Gene Regulation

Positive
Negative

Regulation of protein activity vs. amount

Regulate protein activity
Allostery
Covalent modification
Sequestration

Regulate the amount of protein

Gene transcription
RNA processing
RNA turnover
mRNA translation
Protein processing, assembly, turnover

Operons
An operon is a cluster of coordinately
regulated genes. It contains:
Structural genes: encode enzymes
Regulatory genes: encode repressors or
activators of expression
Regulatory sites: e.g. promoters, operators

Positive vs negative control

Mutate
regulatory
gene to lose
function

Regulatory
protein is
present

Example of
regulatory
protein

Positive control

Operon ON

Activator

Operon OFF

Negative control

Operon OFF

Repressor

Operon ON

Catabolic vs. biosynthetic operons

Operon
encodes

Absence of

Catabolic
enzymes

Substrate

Biosynthetic
enzymes

Product

Effect
Repressed
Induced

Presence of

Effect

Substrate

Induced
(derepressed)

Product

Repressed

Inducible vs. repressible operons

Defined by response of operon to a metabolite (small molecule).
Type of
operon

Examples
Metabolite Operon

Presence of

Effect

metabolite

ON

lactose

lac

Repressible metabolite

OFF

Trp

trp

Inducible

Negative control of the lac operon

Induced (derepressed) lac operon

Promoter
Operator

lacZ

lacY

lacA

transcription
AUG

UAA AUG

UAA AUG UAA

translation

-galactosidase

lactose
permease

Structural
genes &
regulatory
sites in
operon
Polycistronic
mRNA

-galactoside
transacetylase

Repressed lac operon

lacI

Promoter
Operator lacZ

lacY

lacA

lac repressor
Repressor binds to the operator in the absence of the inducer
(a metabolite of lactose), and blocks transcription of the lac
operon.

Induction of the lac operon by derepression

lacI

Promoter
Operator lacZ

lacY

lacA

Inducer (allolactose)
lac repressor no longer
Binds operator

lacI

Promoter
Operator lacZ

lacY

Operon is expressed

lacA

Inducers of the lac operon

Lactose, the substrate for the operon, is
converted to its isomer allolactose.
Allolactose is the natural inducer.
A gratuitous inducer induces the operon
but is not metabolized itself.
e.g. isopropylthiogalactoside= IPTG

Regulatory mutations in the lacI gene

Genotype
I+Z+A+
I+Z-A+

(lac Z )
(lac A )
-galactosidase transacetylase
-IPTG +IPTG -IPTG +IPTG
<0.1 100
<1
100
<0.1 <0.1
<1
100

I -Z +A +
100
I+Z -A+ /F' I -Z +A+ <0.1
I sZ +A+
<0.1
I sZ +A+ /F' I+Z +A+ <0.1

100
100
<1
1

100
<1
<1
<1

100
200
<1
1

Conclusion
Inducible

Constitutive
I+ >I - in trans
Noninducible
I s >I+ in trans

The lacI gene encodes a trans-acting factor (protein)

needed for repression.
Most lacI - mutants are constitutive.
The lacI S allele is noninducible.

Regulatory mutations in the operator

Genotype
I +o + Z +
I +o C Z +
I +o C Z + / F' I +o + Z I +o C Z - / F' I +o + Z +

-galactosidase
-IPTG +IPTG
<0.1 100
100 100
100 100
<0.1 100

Conclusion
Inducible
Constitutive
Constitutive
Inducible

Loss-of-function alleles of the operator confer a constitutive

phenotype on the operon. They are called oC.
The operator acts in cis, i.e. it affects the allele to which it is
linked.
The allele of the operator that is in cis to the active reporter
gene is the dominant allele. The operator shows cis dominance.

Interactions between operator and

repressor
Constitutive mutations

A TGTTA
T ACAAT

C
G

T
A

-10
+1
+10
+20
5TGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACA
3ACAACACACCTTAACACTCGCCTATTGTTAAAGTGTGT
Dyad axis
Nucleotides in contact with
repressor
Promoter

lac repressor
lacI

Promoter
Operator lacZ

lacY

lacA

Inducer (allolactose)
lac repressor no longer
Binds operator

lacI

Promoter
Operator lacZ

lacY

Operon is expressed

lacA

Picky eater?

Positive control: catabolite repression

Glucose is the preferred carbon source for
E. coli.
Glucose causes repression of operons
whose products catalyze the metabolism of
other carbon sources, e.g. lac operon and
lactose.
This is called catabolite repression.
In the absence of glucose, operons needed
for metabolism of other carbon sources are
induced.

Catabolite repression is mediated by

cAMP and CAP
cAMP
3, 5-cyclic adenosine monophosphate
In presence of glucose, [cAMP] is about 10-7 M.
In absence of glucose, [cAMP] increases to
about 10-4 M.

Catabolite activator protein = CAP

Is a dimer
Binds cAMP

cAMP-CAP binds DNA adjacent to promoter

and stimulates transcription

Binding site for cAMP-CAP

A
Mutations that make
promoter nonresponsive T
to CAP

T
A

-70
-60
-50
5ATGTGAGTTAGCTCACACATT
3TACACTCAATCGAGTGTGTAA
Dyad axis
Nucleotides in contact
with cAMP-CAP
Promoter

lac regulatory region

Activator binding site

Promoter

Operator

UV5 mutation, up TATAAT

-72

-52

TTTACA

TATGTT

-35

-10

cAMP-CAP

RNA polymerase

+1

'

+11

Repressor

Some generalities
Repressors, activators and polymerases
interact primarily with one face of the DNA
double helix.
Regulatory protein are frequently
symmetrical and bind to symmetrical sites
on the DNA.
RNA polymerases are not symmetrical, and
bind to asymmetric sites. This helps
establish the direction of transcription.

cAMP-CAP helps RNA polymerase

bind to promoter by interacting with
the alpha subunit
More in chapter II of Part Four

Contacts between CAP and DNA

CAP bound to DNA

cAMP-CAP bends the DNA

Assays for DNA bending

Problem 4.19,p. 674

Consider a hypothetical regulatory scheme in which citrulline
induces the production of urea cycle enzymes. Four genes
(citA, citB, citC, citD) affecting the activity or regulation of the
enzymes were analyzed by assaying the wild-type and
mutant strains for argininosuccinate lyase activity and
arginase activity in the absence (-cit) or presence (+cit) of
citrulline. In the following table, wild-type alleles of the
genes are indicated by a + under the letter of the cit gene
and mutant alleles are indicated by a - under the letter. The
activities of the enzymes are given in units such that 1 = the
uninduced wild-type activity, 100 = the induced activity of a
wild-type gene, and 0 = no measurable activity. In the
diploid analysis, one copy of each operon is present in
each cell.

4.19: Haploid analysis

Strain
Number
Haploid:
1
2
3
4
5

genes
A B
C
+ +
+
+
+
+ +
+ +
+ +
+

lyase activity arginase act.

- cit + cit - cit + cit
D
+
+
+
+
-

1
100
0
100
1

100
100
0
100
100

1
100
1
100
0

100
100
100
100
0

Strain 1 (wt) : operon is inducible by citrulline.

Strains 2 &4: Mutation in A and C make the operon constitutive.
Strains 3 & 5: Genes B and D encode enzymes.

4.19: Diploid analysis

Strain
Number
Diploid: A
6
+
7
8
+
9
+

genes
BCD/A
+ + - /+
+ + +/+
+ - +/+
- - +/+

lyase activity arginase act.

- cit + cit - cit + cit
B
+

C
+
+
+
+

D
+
+
-

1
1
100
1

100
100
100
100

1
2
100
100

100
200
100
100

Strain 6: B- complements D-; the genes encode enzymes.

