Vol. 62, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A COMPARISON OF LAC REPRESSOR BINDING TO OPERATOR AND TO NONOPERATOR DNA Syr-yaung Lin and Arthur D. Riggs City of Hope National Medical Center Duarte, California 91010 Received

December

12,1974

SUMMARY. We have compared the operator and nonoperator DNA bind, mg activities of the lac repressor with respect to inactivation or inhibition by trypsin, heat, actinomycin, and isopropylthiogalactoside. The two DNA binding activities were found to differ only in their sensitivity to the inducing ligand isopropylthiogalactoside. Repressor binding to poly(dT-dT-dG).poly(dC-dA-dA) was shown not to be affected by isopropylthiogalactoside. INTRODUCTION. The first is

is

strong

relatively

purpose

of this

of

of

the

Until

recently,

DNA could

not the

repressor

that

the ator

covalent

assays

report

here similar

directly

of that in

their

(4,9)

of repressor

binding

activity

operator

and nonoperator

sensitivity

to trypsin,

Copyrikh t o 19 75 by A eademie Press, Inc. AN rights of reproduction in any form reserved.

704

as-

kinetics

assays filter

re-

any treatment

activity

photochemical

vivo

filter

but

to both

in

of

to nonoperator

efficient

complexes

the

certain

of

rendered

Fortunately

impossible.

second

is

fast

repressor

binding

[BrdU]DNA-repressor

both

lac

done

operator

nonoperator

the

explaining

weak to cause

assayswere

this

for

by nitrocellulose

was too

By using

It

paradoxically

of

attachment

DNA (10).

direct

are

of

and for

the

binding

the

The binding

relevant

(3,5,7,8).

competition

UV irradiation

(5,6)

(l-3);

activities.

such as the

inhibited

of

these

activities.

DNA (4,5).

to operator

interaction

Competition

measurement

operon

be studied

tention.

operator

DNA is highly

studies,

binding

lac

to nonoperator

to compare

lac

in vitro

repressor

because

paper

has two DNA binding

to the

binding

to nonoperator

regulation pacts

repressor

binding

weak

repressor

of

The lac

the

we found in

low salt

that causes

operator

and nonoper-

attachment

procedure,

can be done binding heat,and

and we

activities actinomycin.

BIOCHEMICAL

Vd. 62, No. 3,1975

but

they

differ

MATERIALS strain

of

response

M96 according

repressor 32P]DNA

BrdU-substituted)

band

and all

has been

(IPTG)

(isuperq)

procedures

in a glycerol

and wild

RESEARCH COMMUNICATIONS

isopropylthiogalactoside

to published

gave a single

as lac

hh80dlac[

to

Lac repressor

AND METHODS.

trophoresis mented

in

AND BIOPHYSICAL

was prepared SDS-gel

(10).

DNA binding

Ah80[32P]DNA

described

(lo),

.

from elec-

sedi-

The preparation

gradient.

type

activity

t

(both

normal

and

Poly[d(TTG).d(CAA)

1

O 06aT 0 2

04-

u 02-

I 5

0

I 10

I 15

0 20

MINUTES

Figure 1. Inactivation of operator, nonoperator, and IPTG binding activities of lac repressor by trypsin. Lac repressor (930 pg/ml protein) was treated with trypsin (0.14 pg/ml) at 37O C as in reference 11. At the indicated times, aliquots of the treated repressor were removed and trypsin action stopped by phenylmethylsulfonyl fluoride and bovine serum albumin as in Platt, et al. (11). Operator, nonoperator, and IPTG binding activities were measured as described in Materials and Methods. For operator binding, LO-u1 aliquots were diluted 30 times and then 20 ul added to 0.5 ml of buffer I containing 50 ng of Xh80dlac[32P]DNA. For nonoperator DNA binding, 50 ~1 of the diluted repressor was added to 0.5 ml of buffer I containing 50 ng of BrdU-substituted hh80 wild type [32P] DNA. For both operator and nonoperator DNA binding, the concentration of repressor was chosen, on the basis of complete binding curves, to be 20 times that needed to give half maximum filter retention. For IPTG binding, 40 ~1 of the treated repressor was added to 140 ~1 of buffer I containing 0.26 M KC1 and 4.6 X 10m6M of ["C]IPTG. The data were normalized by CPM,,, the filter-bound counts obtained with no trypsin treatment. A-A, [14C]IPTG; O-O, Xh80dlac[32P]DNA: O-O, BrdU-substituted Xh80 wild type L3*P]DNA. t Abbreviation:

IPTG,

isopropyl-1-thio-8-D-galactoside.

