Genes: Expression and Regulation

Terms and concepts

clone (pure culture)
population of cells that are genetically identical
genome
all genes present in a cell or virus
haploid one set of genes
diploid two sets of genes
partially diploid cell is termed merodiploid
genotype
specific set of genes an organism possesses
phenotype
set of observable characteristics

1
 Genes: Expression and Regulation
)( =
,

. ,

,

. ,pH - ,

Transcription in Procaryotes
polygenic (polycistronic) mRNA
contains directions for > 1 polypeptides

1 2 3 4

An operon is a multigenic unit including an operator, a common promoter, and

one or more structural genes that are controlled as a unit to produce mRNA

2
 Two major modes of regulation in the cell:
enzyme activity and/or enzyme synthesis

( transcriptional regulation) .1
( translational regulation) .2
( post translational regulation) .3

Regulation of mRNA Synthesis

Regulation of gene expression serves:

to conserve energy and raw ( )materials

to maintain balance ( )between the amounts

of various cell proteins

allows organism to adapt to long-term ()

environmental change

Thus control of gene expression complements

the regulation of enzyme activity

3
 Negative vs. positive controls

)(

)(

Induction ( )and Repression ()

inducible enzyme (e.g. -galactosidase)

level increases in presence of inducer (lactose)
small molecule, usually substrate of catabolic pathway
in which enzyme functions

repressible enzyme (e.g. tryptophan synthetase)

level decreases in presence of corepressor
(tryptophan)
usually end product of biosynthetic pathway
in which the enzyme functions

4
Negative Control

presence of regulatory protein (repressor)

at regulatory site (operator) decreases
mRNA synthesis

repressor proteins
exist in active and inactive forms

inducers and corepressors alter activity of

repressor

The -Galactosidase Reaction

5
 Gene Induction by Negative control
of catabolic pathway
the goal an operon
make enzymes
operator
of pathway promoter
only when
substrate of
pathway is
available

usually substrate
of pathway

An The
operon is a multigenic unit including an operator,
a common promoter, and one or more structural genes
that are controlled as a unit to produce messenger RNA

lacA encodes -galactose

transacetylase (LacA), an
enzyme that transfers an
acetyl group from acetyl-
CoA to lactose.

6
 Induction of the enzyme -galactosidase on the
addition of lactose to the medium

~3,000
molecules

~3 molecules

Gene Repression by Negative control

of a biosynthetic pathway

the goal
only make
enzymes of
pathway when
end product of
pathway is not
available usually end product
of pathway

aporepressor

7
Gene Repression by Negative control of a biosynthetic pathway

.(Corepressor) -
. ,

Repression of enzymes involved in arginine synthesis

by addition of arginine to the medium

,

.

?

8
 Positive Control
presence of a regulatory protein (activator protein)
at a regulatory region promotes transcription

Complex CAP+cAMP binding to DNA and bends it about 90o.

Interaction of CAP with RNA polymerase stimulates transcription.

The activator binding site is near or

several hundred base pairs from promoter

9
 Positive Control of Lactose Operon
 regulated by catabolite activator protein (CAP) and
inducer - cyclic AMP (cAMP)
- CAP also called, cyclic AMP receptor protein (CRP)

levels of cAMP
vary depending
on environmental
conditions

P O structural genes

The activator protein,

helps the RNA polymerase
either recognize the promoter operator
or begin transcription

Global Regulatory Systems

affect many genes and pathways simultaneously
regulon
collection of genes or operons controlled by a
common regulatory protein

10
 Positive Control of the maltose regulon
Regulon - several operons
under control of the same
regulatory (activator) protein.
Therefore, the enzymes
for maltose utilization are
encoded by the
maltose regulon.

The maltose activator

protein cannot bind
to the DNA unless the
protein first binds maltose,
the inducer.

The SOS response

, )DNA
( UV
.
.SOS response

:
SulA .1
. DNA .

11
 UmuC .2 (Umu=U.V. Mutagenesis) UmuD -
.DNA polymerase V DNA polymerase
DNA ).(lesions

DNA poly V " DNA pol V : "

.DNA poly III
.Error prone TLS
)(TLS = trans lesions

DNA
)( template-
DNA poly III
.

Poly V

-
template
.

TLS - Poly V :
! fitness -

12
 RecA .3 DNA -- ) DNA -
-( . RecA -
) (.

SOS - RecA -

?SOS response-
SOS genes - .LexA
LexA ) (SOS box
SOS genes - .

13
 SOS genes - .
:
) ( LexA .
).(regulon

14
 , DNA DNA . RecA
RecA DNA -- , . LexA
. SOS genes

, ?

DNA RecA .LexA

LexA .SOS boxes -
SOS genes -.

, ,
SOS .SOS
, .
:
SulA !
DNA Poly V !
ATP ,
, .