Strain 7: B- complements A-, so A encodes a trans-acting
regulatory factor. A+ > AStrain 8: B- does NOT complement C-. citC shows cisdominance, and thus is a regulatory site on the DNA.

Regulatory scheme for 4.19

Gene citB encodes argininosuccinate lyase.
Gene citD encodes arginase.
Gene citA encodes a diffusible, regulatory
molecule, such as a repressor.
Gene citC is a site on DNA at which the
repressive effect of CitA is exerted. e.g. the
operator at which CitA repressor binds.
In the presence of the substrate citrulline,
the repressor no longer binds the
operator,and the operon is induced.

Regulatory effects on RNA

polymerase
Binding constants
Rate constants
Measuring KB

Multiple steps in initiation and elongation

by RNA polymerase are targets for
regulation
RNA Polymerase has to
* bind to promoters,
* form an open complex,
* initiate transcription,
* escape from the promoter,
* elongate
* terminate transcription.
All are potential targets for regulation.

Steps at which RNA polymerase is regulated

Factors associated with each step

Effects on KB, kf, kr

Summarizing a lot of work, we know that:
strong promoters have high KB, high kf, low kr, and high rates
of promoter clearance.
weak promoters have low KB, low kf, high kr, and low rates of
promoter clearance.
moderate promoters have one or more "weak" spots.

lac regulatory region

Activator binding site

Promoter

Operator

UV5 mutation, up TATAAT

-72

-52

TTTACA

TATGTT

-35

-10

cAMP-CAP

RNA polymerase

+1

'

+11

Repressor

Measurement of KB

Synonymous and related terms

KB = Kb = Keq = equilibrium constant for binding
KS = KB for binding of protein to a specific DNA sequence
KNS = KB for binding of protein to nonspecific DNA
[P] = [P2] = molar concentration of protein
[R4] = molar concentration of repressor
[D] = molar concentration of free DNA
[DS] = concentration of free specific DNA
[DNS] = concentration of free nonspecific DNA
[DP] = molar concentration of DNA-protein complex
[R4DS] = concentration of repressor-operator

Techniques to measure amount of

protein bound to DNA
Need:
Radioactively labeled DNA (usually a specific
sequence)
Purified DNA-binding protein

Combine them and measure the amount of

protein-DNA complex and free DNA by:
Electrophoretic mobility shift assays
DNase I footprinting
Retention of protein-DNA complexes on filters

Measure KB by EMSA
0
DP

[P]

DP

D+P
[DP]

KB =

When

[D] [P]
[DP]
[D]

KB =

=1, then
1
[P]

Measure KB from [DP]/[D]

DP

D+P
[DP]

KB =

[D] [P]

[DP]
= KB [P]
[D]
When

[DP]
[D]
KB =

=1, then
1
[P]

If you could measure [DP] and

[D] at each [P], you could
measure KB:
[DP]
[D]

1.0

1
KB

slope = KB

[P]

Measure KB from [DP]/[D]tot

It is more reliable to measure the fraction of labeled
DNA in complex with protein, i.e. [DP]/[D]tot
Substitution of [D]=[D]tot - [DP] into equation for KB gives:
[DP]
KB [P]
=
[D]tot
1 + KB [P]
1.0

[DP]
[D]tot
10

[P]

50 nM

Protein binding assayed by DNase I footprinting

Need to use many orders

of magnitude of [P].
Fr. Dr. Tracy Nixon

What value for KB provides the best fit?

Classical methods:
Transform the data into a line
Or, e.g., at [DP]/[D]tot = 0.5, then KB=1/[P]
Problems:
Where to draw the line?
No accurate estimate of error

Nonlinear, least squares regression analysis

= NLLS
A computer program calculates the goodness of
fit for many values of KB, then one can choose
the best fit (least error).

Modeling binding reactions for NLLS

We can model binding reactions by
1. tabulating the different states that exist in a system,
2. associating each state with a fractional probability based on
the Boltzmann partition function and the Gibb's free energy
for that state (Gs),
and
3. determine the probability of any observed measurement by
the ratio of
a) the sum of fractional probabilities that give the
observation, and
b) the sum of the fractional probabilities of all possible
states.

Fractional occupancy from fractional probabilities

Where j is the number of ligands bound, the fractional
probability of a particular state is:

e Gs / RT [P2 ]j
fs =
Gs / RT
j
e
[P
]

2
sj

For a single protein binding to DNA (one site), the

fractional occupancy, Y, is:

f1
Y=
fs

and

Y=

G / RT
G / RT

1+ e

[P2 ]
[P2 ]

Fractional occupancy in terms of KB

Since
then

Same as:

G = -RT ln (Keq)

K B [P2 ]
Y=
1 + (K B [P2 ])
[DP]
KB [P]
=
[D]tot
1 + KB [P]

Data analysis by NLLS

After collecting binding data, one uses a nonlinear,
least squares regression analysis to find the
values of G (or KB) that generate a function that
best predicts the data.
Uses maximum likelihood theory to find the value
most likely to be correct.
Produces plot of the variance of fit (or error) over
a wide range of possible values for the parameter
being measured, e.g. G.
Reproducible by different investigators
Provides a rigorous estimate of error.

Variance of fit plotted vs. free energy

The G value with the smallest error is the most accurate.

Example of calculating KB from plot of

variance of fit vs. G
G1= -9.5 kcal/mol gives the minimum variance (or error).
G = -RT ln (Keq)

ln KB = -G/RT = -(-9.5 kcal/mol)

0.59 kcal/mol
KB = 9.8 x 10 6 M -1
R = 1.98 x 10-3 kcal deg-1 mol-1
T = 298 K
RT= 0.59 kcal/mol

= 16.1017

What do you learn from binding

constants and rate constants?

Regulatory effects on RNA

polymerase
Inducers
Repressors
Activators

Inducer lowers the KB for repressor binding

to operator
lac repressor
nonspecific site

lac repressor
Operator

+
KB = KS =

+
2x1013

M-1

KB = KNS = 2x106 M-1

In the presence of inducer:

lac repressor

nonspecific site

+
KB = KS =

+
2x1010

M-1

KB = KNS = 2x106 M-1

Specificity parameter (See also Appendix A)

Ratio of KS to KNS decreases 1000 fold in presence of
inducer.
Specificity =

KS

= 107 in absence of inducer

KNS
KS

= 104 in presence of inducer

KNS
Nonspecific sites are in vast excess over operator
sites. Thus, in the presence of inducer, the repressor
is redistributed so that the operator is not occupied.

Virtually all the repressor is associated with DNA

10 molecules of repressor/cell means [P] = [R4] = 1.7x10-8 M
4.6x106 nonspecific sites on DNA/cell means
total [D] = total [DNS] = 7.64x10-3 M
KB =
[R4]
[R4DNS]

[DP]

[D] [P]

1
KNS [DNS]

KNS =

[R4DNS]

= 2x106 M-1

[R4] [DNS]
1

= 6.5x10- 5

(2x106 M-1 )(7.64x10-3 M)

Only about 1 in 15,000 repressor molecules is NOT bound to

DNA.

Can calculate bound to free DNA while

considering both specific and nonspecific binding
[R4DS]
Specificity =

KS

KNS

[R4] [DS]
[R4DNS]

[R4DS]
[DS]

[DNS]
[R4DNS]

[R4] [DNS]

KS
KNS

Measured

[R4DS]
[DS]

[DNS]

[R4] total - [DS]total

since [R4]free is negligible,
Ds is saturated with R4

[R4] total - [DS]total

Want to know

Constants

Inducer shifts the distribution of repressor so

that more is bound to nonspecific DNA
KS
KNS
KS
KNS

KNS

[DS]

[DNS]
[R4] total - [DS]total

= 107 in absence of inducer

[DS]
[R4DS]

KS

[R4DS]

= 0.05

About 95% of operators

are bound by repressor.