705

Vol. 62, No. 3, 1975

BIOCHEMICAL

0

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5

IO

A

A

!, 15

R 20

MINUTES

Figure 2. Heat inactivation of lac repressor activities. 35Slabeled lac repressor (49 pg/ml protein, 1500 cpm/ug) was heated at 60' C in O.OlM Tris HCl, pH 7.4. Aliquots were taken at various times, diluted in buffer I, and operator and nonoperator DNA binding activities measured. For operator binding assays, 33 ng of repressor protein was added to 50 ng of hh8Odlac[ 32PlDNA in 0.52 ml of buffer I. For nonoperator binding assays, 85 ng of repressor protein was added to 50 ng of BrdU-substituted Xh80 wild type t3'PlDNA in 0.55 ml of buffer I. The 35S counts in the repressor were sufficiently low as not to interfere with the DNA binding assays. [35Slrepressor was used for the control to show that heat treatment was not reducing the retention of repressor protein by nitrocellulose filters. e-e, Xh80dlac[32P]DNA; O-O, BrdU-Xh80 wild type [32P]DNA; A-A, heat treated [ 35S]repressor mixed with Xh8Odlac DNA and filtered.

was a gift trypsin

from

were

repressor

from

Merck

was treated

Operator standard used.

R. D. Wells

and Sigma with

The binding

of

DNA (BrdU-Xh80[32PlDNA)was

from

the

RESULTS. the

lac

(10). lamp. Platt, repressor

UV irradiation Buffer et

destroys

et al.

were measured

(12,13)

by the

Lac

except

that

(11). by

O" C was

nonoperator

photochemical

attachment

was at 0" C and at 9 cm distance

I has been al.

to Platt,

to BrdU-substituted

measured

D and

respectively.

activities

assays

repressor

Actinomycin

according

binding

filter

9).

Companies

trypsin

and [ 14ClIPTG

nitrocellulose

technique

(see ref.

(11)

have

described

(10).

shown that

trypsin

operator

706

binding

activity

treatment much more

of

BIOCHEMICAL

Vol. 62, No. 3, 1975

AND BIOPHYSICAL

Actinomycln

Cl (~10~

RESEARCH COMMUNICATIONS

M)

inhibition of operator and nonoperator DNA Figure 3. Actinomycin binding activities. Repressor (23 ng protein) was mixed with 25 ng of BrdU-DNA (either wild type or dlac) in 0.5 ml of buffer I binding and the indicated concentration of actinomycin D. Operator and nonoperator binding were then ascertained as in Materials and O-O, BrdU-subMethods. O-O, BrdU-substituted XhB0dlac132PlDNA. stituted Xh80 wild type [32P1DNA.

rapidly

than

confirm

these

activates

IPTG

binding

observations.

operator

rates.

Figure

vities

are

operator ing bone)

and not

bition

studies

differential operator

operator

in

at the

the

inhibit

minor

sequence

repressor

binding

only

sensitive with

to

response. and nonoperator

activities

at

(14).

might

We report

here

binding

activities 707

that

acti-

operator

and actinomycin (12). than

with

have

similar

binding

The lac

to operator

in the

in-

of double-stranded

mechanism

changes

1

treatment.

site,

interactions

actinomycin

trypsin

groove

binding

figure

shows that

by heat

dG-dC

were by a different

involving

shown in

and nonoperator rates

has one actinomycin

binding

(perhaps

binding

at similar

D binds

DNA preferentially

is known to

1 also

and nonoperator

inactivated

(15)

The data

Figure

2 shows that

Actinomycin

sequence

activity.

the

minor

If

operator phosphate

groove,

then

nonbindbackinhi-

shown an interesting such is not

the

are

sensitive

equally

case:

Vol. 62, No. 3, 1975

BIOCHEMICAL

0.8 \

RESEARCH COMMUNICATIONS

0 \ 8

r"O.69

\ j$

AND BIOPHYSICAL

0.4-

0 l

A

l

c,

A \

0.2 -

r--s_:__ I 0.1

0

O----i

I 02

I 03

I 0.4

I 05

DNA igg/ml)

Figure 4. The effect of IPTG on competition b synthetic DNAs. Competing DNA was mixed with 25 ng of BrdU-Ah80[ Y 'PIDNA in 0.4 ml of buffer I and then sufficient repressor was added to give maximum retention in the absence of competing DNA. After at least 15 min to reach equilibrium, the solution was W irradiated as in Materials and Methods for 15 min. Aliquots (0.15 ml) were then filtered in duplicate. When present, IPTG was at 1 mM through the procedure. (A), poly(dA-dT).poly(dA-dT), no IPTG. (A), poly(dA-dT) p;.y(dA-dT) plus IPTG. (o), poly(dT-dT-dC)*poly(dC-dA-dA), no IPTG. , poly(dT-dT-dG) l poly(dC-dA-dA),plus IPTG.