15
 Catabolite Repression
occurs when operon is under control of
catabolite substance
allows preferential use of one carbon source
over another when both are available in
environment
e.g., catabolite repression of lactose and
other operons by glucose
glucose decreases cAMP levels, thereby blocking
CAP binding and decreasing mRNA synthesis

cAMP

Glucose entry into the cell:

1. Inhibits the synthesis of cyclic AMP
2. Stimulates cyclic AMP transport
out of the cell

16
 Catabolite Repression Glucose effect

Attenuation
( , ,)

regulation of transcription by the behavior

of ribosomes (translation)

observed in bacteria, where transcription

and translation are tightly coupled
translation of a mRNA can occur as the
mRNA is being synthesized

17


RNA polymerase

Tryptophan operon

18
 e.g., the tryptophan operon

operon that
encodes enzymes
for the tryptophan
biosynthetic
pathway
leader of
operon
the goal
terminate
transcription if
tryptophan is
available

t-RNATrp

t-RNATrp

t-RNATrp

t-RNATrp

Trp-Trp
t-RNATrp
codons

19
Trp-Trp

20
 Riboswitch -
 A riboswitch is a part of an mRNA molecule that can directly bind a small target
molecule, and whose binding of the target affects the gene's activity.
 Thus, an mRNA that contains a riboswitch is directly involved in regulating its own
activity, in response to the concentrations of its target molecule.

Transcription regulation by riboswitches

(A) When the riboswitch ligand is unavailable, transcription of the downstream gene is permitted due to the
formation of an anti-terminator stem. However, when the aptamer is able to bind the ligand, the anti-
terminator is unable to assemble, and transcription termination occurs via formation of a terminator stem.

21
 Translation regulation by riboswitches

, , mRNA -

, (5 UTR =) 5 untranslated region - mRNA -

Riboswitch

.

:
thiamine B. subtillis -
thiamine 5 UTR -
mRNAs
. thiamine

mRNA

22
 Regulation of Transcription by Sigma Factors

 different sigma factors recognize different promoters

 substitution of sigma factors changes gene
expression of many genes and operons

Regulation of Transcription by Sigma Factors

DNA-dependent RNA polymerase

2 : Core RNA-polymerase -

: core RNA-polymerase -

.alternating factor

RNA poly -
.

23
 Sigma Factors
sigma factors - "
10 - 35 - .

consensus sequence .70

Sigma Factors
factor - RNA polymerase
.

RNA polymerase factors -

. , factor-
"" ,
. factor -

24
 Sigma Factors
factors E. coli
.

Sigma factor Function

70 principal sigma factor
54 nitrogen-regulated gene transcription
32 heat-shock gene transcription
S gene expression in stationary phase
F expression of flagellar operons
E Envelope stress response
FecI regulates the fec genes for iron dicitrate transport

70 the principal sigma factor

" , sigma factors -

.

, .70 E. coli - ,
.70 E. coli -

,70 ",
!

. ,

25
 Sigma Factors
consensus sequence -
70 - ,
.

, Lac repressor - )
(.
lacZ , 70
.

,
,
.

Regulation by Sigma Factors

and Control of Sporulation

 e.g., Bacillus subtilis sporulation sigma factors

 synthesized only as cell switches from
vegetative growth to sporulation
 lead to transcription of sporulation-related genes

26
Regulation of Transcription by Sigma Factors

Sigma factor cascades in B. burgdorferi (A) and Salmonella typhimurium (B).

)The heat-shock proteins and sigma factor (32

:
.

heat-shock proteins.Hsps :

27
 Hsps - :

' ) :(chaperones
.

) :(proteases
.

Chaperones
' .DnaK'
.Hsp70
.ATP
DnaK - protein quality control -.

ATPase
domain

Substrate binding
pocket

28
 , )(
.GroESL
Chaperone Hsp60
,
.

ATP dependent proteases

"
.
.

,
.ATP

29
 Chaperones vs. Proteases
'
) (
.

.

-
'.

"
.

Response to heat-shock

E. coli -

heat-shock genes 32

32 70

heat-shock genes -
32

) Heat shock factor 1 (HSF-1
heat-shock

heat-shock genes
, ?

30
 32 E. coli -

32 ,
"" DnaK
32 DnaK - .FtsH
" .FtsH
,
.heat-shock genes

,
.

DnaK
32 '.
, -
32 .DnaK

32 DnaK -
RNA poly -
.heat-shock genes

31
 ?

) , , '(.

)(.

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.

Two-component systems

.
:
Sensor kinase -1-
Response regulator -2

.1 Sensor kinase -
.2 Sensor kinase -
Response regulator
.3 Response regulator -

32


, signal transduction
, .

Two-component
.

33
 Antisense RNA and the Control of
Porin Proteins

antisense RNA
has sequence complementary to some RNA
component necessary for gene expression
(target RNA)
binds target RNA and blocks its function

34