= 104 in presence of inducer

[R4DS]
[DS]

= 0.02

About 2% of operators
are bound by repressor.

Operator mutants decrease the

affinity of repressor for operator
Appendix B

Effect of repressor on RNA

polymerase at the promoter

Repressor increases affinity of polymerase for

promoter
RNA polymerase

'

'

+
-35
-10 +1 +11
Promoter Operator

KB = KS = 1.9x10 7 M-1

Repressor

'

'

KB = KS = 2.5x10 9 M-1

Repressor decreases rate of transition

from closed to open complex

Events at
initiation of
transcription

Abortive initiation assay

Let R = RNA polymerase, P = promoter (closed), and Po=
promoter (open)
R+P

KB

RP

kf
kr

[ApUp*U]

ATP + UTP*
RPo
ApUp*U

lag

time

Abortive transcripts

Measure kf and KB from lag time vs. 1/[R]

Lag time in abortive initiation assay is inversely proportional to [R].
Lag time =

KB kf

x 1
[R]

Lag time

Y-intercept = 1
kf

+ 1
kf

Slope =
1

[R]

KB kf

Activation of transcription by CAP

cAMP-CAP interacts directly with RNA
polymerase
Binds to alpha subunit of RNA polymerase
For lac operon, this increases KB for binding
of polymerase to promoter.
At other operons, CAP binds to a different
surface of the alpha subunit and increases
kf for isomerization from closed to open
complex.

CAP illustrates the fact that a single

protein can interact with RNA Pol via
different contact surfaces

The CTD of the alpha subunit of RNA

Pol can bind to UP sequences

UP sequences are
in the promoters of
genes for tRNAs
and rRNAs.
(-57) 5 AAAATTATTT 3 (-35)

The CTD of the alpha subunit of RNA

Pol can interact with activators
Class I promoters:
CAP binding sites
upstream of -35,
E.g. centered at -62,
-83, -93.
Class II promoters:
CAP binding sites
centered at -42,
Overlaps -35 box.

CTD of alpha subunit of RNA Pol has

multiple contact sites for activators

CAP has 2 activation regions

Interactions of CAP with alpha subunit of RNA

polymerase
AR1 (residues 156-164) of CAP:
At class I promoters, AR1 in the downstream subunit
"sees" residues 258-265 of CTD of alpha; increases KB .
AR2 (residues 19, 21, 96, 101)
At class II promoters, AR2 in the downstream
subunit "sees" alpha NTD residues 162-165,
increasing kf for isomerization from closed to open
complexes.

Bacteriophage lambda ()
Transcriptional switches can
regulate cellular decisions

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

Bacteriophage lambda can do either.

Infection by temperate phage leads to

lysis or lysogeny
E. coli cell

E. coli cell

E. coli chromosome

phage
+

cell undergoes lysogeny

lytic growth of phage

prophage

Temperate and lytic phage have a

different plaque morphology
Mutants of phage that
have lost the capacity to
lysogenize form clear
plaques

Temperate phage
generate turbid
plaques

lysogenized cells
lysed cells

lysed cells

uninfected cells

Lytic phage: clear plaques

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.

Induction and immunity of lysogens

A lysogen
Spontaneously,
1/1000 lysogens will
induce, i.e. the l
prophage will
excise, replicate and
lyse the cell.

+
UV treatment leads
to induction of
virtually all lysogens
in a culture.

Lysogens are immune to

further infection with similar
(lambdoid) phage

Regulatory mutants of lambda

Clear plaque mutants
Need wild type for lysogeny:
Establishment
Maintenance
cI
Yes
Yes

cII

Yes

No

cIII

Yes

No

Act in trans
Virulent mutants (vir)
Act in cis : are double mutants in oR &/or oL

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
N

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
+

CII

att

int

gam
red
xis cIII N

tint Pint

Int

CIII

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

 operators overlap promoters

oR :

oR3

oR2

oR1
PR
-35
TTGACT

-10
GATAAT

cro

N
TTAGAT 5
-10

ATAGAT 5
-35

PRM

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

Cro structure
Cro is a dimer
Monomer has 66 amino acids
Has only one protein domain
Does NOT display cooperativity
DNA binding; Helix-Turn-Helix motif; also dimerization

operator
oR3

Competition between repressor and Cro

for operator sites
PR
-35
OL1
N

-10

OL2

OL3

OR3
cI

-35
PL

-10

OR2

-10

cro

OR1

-35
PRM

OL1

OL2

OL3

OR3

OR2

OR1

Affinity for

High

High

Low

Low

High

High

Repressor

Low

Low

High

High

Low

Low

Cro

Use hybrid genes to dissect regulatory

schemes
Place a convenient reporter gene under
control of the regulatory elements being
studied
Use a known regulatory region to control the
trans-acting regulatory element

/lac hybrid genes

Place cI gene under lac control.

lac p, o

cI

Use lacZ as a reporter.

pR , OR

lacZ

321

Control amount of
repressor by [IPTG].

E. coli with lac repressor,

no lacZ.

See effect of repressor

by -galactosidase activity

 repressor will turn off expression from PR & PL

lac p, o

cI

pR , OR

-galactosidase

[IPTG]
repressor acts cooperatively.

lacZ

repressor

Mutation of oR1 decreases affinity for repressor

lac p, o

cI

pR , OR

lacZ
LOF mutation
at oR1

-galactosidase

repressor

[IPTG]

Repressor will stimulate transcription from PRM

lac p, o

cI

pRM , OR lacZ
123

-galactosidase

repressor

[IPTG]
repressor at oR1 and oR2 stimulates transcription from pRM.

Binding of repressor blocks transcription

from pR but activates pRM
PR
-35

-10

oR3

cro

RNA Pol
-10

2 dimers of
Repressor,
bound
cooperatively

oR2

oR1

-35

= operator

PRM
-35

-10

= promoter

Bacteriophage : Events leading to lysis

lysis or lysogeny (cI or Cro?) ?
Both lysis and lysogeny:
PR, PL, PR active : synthesize N, Cro
antitermination by N : synthesize cIII, cII, Q

Lysis:
Low [Cro] : binds OR3, shuts off PRM (cI)
High [Cro] : shuts off PR and PL
antitermination by Q + activation of PR by Cro

Bacteriophage : Events leading to lysogeny

lysis or lysogeny (cI or Cro?) ?
Lysis and lysogeny :
PR, PL, PR active : synthesize N, Cro
antitermination by N : synthesize cIII, cII, Q

Lysogeny:
cII stimulate expression from PRE (cI repressor)
and PINT (integrase)
cIII stabilizes cII
cI repressor shuts off PR, PL, PR (no lytic
functions), stimulates PRM

Factors favoring lysogeny cause increased

concentrations of repressor vs. Cro
High multiplicity of infection
More templates produce more of the CII protein, which
stimulates PRE.
Phage sense that it is too crowded.

Poor nutrient conditions for host

Low [glucose] leads to increase in [cAMP].
Increased [cAMP] will repress the host gene hflA.
Less HflA (a protease) leads to less degradation of the
CII protein.

Problems 4.35-4.39 provide a

quantitative approach to the
competition between Cro and
repressor for the operators.

Regulation after initiation

Antitermination of transcription:
Attenuation in biosynthetic
operons: trp

lac regulatory region

Activator binding site

Promoter

Operator

UV5 mutation, up TATAAT

-72

-52

TTTACA

TATGTT

-35

-10

cAMP-CAP

RNA polymerase

+1

'

+11

Repressor

The CTD of the alpha subunit of RNA

Pol can interact with activators

CTD

Class I promoters:
CAP binding sites
upstream of -35,
E.g. centered at -62,
-83, -93.
Class II promoters:
CAP binding sites
centered at -42,
Overlaps -35 box.

