGACA

TATAAT

Deviation from consensus -10 , -35 sequence leads to


weaker gene expression

Bacterial sigma factors

Sigma factors are transcription factors


Different sigma factors bind RNAP and recognize
specific -10 ,-35 sequences
Help melt DNA to expose transcriptional start site
Most bacteria have major and alternate sigma factors
Promote broad changes in gene expression
E. coli 7 sigma factors
B. subtilis 18 sigma factors
Generally, bacteria that live in more varied
environments have more sigma factors

Sigma factors
Sigma subunit

Type of gene controlled

70 RpoD

Growth/housekeeping

54 RpoN

N2; stress response

~15

RpoS

Stationary phase, virulence

~100

RpoH

Heat shock

~40

RpoF

Flagella-chemotaxis

~40

Extreme ?heat shock, unfolded proteins

~5

Ferric citrate transport

~5

32 RpoE
FecI

# of genes controlled
~1000

E. coli can choose between 7 sigma factors and about 350


transcription factors to fine tune its transcriptional output
An Rev Micro Vol. 57: 441-466 T. M. Gruber

What regulates sigma factors

Number of copies per cell (70 more than


alternate)
Anti-sigma factors (bind/sequester sigma
factors)
Levels of effector molecules
Transcription factors

Bacterial RNAP numbers

In log-phase E. coli:
~4000 genes
~2000 core RNA polymerase molecules
~2/3 (1300) are active at a time
~1/3 (650) can bind subunits.

Competition of for core determines much of a cells


protein content.

The alternative

54

factor

Most alternative sigmas are related in


sequence and structure to 70.
2nd distinct type of called the 54 family
Differences between the families

54 family shares no sequence homology with


the 70 family
Whereas 70 holoenzymes carry out this
process of open complex formation on their
own, 54 holoenyzmes require both an
enhancer and ATP to perform this process.

Lysis or Lysogeny
Lysis: Infection by phage produces
many progeny and breaks open (lyses)
the host bacterium
Lysogeny: After infection, the phage
DNA integrates into the host genome
and resides there passively
No progeny
No lysis of the host
Can subsequently lyse (lysogeny)

Bacteriophage lambda can do either.

UV
Induction

Lysis

Lysogeny

Elements of lysogeny
The phage genome integrated into the
host bacterial genome is a prophage.
Bacterium carrying the prophage is a
lysogen.
Lysogens are immune to further infection
by similar phage because the phage
functions are repressed in trans.
Induction of the lysogen leads to
excision of the prophage, replication of
the phage DNA, and lysis of the host
bacterium.

Genes are clustered by function in


the lambda genome
Late control
Recombination
att

int

Control region Replication

gam
red
xis cIII N

Pint

tL1

cI

cro

cII O P Q

Virus head
Lysis &tail
SR

AJ

PL oL PRM PR tR1 PRE tR2 PR t6S cos


tR3
oR
origin

promoter
operator
terminator

Not to scale!

Immediate early
transcription

Transcription by E. coli RNA polymerase initiates at strong


promoters PR , PR, and PL , and terminates at ts.
att

int

gam
red
xis cIII N

Pint

tL1

cI

cro

cII O P Q

PL oL PRM PR tR1 PRE


oR

SR

tR2 PR t6S
tR3
6S RNA

Cro

AJ

Antitermination by N protein leads to


early gene expression
N
att

int

gam
red
xis cIII N

Pint

tL1

PL

N
cI

cro

N
cII O P Q

PRM PR tR1 PRE

SR

AJ

tR2 PR t6S
tR3
6S RNA

N protein
CIII
Recombination proteins

Cro
CII

Q protein
Replication proteins

Lytic cascade: Cro turns off cI, Q


protein action leads to late gene
expression
Cro
att

int

gam
red
xis cIII N

Pint

tL1

Cro
cI

cro

Q
cII O P Q

PL oL PRM PR tR1 PRE


oR

SR

AJ

tR2 PR t6S
tR3

Lytic functions
Replication proteins
Viral head & tail proteins

Late stage of lytic cascade


High concentrations of Cro turn off PR and PL .
Abundant expression from PR.
Cro
att

int

gam
red
xis cIII N

Pint

tL1

Cro
cI

Q
cro

cII O P Q

PL oL PRM PR tR1 PRE


oR

SR

AJ

tR2 PR t6S
tR3

Lytic functions
Viral head & tail proteins

Lysogeny: CII and CIII stimulate


expression of cI to make repressor
+

att

int

gam
red
xis cIII N

tint Pint

Int

CIII

CII

tL1

cI

cro

CII

cII O P Q

PL oL PRM PR tR1 PRE


oR

CI

Repressor

SR

AJ

tR2 PR t6S
tR3
PRE = promoter for
repression
establishment

Lysogeny: Repressor turns off


transcription
CI

att

int

gam
red
xis cIII N

Pint

tL1

CI

cI

cro

cII O P Q

PL oL PRM PR tR1 PRE


oR

CI

Repressor

SR

AJ

tR2 PR t6S
tR3
PRM = promoter for
repression
maintenance
Activated by Repressor
binding to oR1 & oR2

Repressor structure
repressor is a dimer; monomer has 236 amino acids.
C-terminal domain: protein-protein interaction;
dimerization and cooperativity
Connector
N-terminus: DNA binding; Helix-Turn-Helix motif

operator

repressor can bind cooperatively


to operator sub-sites.

operator
oR2

operator
oR1

Thank you