Binding of repressor blocks transcription

from pR but activates pRM
PR
-35

-10

oR3

cro

RNA Pol
-10

2 dimers of
Repressor,
bound
cooperatively

oR2

oR1

-35

= operator

PRM
-35

-10

= promoter

Antitermination occurs at two stages

in the life cycle

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
N

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

Review of -dependent termination

of transcription

Termination of transcription in E. coli:

Rho-dependent site
5' ...AUCGCUACCUCAUAUCCGCACCUCCUCAAACGCUACCUCGACCAGAAAGGCGUCUCUU

Termination occurs at one

of these 3 nucleotides.

Little sequence specificity: rich in C, poor in G.

Requires action of rho ( ) in vitro and in vivo.
Many (most?) genes in E. coli have rho-dependent
terminators.

Rho factor, or

Rho is a hexamer, subunit size is 46 kDa

Is an RNA-dependent ATPase
Is an essential gene in E. coli
Rho binds to protein-free RNA and moves
along it (tracks)
Upon reaching a paused RNA polymerase,
it causes the polymerase to dissociate and
unwinds the RNA-DNA duplex, using ATP
hydrolysis. This terminates transcription.

 hexamer binds to protein-free

RNA and moves along it.

Model
for
action
of rho
factor

-dependent site

'

RNA polymerase transcribes along the

template, and moves along the RNA.

RNA polymerase pauses at the

-dependent terminator site,
and catches up

Structure in RNA that causes pausing

unwinds the RNA-DNA hybrid
and transcription terminates

Components needed for antitermination

Sites on DNA
nut sites (N utilization sites) for N protein, qut sites for Q protein
Are found within the transcription unit
nut sites are 17 bp sequences with dyad symmetry

Proteins
Antiterminators: N protein and Q protein encoded by
Host proteins (encoded by E. coli)
Nus A (encoded by nusA, N-utilization substqance)
Rho protein

Arrangement of nut sites in

transcription units
delayed
early txn (N
present)
immediate
early txn

PPRR
tL1
... cIII

nutL
N

PL

PL
cI

immediate
early txn
delayed
early txn (N
present)

cro
nutR

cII
tR1

Q
tR2

...

Model for antitermination by N protein

core

nut
Will readthrough
rho-dependent
termination sites

RNA
polymerase
holoenzyme =
core + sigma

NusA

-dependent site

N plus
Nus
factors
block
rho
action

N NusA

RNA polymerase (with N and NusA )

transcribes along the template, and
moves along the RNA.

RNA polymerase does NOT pause at

the -dependent terminator site, and
never catches up

Transcription continues past the

terminator

NusG and elongation

NusG is another E. coli protein needed for lambda N to
prevent termination
Homolog of a family of proteins involved in elongation in
prokaryotes and eukaryotes
Eukaryotic DSIF
DRB-sensitivity inducing factor (Flies and mammals)
DRB is a drug that blocks transcriptional elongation

Two subunits
160 kDa, homolog to yeast Spt5
14 kDa, homolog to yeast Spt4

Implicated in positive and negative control of elongation

Regulation of E. coli trp operon by

attenuation of transcription

Organization of the E. coli trp operon

trpE

p,o

trpD

trpC

trpB

trpA

t t

leader
attenuator

Chorismic
acid

tryptophan

COOH
CH2

CH2
O
OH

C
COOH

CH

NH2

COOH

The trp operon is regulated in part by an

apo-repressor
p,o

p o

trpE

trpD

trpE

trpC

trpB

trpA

t t

Operon ON
p

trpE

+ trp
Apo-repressor

Repressor
(with trp bound)

Operon OFF

The trp operon is also regulated by attenuation

leader atten. trpE
p,o

RNA

27
AUG

trpD

trpC

54

70

UGGUGG
trp trp

Leader peptide:
14 amino acids,
2 are trp

trpB

90

UGA

trpA

t t

114 126

140

txn
pause

attenuator

Rho-independent terminator
of transcription.
Conditional :
Terminates in high [trp],
Allows readthrough in
low [trp]

Termination of transcription in E. coli:

Rho-independent site
AG U U
U
A
G
G
A
A
UG
GC
GC
C GA
C U
UA
UA
GC

G
CG
AU
AU
AU
GC
CG
CG
CG
UA
AU
A U U U U U ...3'
5' ... G C
A

G+C rich region in stem

Run of U's 3' to stem-loop

How attenuation works

The [trp] determines the [trp-tRNA].
The [trp-tRNA] determines whether a translating ribosome
will add trp to the leader peptide.
If trp is added:
The ribosome moves on to the translation stop codon.
This places the attenuator in a secondary structure that causes
termination of transcription (OFF).

If trp is not added:

A different secondary structure forms in the leader RNA
Allows readthrough transcription into the structural genes (ON).

Basic components for attenuation in trp

translation
[trp-tRNA] of trpL
High
complete
Low
stalls at
trp codons

secondary structures
formed in RNA
Attenuator Operon
3-4 stem
terminate txn OFF
2-3 stem
allow readON
through txn

Requirements for attenuation in trp

operon
Simultaneous transcription and translation.
A segment of RNA that can serve as a
terminator because of its base-paired
(secondary) structure.
An alternative secondary structure in the
RNA that does not allow termination of
transcription.
Does NOT need an additional protein, such
as a repressor.

Alternative base-paired structures in leader RNA

1

27

54

AUG

70

UGGUGG
trp trp

UGA

114 126

txn
pause

140

attenuator
3 4

2
3

Termination
of transcription

90

4
1

No termination

Progress of ribosome determines

secondary structure of trp leader RNA
attenuator
3

High [trp],
termination
of transcription

UGGUGG 2
1

Low [trp],
No termination

trp trp
ribosome
UGGUGG UGA
1

Examples of mutational analysis of trp

Translation of trp leader is needed for regulation
Mutation of AUG prevents transcription past the attenuator
Without translation, the 1:2 and 3:4 stem-loops form, and thus
causing termination

Specific secondary structures are needed

Mutations that decrease the number of base pairs in the 3:4 stemloop increase expression (less termination) in high [trp].
Compensatory mutations that restore the wild-type number of base
pairs allow termination in high [trp].

Many biosynthetic operons are

regulated by attenuation
Amino acid biosynthetic operons
E.g., his, phe, leu, thr, ilv
In each case, a short leader RNA and
polypeptide precede the structural genes.
This leader polypeptide is rich in the amino
acid that is the product of the pathway.

Regulation of eukaryotic genes

Gene silencing
Enhancers
Activators
Functional domains of activators

States of eukaryotic genes

Inactive:
Closed chromatin
Open chromatin, but repressors or lack of
activators prevent initiation
Open chromatin, transcription has initiated, but
polymerases will not elongate.

Active:
Open chromatin, basal transcription: requires
TATA + Inr
Open chromatin, activated transcription:
requires enhancer or upstream activator
sequences

Silencing Mechanism

Silencer
Cis-acting sequences that cause a
decrease in gene expression
Similar to enhancer but has an opposite
effect on gene expression
Gene repression - inactive chromatin
structure (heterochromatin)
Examples
Telomeric silencing
a or genes - silent loci of mating type
switching in yeast

Silencer binding proteins

Silencer binding protein serve as anchors
for expansion of repressed chromatin
Rap1 protein binds to silencer elements
SIR proteins (Silent Information Regulators)
Nucleates assembly of multi-protein
complex hypoacetylated N-terminal tails of
histones H3 and H4
Experiments: Condensed chromatin
Resistant to DNaseI digestion
Delete silencer - genes are derepressed

Gene Silencing

Silencing Mechanism

Enhancers
Cis-acting sequences that cause an
increase in expression of a gene
Act independently of position and
orientation with respect to the gene.
Can act to:
Increase the rate of initiation at a promoter
Increase the fraction of cells in which a
promoter is active

SV40 Control region

Origin of replication
Promoter and upstream activator sequences
for early transcription
Promoter for late transcription
Enhancer

Stimulation of transcription by enhancer is

independent of orientation and position
SV40:
Early

Late
wt

T-Ag

pos

T-Ag

orien

T-Ag

Enhancer

Enh-

T-Ag

Enhancer contains multiple binding sites

for transcriptional activators
SV40:

Early

Late

Enhancer

T-Ag

wt

A C B

high level

deletion

C B

low level

revertant

C C B

high level

An enhanson

Enhancers can occur in a variety of

positions with respect to genes
Enhancer
Upstream

Enhancer

P Transcription unit

Adjacent
Downstream
Internal
Distal

Ex1

Ex2

Activator proteins

Modular nature of activator proteins

DNA binding domain: recognition and
binding to specific DNA sequences
Multimerization domain: allows formation
of homo- or hetero-multimers
Activation domain:
Needed for increase in expression of
responding gene
Targets are still under investigation
General transcription factors
Histone modifying enzymes
Nucleosome remodeling complexes, etc

Modular structure of GAL4

1

98 148 196

768

881

DNA
binding

Activation

Dimerization

Activation
GAL80
binding

Induction by galactose exposes an

activation surface
In the presence of galactose, GAL4 activates several
genes whose products are required for galactose
metabolism.
GAL4 binds to a DNA sequence called UASG.
In the absence of galactose, GAL80 blocks GAL4
activation.
Binding of the sugar causes GAL80 to move.
This exposes the activation domain of GAL4.

Domain swap experiments show the

domains are interchangeable
Fuse an DNA-binding domain (DBD) from one transcription
factor to the activation domain (AD) of a different one.
DBD from LexA (E. coli)
AD from GAL4 (yeast)
Now a target gene can be placed under control of the DNA
binding site for the first factor
GAL1 gene with oLex (LexA binding sites) can be
activated by the fusion protein.
Basis for 2-hybrid screen for any interacting proteins

Two
Hybrid
Screens
(Interaction
Cloning)

DNA binding domains and

Activation domains
of transcription factors

A survey of DNA binding domains

Zn -containing domains
6 Cys and 2 Zn: Gal4
Zn fingers
Many eukaryotic transcription factors

Basic-leucine zipper proteins

(hetero)Dimers, eukaryotic activators

Helix-turn-helix
Many bacterial regulators, e.g. repressor
Homeodomain proteins involved in segment determination in
eukaryotes

Basic-helix-loop-helix proteins
(hetero)Dimers, differentiation factors

Zinc fingers
Cys or His amino acids donate electron pairs to a
tetrahedral configuration organized by a Zn++ ion
Several different types TFIIIA

C2H2 (e.g. TFIIIA)

C2C2 (e.g. Glucocorticoid receptor)
C6
GATA

Different functions
DNA binding
Protein-protein interactions

Each finger contacts 3 consecutive bp in major groove

C2H2 Zn finger

C2-C2 Zn Finger
Found in steroid receptors
Glucocorticoid receptor (p. 644)
Three functions in central domain
DNA binding (Zn finger)
Dimerization (Zn finger)
Activation domain
C terminus binds steroid hormone
N terminus activates transcription
N

C
403

491

777

Basic-leucine zipper proteins

View down long axis of DNA

Lateral view of DNA

S. Harrison lab: cFos-cJun heterodimer, DBD, 2 complexes

Nature 1995 Jan 19;373(6511):257-61

Images via NCBI and their Cn3D program.

Helix-turn-helix (HTH)

View Chime tutorial of lambda

repressor-operator co-crystals

Helix-loop-helix proteins
Ma PC, Rould
MA, Weintraub
H, Pabo CO:

MyoD bHLH
domainDNA
complex
Cell 1994 May
6;77(3):451-9

Use Dr. T. Nixons Chime tutorials

http://www.bmb.psu.edu/pugh/514
/mdls/default.htm

Transcriptional activator domains

(ADs)

3 general types of activator domains

Acidic
Amphipathic helix, acidic amino acids on one
face
No consistent secondary or tertiary structure
has been identified

Glutamine-rich (Q-rich)
Pro-rich (P-rich)

No correspondence between type of

DBD and type of AD
Examples of proteins with acidic AD
GAL4 (Zn2Cys6)
AP1 (bZIP)
VP16 (no DBD)
repressor (HTH)

Examples of proteins with Q-rich AD

Sp1 (Zn finger)
Antp (homeodomain)
Oct (POU-homeo)

Lack of fixed structure in activator domains

DBDs of transcription factors form discrete structures that
can be analyzed by X-ray crystallography and NMR
The ADs do not generate identifiable electron density in
the crystallographic analysis.
This indicates that they do not form discrete structures.
One hypothesis is that the ADs are unstructured until they
interact with their targets.
This is an induced fit model.

Mechanism of activation

3 general types of activator domains

Acidic
Amphipathic helix, acidic amino acids on one
face
No consistent secondary or tertiary structure
has been identified

Glutamine-rich (Q-rich)
Pro-rich (P-rich)

No correspondence between type of

DBD and type of AD
Examples of proteins with acidic AD
GAL4 (Zn2Cys6)
AP1 (bZIP)
VP16 (no DBD)
repressor (HTH)

Examples of proteins with Q-rich AD

Sp1 (Zn finger)
Antp (homeodomain)
Oct (POU-homeo)

Lack of fixed structure in activator domains

DBDs of transcription factors form discrete structures that
can be analyzed by X-ray crystallography and NMR
The ADs do not generate identifiable electron density in
the crystallographic analysis.
This indicates that they do not form discrete structures.
One hypothesis is that the ADs are unstructured until they
interact with their targets.
This is an induced fit model.

Models for mechansim of activation

Direct contact between an activator and RNA polymerase
or GTF
Indirect interactions

Adaptor
Mediator
Histone modifier complexes
Nucleosome remodelers

No contact between enhancer bound proteins and the

target promoter
Open a chromatin domainbut not target a promoter
Linking via enhancerfacilitators

Direct contact in activation

Demonstrated in bacteria and yeast (some
genes)
Upstream activation sequences are adjacent
to minimal promoters
Examples
lambda repressor activates RNA polymerase at
PRM.
cAMP-CAP activates RNA polymerase at lac.
Direct contact between cAMP-CAP and the Ctreminal domain of the alpha subunit of RNA
polymerase

Suppression is strong evidence for direct

contact
Hypothesis: an AD makes direct contact with a component
of the transcriptional apparatus
Prediction: LOF mutations in the activation domain should
be suppressed by appropriate mutations in that
component.
E.g. mutations in CAP can be suppressed by mutation in
the subunit of RNA Pol.

How do distal enhancers work?

Does activation require communication

between an enhancer and a promoter?
If so, expect
An effect on rate of transcription
Specific binding between activator or coactivator and the transcription complex
Mutations in target of binding should abolish
activation
Find targets in suppressor screens

If so, is it by looping vs. tracking?

Direct interaction?
Interact via another component?
Tracking?

Some enhancers increase the rate of

transcription initiation
With Enhancer

Enh

gene
promoter

Without Enhancer

polymerase &
transcript

Polymerase
density and
amount of
transcription
increases in
all cells in a
population

Looping vs. tracking

Communication between enhancer and
promoter can be via direct contact
Contact between proteins bound to adjacent
sites
Contact between proteins at distal sites, with
DNA between them looped out.

Communication can be via tracking

Some component(s) of the transcriptional
apparatus enter the chromosome at an
enhancer and move along (track) until they act
at a distal promoter.

Direct contact for activation

Enhancer

IID

Pol IIa

PolII

Promoter
GTIF?

Coactivator?

Tracking for activation

Enhancer

PolII
IID

Promoter

Interactions may be facilitated by DNAbending proteins

Many proteins that bind in the minor groove
of DNA also bend the DNA.
TBP, YY1, HMG I(Y)

interferon- gene enhancer

binding sites for 3 conventional txn factors
binding sites for HMG I(Y)
requires binding and bending of DNA by
HMGI(Y) for activation by the other proteins
bound to the enhancer.

Can activation occur without

communication between an enhancer
and a promoter?
If so, expect no specific binding between
activator and the transcription complex
Possible models:
Open a chromatin domain so that it is more
likely to be expressed
Affect probability that gene is in a
transcriptionally competent region

Some enhancers increase the probability that a

gene is in a transcriptionally competent region
With enhancer
Enh

Enh

Enh

Enh

Without enhancer

Increase in fraction
of cells expressing
the reporter gene.

Amount of
expression per
expressing cell is
same with and
without enhancer.

Communication or not?
An increase in rate of initiation by an enhancer can be
explained by some kind of communication between the
enhancer and the promoter
Direct or indirect?

An increase in the probability that a gene is in a

transcriptionally competent region does not require
communication between the promoter and the enhancer.
It could be exerted by making the chromatin structure in that
domain accessible to transcription factors in a greater fraction of
cells.

Experiments to look for targets of

activators

What proteins bind to the activation

domain?
Use affinity chromatography, with AD as
the ligand
Determine the nuclear proteins that bind
specifically to that activation domain.
Find that some GTFs, especially TAFs, bind
to either acidic or Q-rich ADs
E.g. the acidic AD of VP16 will bind to TBP,
TAFII40 and TFIIB
Q-rich AD of Sp1 binds TAFII130

GTFs for RNA polymerase II

Modulates helicase
IIA

IIB

TAFs
TBP

IIE

IIH

Targets Pol II
to promoter

IIA

protein kinase
CTD protein
kinase

IIF

IIE
IIB

IIF

TFIID

Recognize core promoter

Helicase
helicase

Pol IIa
Inr

IIH

TBP
CTD of large subunit of Pol II

Many GTFs are possible targets for activators of transcription.

Are TAFs required for transcriptional activation?

Construct conditional (ts) loss-of-function (LOF) alleles in
genes for TAFs in yeast.
Examine the level of expression of various target genes
before and after temperature shift (active vs. inactive TAF).
See that many genes are still activated in the absence of
TAF function!
TAFS are not required for all activation.
TAFs are important - LOF alleles are lethal. Other
functions include cell cycle progression.

Co-regulators
Some sequence-specific activators (or
repressors) do not regulate transcription by
themselves.
Sp1 + TBP + RNA Pol II + other GTFs +
promoter DNA: only basal transcription

Co-activators and co-repressors are also

needed
Sp1 + TBP + TAFS + RNA Pol II + other GTFs
+ promoter DNA: get activated transcription

Activators and Co-rgulators

Lemon
& Tjian
(2000)
Genes
&
Devel.
14:2551

Mediator is a co-activator
-30

+1

TATA

Inr

TBP

Yeast RNA Pol II does not respond

} to
activators, but the RNA Pol II holoenzyme
does respond to activators
TAFs

or

TFIID

TBP

Inr

TATA

Mediator
(SRBs,
Rgr1, Gal11, Med 1, 2, 6, 7,
IIA ?
SRB
IIF
IIE
etc) is a type of co-activator
Holoenzyme
IIB

Pol IIa

IIH

activator
?

SRB

IIF

IIE
IIB
TATA

IIA

preinitiation complex
Pol IIa
Inr

IIH

ATP hydrolysis

Some co-regulators work on chromatin

Transcriptional activation in vitro from some
promoters requires a chromatin template
Some co-activators and co-repressors
covalently modify histones and transctipion
factors:
Acetyl transferases
Deacetylases
Kinases, Methylases, ADP-ribosyltransferases

Some co-activators use ATP hydrolysis to

modify nucleosomes
SWI/SNF, ISWI, etc

Classes of co-regulators
Class I: activator and repressor targets in polymerase and
GTFs
TAFs, TFII

Adapters that bind to the activators

VP16, OCA-B

Mediator
SRBs, etc. 3 in mammals: CRSP, SRC, NAT

Complexes that covalently modify nucleosomal histones

and transcription factors
HATS, HDACs, kinases, methyl transferases, etc

Complexes that remodel chromatin in an ATP-dependent

manner: SWI/SNF

Regulating the regulators

Regulation of activator proteins

Tissue-specific synthesis of activator
proteins
Covalent modification
Phosphorylation of HSTF will activate it.
Phosphorylation of AP1 at some sites will
activate it, at other sites will inhibit it.

Active form of the transcription factor can be

imported to the nucleus after dissociation of
an inhibitor in cytoplasm
Exchange heterodimeric partners

Example of steroid-hormone receptors

In the absence of ligand (steroid), the receptor is
in an inactive form in the cytoplasm.
Complexed with Hsp90 and other proteins.

When steroid binds, Hsp90 dissociates, and the

hormone-receptor complex is imported into the
nucleus.
In the nucleus, the hormone-receptor complex
associates with an additional protein, binds to
specific sites and activates target genes.

Regulation by changes in
chromatin structure
Active chromatin

Chromatin Structure

Principal proteins in chromatin are

histones
H3 and H4 : Arg rich, mostly conserved sequence
H2A and H2B : Slightly Lys rich, fairly conserved
H1 : very Lys rich, most variable in sequence
between species

Histone structure and function

Histone structure and function
"Minimal" structure for a core histone, e.g. H4. Others have one additional alpha helix.

1
N

K5 K8 K12 K16

Highly charged
N-terminal tail.

L1

L2

3
C

Globular, hydrophobic domain for histone-histone

interactions and for histone-DNA interactions.

Histone interactions via the histone fold

The alpha-helical regions of the core histones mediate dimerization.
N
L2
L1

L2

3
2

C
L1
L1

N
The histone fold flanked
by N and C terminal tails.

L2

N
Dimer of histones joined by interactions at the
histone fold.

Nucleosomes are the subunits of the

chromatin fiber
Experimental evidence:
Beads on a string in EM
Micrococcal nuclease digestion
Nuclei

Chromatin

Micrococcal
nuclease

View in electron
microscope
Measure size on gels
ca. 140 bp, 280, 420 bp

Nucleosome components
Nucleosome core + histone H1 (in higher eukaryotes) +
linker DNA (0-50bp)
The nucleosome core contains
an octamer of 2 each of the core histones (H2A, H2B,
H3 and H4) and
146 bp of DNA wrapped 1.75 turns.
Core histones dimerize through their histone fold motifs
generating H3/H4 dimers and H2A H2B dimers
Each histone pair bends approximately 30bp of DNA
around the histone octamer.

General model for the nucleosomal

core

A string of nucleosomes

H3-H4 dimer bound to DNA

Nucleosome core particle

Side view of nucleosome

Chromatin higher order structure

Arrays of nucleosomes condense into higher order
chromatin fibers.
Despite over 2 decades of investigation the
structure of the 30nm chromatin fiber is not
known.
This may be due to irregularity or instability of the
structure.
This level of structure has been implicated in
mechanisms of chromatin repression, thus, the
lack of structural information at this level is
particularly troublesome.

Higher order chromatin structure

Histone H1 associates
with the linker DNA,
and may play a role in
forming higher order
structures.

Solenoid model for 30 nm chromatin fiber

Solenoid of nucleosomes

Path of DNA between

nucleosomes is unknown

Transcriptionally active chromatin is

more open
Direct assays show that it is more
accessible to DNases.
We infer that it is more accessible to
components of the transcriptional apparatus.
This inference is now being verified by in vitro
experiments.

Classical evidence that chromatin structure

can regulate genes
Radiolabeled UTP is incorporated into RNA in regions of
euchromatin, not heterochromatin
Cells that are actively expressing their genes have larger
nuclei than do quiescent cells.
Activation of particular sets of genes in Drosophila
generates visible puffs at defined loci on the polytene
chromosomes.
Lampbrush chromosomes show transcription in the more
extended, open regions of the chromosomes.

Condensed chromatin is
transcriptionally inactive
Metaphase (mitotic) chromosome

heterochromatin
Interphase nucleus

nucleolus

euchromatin
nuclear membrane

Coil of the solenoid

Transcriptionally
inactive;
nuclease
insensitive

Heterochromatin is not transcribed

Position effect variegation
Wild-type w+ gene produces red eyes in
Drosophila when it is at its normal location.
Movement of the w+ gene close to the centromere
causes it to not be expressed in some of the
sections (ommatidia) of the eyes, generating white
patches.
This variegation in the pattern of expression is
explained by whether the w+ gene is in
heterochromatin (OFF) or euchromatin (ON).

Silenced chromatin at telomeres

More open chromatin can be

transcriptionally active
Coil of the solenoid

Compact solenoid with

6 nucleosomes per turn.
Is this the 30 nm fiber?

Less compact solenoid

+H1

-H1

String of nucleosomes
with exposed linker
region. 10 nm fiber.

Transcriptionally
inactive;
nuclease
insensitive

??

Direct measurement of accessibility

of chromatin
Nuclease sensitivity

Nuclease sensitivity assays

The overall sensitivity of a gene to DNase I
is increased about 3 to 10 fold when it is
expressed.
Can measure this by
Isolating nuclei from cells expressing or not
expressing the gene.
Digest nuclei (chromatin) with DNase I
Measure how much DNA from that gene
survives nuclease treatment.

DNAse I digestion of nuclei reduces the

concentration of actively transcribed DNA
Hybridize chick beta-globin
probes to:

Total DNA,
DNA from erythroid
nuclei digested with
micrococcal
nuclease

Hybridize chick ovalbumin

probe to DNA from
erythroid nuclei
digested with micrococcal
nuclease or DNase I

DNA from
erythroid nuclei
digested with
DNase I

Stalder et al. (1980) Cell 20:451-460, Fig. 5

Map the extent of the region around a

gene that is accessible to nucleases
Combine nuclease treatment of chromatin
with restriction digestion
Assay by blot-hybridization

DNAse I digestion of nuclei preferentially

cuts restriction endonuclease fragments
containing actively transcribed DNA
Lanes 1 and 3: nuclei digested
with DNase I
Lane 2: not digested
Lanes 1 and 2: nuclei from
erythroid cells
Lane 3: nuclei from lymphoid
cell line.
DNA from nuclei was digested
with BamHI, run on gel
and hybridized with chick alphaglobin (left) or ovalbumin
(right) probes.
Stalder et al. (1980) Cell 20:451-460, Fig. 2

Domains as loops
relaxed

Condensed/
constrained

"Interpretation of moderate sensitivity to DNAase I

in terms of "lampbrush chromosome-like loops or
domains."
Stalder et al., 1980, Cell 20:451

Map DNase hypersensitive sites = HSs

Use indirect end-labeling to find the sites
of discrete, double-strand breaks caused
by nuclease digestion of chromatin.
These correspond to discrete regions of
substantially altered chromatin structure
In some cases they lack nucleosomes

Landmarks to functional sites on the DNA

Sites for binding of other proteins
Transcriptional activators at enhancers
Replication proteins at origins

Indirect end-labeling to see DNAse HSs in

gamma globin genes
Nuclei from human fetal
erythroblasts were
digested with DNase I.
DNA was purified,
digested with the indicated
restriction endonuclease,
run on a gel and blotted. A
fragment from the gammaglobin gene was used as a
hybridization probe.
DNase HSs are revealed as
new fragments smaller
than the parental bands.

Groudine et al. (1983) PNAS 80:7551-7555.

Example of indirect end-labeling to see

multiple HSs
K562 nuclei:
HS4
HS3.5
HS3

DNase

time

0 0 HS4 HS3.5 HS3

7.8 kb
6.6 kb
4.0 kb
3.1 kb

probe
H. Petrykowska

Features of active chromatin

Accessible to nucleases
DNA is less methylated
Less histone H1
Core histones are acetylated at discrete
sites
Presence of nonhistone proteins HMG14
and HMG17
Nucleosome phasing

Biochemically defined domain can correspond

to a set of coordinately expressed genes
Chicken HBB

HSA

FOLR

DNase Sensitive

Histone Acn
Histone H1
DNA methylation
Expressed:

Enh
HSs
H A

4 3 2 1

Progenitors

+
+

+
+
Maturing erythroblasts

ORG

HS4 from chick HBB complex

Marks a boundary in chromatin: open to closed
Acts as an insulator: Blocks activation of promoter by an
enhancer
InsuPr neoR lator Enhancer

Silencer

Neo-resistant colonies
% of maximum
10
50
100

Cis-regulatory elements that act in chromatin

Generate an open, accessible chromatin
structure
Can extend over about hundreds of kb
Can be tissue specific

Enhance expression of individual genes

Can be tissue specific
Can function at specific stages of development.

Insulate genes from position effects.

Enhancer blocking assay

Human -globin gene cluster

0

20

40

60

80 kb

DNase HSs

Domain opening?

G A

LCR

Embryonic Fetal >

Adult
Embryonic
Locus control
Locus region:
Control Region is needed to:
openglobin
a chromatin
in erythroid
cells cells.
Activate linked
gene domain
expression
in erythroid
express of linked globin genes at a high level
Overcome position
effects effects
at many
integrationmice
sites
override position
in transgenic

in transgenic mice.
Role in switching expression?

Yes

HBB LCR will activate expression at many

chromosomal locations
Expressed in
red cells

DNase HSs

G A

Hispanic
() thalassemia

G A

Erythroid
Chromatin

LCR

Developmental Position
Regulation
Effects

Yes

Yes

No

Open

No

No

Yes?

Closed

In transgenic mice:

Sometimes

Yes

Yes

Sometimes
open

Yes

Precocious
expression

No

Open

Yes

Yes

Yes

Open at
some sites

LCR

G A

Domain
opening is
associated
with
movement
to nonheterochromatic
regions

Domain opening and gene activation are

separable events
wildtype
N-MEL
ORGs

LCR
HSs

Location,
DNase heterosensi- chromtive
atin

Human
HBB
complex

Del. HS2-HS5

General
histone H3
hyper- hyper
Acn
Acn Txn

away

away

close

T-MEL, Hisp. del.

Reik et al. (1988) Mol. Cell. Biol. 18:5992-6000.

Schbeler et al. (2000) Genes & Devel. 14:940- 950

Proposed sequence for activation

1. Open a chromatin domain
Relocate away from pericentromeric
heterochromatin
Establish a locus-wide open chromatin
configuration
General histone hyperacetylation
DNase I sensitivity

2. Activate transcription
Local hyperacetylation of histone H3
Promoter activation to initiate and elongate
transcription

Regulation by changes in histones,

nucleosomes and chromatin
Opening and activation
Movement from heterochromatin to euchromatin
Nucleosomes and transcription factors
Chromatin remodeling activities
Histone acetyl transferases and deacetylases
Thanks: Dr. Jerry Workman

Human -globin gene cluster

0

20

40

60

80 kb

DNase HSs

Domain opening?

G A

LCR

Embryonic Fetal >

Adult
Embryonic
Locus control
Locus region:
Control Region is needed to:
openglobin
a chromatin
in erythroid
cells cells.
Activate linked
gene domain
expression
in erythroid
express of linked globin genes at a high level
Overcome position
effects effects
at many
integrationmice
sites
override position
in transgenic

in transgenic mice.
Role in switching expression?

Yes

Domain opening and gene activation are

separable events
wildtype
N-MEL
ORGs

LCR
HSs

Location,
DNase heterosensi- chromtive
atin

Human
HBB
complex

Del. HS2-HS5

General
histone H3
hyper- hyper
Acn
Acn Txn

away

away

close

T-MEL, Hisp. del.

Reik et al. (1988) Mol. Cell. Biol. 18:5992-6000.

Schbeler et al. (2000) Genes & Devel. 14:940- 950

Chromosome localization in interphase

In interphase, chromosomes appear
to be localized to a sub-region of the
nucleus.

Gene activation and location in the nucleus

Condensed chromatin tends to localize
close to the centromeres
Pericentromeric heterochromatin

Movement of genes during activation and

silencing
High resolution in situ hybridization
Active genes found away from pericentromeric
heterochromatin
Silenced genes found associated with
pericentromeric heterochromatin

Domain
opening is
associated
with
movement
to nonheterochromatic
regions

Proposed sequence for activation

1. Open a chromatin domain
Relocate away from pericentromeric
heterochromatin
Establish a locus-wide open chromatin
configuration
General histone hyperacetylation
DNase I sensitivity

2. Activate transcription
Local hyperacetylation of histone H3
Promoter activation to initiate and elongate
transcription

A scenario for transitions from

silenced to open to actively
transcribed chromatin

From
silenced to
open
chromatin

Movement from hetero- to euchromatin

Nucleosome
remodelers
and HATs
further open
chromatin

Assembly of
preinitiation
complex on
open
chromatin

Transcription factor binding to DNA is

inhibited within nucleosomes
Affinity of transcription factor for its binding site on DNA is
decreased when the DNA is reconstituted into
nucleosomes
Extent of inhibition is dependent on:
Location of the binding site within the nucleosome.
binding sites at the edge are more accessible than
the center
The type of DNA binding domain.
Zn fingers bind more easily than bHLH domains.

Stimulate binding of transcription

factors to nucleosomes
Cooperative binding of multiple factors.
The presence of histone chaperone
proteins which can compete H2A/H2B
dimers from the octamer.
Acetylation of the N-terminal tails of the
core histones
Nucleosome disruption by ATP-dependent
remodeling complexes.

Binding of transcription factors can

destabilize nucleosomes
Destabilize histone/DNA interactions.
Bound transcription factors can thus participate in
nucleosome displacement and/or rearrangement.
Provides sequence specificity to the formation of
DNAse hypersensitive sites.
DNAse hypersensitive sites may be
nucleosome free regions or
factor bound, remodeled nucleosomes which have an
increased accessibility to nucleases.

Nucleosome remodeling

Chromatin remodeling ATPases are large

complexes of multiple proteins
Yeast SWI/SNF
10 proteins
Needed for expression of genes involved in mating-type switching
and sucrose metabolism (sucrose non-fermenting).
Some suppressors of swi or snf mutants are mutations in genes
encoding histones.
SWI/SNF complex interacts with chromatin to activate a subset of
yeast genes.
Is an ATPase

Mammalian homologs: hSWI/SNF

ATPase is BRG1, related to Drosophila Brahma

Other remodeling ATPase have been discovered.

Chromatin remodeling ATPases catalyze

stable alteration of the nucleosome

II: form a stably remodeled dimer, altered DNAse digestion pattern

III: transfer a histone octamer to a different DNA fragment

Covalent modification of histones in

chromatin

Histones are acetylated and deacetylated

+

Histone acetyl

NH 3
CH 2
CH 2
AcCoA
O
CH 2 O
CH 2
... NH CH C NH CH C ...
2
Gly
Lys
Ac
Positive charge on amino group

CH 3
transferases
O C
NH
CH 2
CH 2
CoA
O
CH 2 O
CH 2
... NH CH C NH CH C ...
2

Histone deacetylases

No charge on amide group

Covalent modification of histone tails

N-ARTKQTARKSTGGKAPRKQLATKAARKSAP...- H3
4

9 10

14

23

18

27 28

N-SGRGKGGKGLGKGGAKRHRKVLRDNIQGIT...- H4
1

phosphorylation

12

16

20

acetylation

methylation

Two types of Histone

Acetyltransferases (HATs).
Type A nuclear HATs: acetylate histones in
chromatin.
Type B cytoplasmic HATs: acetylate free
histones prior to their assembly into
chromatin.
Acetylate K5 and K12 in histone H4

Acetylation by nuclear HATs is associated

with transcriptional activation

Highly acetylated histones are associated with actively transcribed

chromatin
Increasing histone acetylation can turn on some genes.
Immunoprecipitation of DNA cross-linked to chromatin with antibodies
against Ac-histones enriches for actively transcribed genes.

Acetylation of histone N-terminal tails affects the ability of nucleosomes

to associate in higher-order structures
The acetylated chromatin is more open
DNase sensitive
accessible to transcription factors and polymerases

HATs are implicated as co-activators of genes in chromatin, and

HDACs (histone deacetylases) are implicated as co-repressors

Nuclear HAT As are coactivators

Gcn5p is a transcriptional activator of many genes in
yeast. It is also a HAT.
PCAF (P300/CBP associated factor) is a HAT and is
homologous to yeast Gcn5p.
P300 and CBP are similar proteins that interact with many
transcription factors (e.g. CREB, AP1 and MyoD).
P300/CBP are needed for activation by these factors, and
thus are considered coactivators.
P300/CBP has intrinsic HAT activity as well as binding to
the HAT PCAF.

HAT complexes often contain several

trancription regulatory proteins.

Example of the SAGA complex components:

Gcn5: catalytic subunit, histone acetyl transferase
Ada proteins
transcription adaptor proteins required for function of some
activators in yeast.
Spt proteins (TBP-group)
regulate function of the TATA-binding protein.
TAF proteins
associate with TBP and also regulate its function.
Tra1
homologue of a human protein involved in cellular transformation.
May be direct target of activator proteins.

Yeast SAGA interacting with chromatin

SAGA Complex

TAF90p

Tra1p

TAF25/23p

Ada3p

Spt7p

Ada1p

TAF68/61p
TAF60p

Spt20/
Ada5
p
Gcn5p
HAT

Ada2p

Act.

TAF20/17p

Spt8p

Spt3p

TBP
Ac

Ac

Ac

Ac
Ac

Ac
Ac

Ac

Roles of histone acetylation

Increase access of transcription factors to
DNA in nucleosomes.
Decondense 30nm chromatin fibers
Serve as markers for binding of non-histone
proteins (e.g. bromodomain proteins).

Histone deacetylases are associated

with transcriptional repression
A mammalian histone deacetylase:

HD1
RbAp48
Histone deacetylases:
Are recruited by inhibitors of transcription.
Are inhibited by trichostatin and butyrate.

Repression by deacetylation of histones

Methylated DNA can recruit HDACs

Connections in eukaryotic
transcriptional activation

Transcriptional activators
Coactivators
Nucleosome remodeling
Histone modification
Interphase nuclear localization

The functions of SWI/SNF and the

SAGA complex are genetically linked.
Some genes require both complexes for
activation.
Other genes require one or the other complex.
Many genes require neither - presumably utilize
different ATP-dependent complexes and/or HATs

The yeast HO endonuclease gene

requires both SWI/SNF and SAGA
The order of recruitment at the promoter:
1. SWI5 activator: sequence recognition
2. SWI/SNF complex: remodel nucleosomes
3. SAGA: acetylate histones
4. SBF activator (still at specific sequences)
5. general transcription factors
Cosma, Tanaka and Nasmyth (1999) Cell 97:299311.

The order is likely to differ at different genes