to inhibition IPTG

by actinomycin is known to greatly

operator

(3,16).

pressor

binding

affinity

for

figure

4,

We have

(K 2 3 X 109M-') in correct

not

tant fected close

affect

by IPTG.

whether As

to the

lac

ref.

repressor

operator

4, to elicit

708

inhibit

re-

Poly(dT-

extremely polymer

in the

Therefore,

provides

was impor-

polymer

polymer

an IPTG

well

symmetrical it

to this

even this

high

as shown in

polymer.

this

bases

binding

figure

4) but

repressor

operator.

for

has a relatively

that six

repressor

does not

to this

to bind

of the

of

IPTG

Repressor

binding

out

shown in lac

that

we noticed

each end of the

to ascertain

enough

four

affinity

= 1 X lO*M”,

Later

(9).

the

(10)

DNA.

was found

sequence at

shown

poly(dA-dT)(K

IPTG does

3).

reduce

to BrdU-Ah80

dT-dG)*poly(dC-dA-dA)

regions

(Figure

is is not

response.

af-

BIOCHEMICAL

Vol. 62, No. 3, 1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DISCUSSION.

An attractive

tion

that

one region

of

for

DNA (e.g.,

electrostatic

is

affinity region

has specific

quences the

unique

high

to the

binding

from

the

tacking

the

treated

repressor

tetrameric

ing

(17).

vity, the

site

sensitive

both

substitution

operator

of these

results

operator

that

similar

and nonoperator

Therefore, nonoperator

is

it

is

DNA, even

affected in the

by IPTG. presence

for

of

(11).

bind-

of

lac

the

DNA binding of the

previous

similarly thymidine

acti-

repressor DNA binding.

and from

equally work

to salt,

tem-

in DNA also

in-

affinities

The simplest binding

Genetic

operator

are

binding

at-

and a

activities

respond

data).

without

and nonoperator

and nonoperator

unpublished

acids

regions

portion

by actinomycin,

BrdU

59 amino

in

nonoperator

DNA binding

and pH. both

terminal

operator

activities

by heat

activity

regions

terminal

both

18 and our

mation

for

amino

we know that

creases

not

abolishes

the

inhibition

amino

rates

to operator

terminal

the

and nonoperator to

perature,

both

of

also

critical

Operator

(4,5)

removal

amino

by the

The trypsin-

binding

bind

se-

and nonop-

repressor chain.

inducer

does not

the

that

peptide

the

generate

binding

known to remove lac

another

supported

at similar

of the

retains

only

appears

is

for would

is not

operator

inactivated

of the

but

by trypsin

it

(ref.

regions still

Since

repressor

is

middle

implicate

that

portion

structure,

studies

here

they

model

general

whereas mainly

Together

treatment

terminal

has a moderate

H-bonding)

This

are

Trypsin

repressor

interac-

interaction),

operator.

We report

amino

lac

repressor-operator

operator.

activities

and trypsin.

for

(e.g.,

lac

for

evidence.

erator

the

affinity

affinity

present

model

tenfold

interpretation

mechanisms

are

used

for

DNA.

interest

repeating

that

the

binding

repressor

poly(dT-dT-dG)*poly(dC-dA-dA),

The lac

repressor

is

of

(19-21).

Apparently

IPTG

of

709

known to change these

to is confor-

conforma-

Vol. 62, No. 3,1975

tional

changes

operator

BIOCHEMICAL

can be tolerated

AND BIOPHYSKAL

for

nonoperator

RESEARCH COMMUNICATIONS

binding

but

not

for

binding.

ACKNOWLEDGMENTS. We are grateful to D. Lin and J. Roberts for their important contributions to this work. Support was by grants from the National Institutes of Health (HD-04420) and the National Science Foundation (GB-26517).

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Gilbert, W. and Mfiller-Hill, B. (1967) Proc. Nat. Acad. Sci. USA 58, 2415-2421. RiggcA.D., Bourgeois, S., Newby, R.F., and Cohn, M. (1968) J. Mol. Biol., 34, 365-368. Riggs, A.D., Bourgeois, S., and Cohn, M. (1970) J. Mol. Biol., 53, 401-417. Lin, S. and Riggs, A.D. (1972) J. Mol. Biol., 72, 671-690. Lin, S. and Riggs, A.D. (1974) Cell, in press. Von Hippel, P., Revzin, A., Gross, C-A., and Wang, A.C. (1974) Proc. Nat. Acad. Sci. USA, in press. Goad, W. (1972) Biophysical Sot. Abst. 12, 248a. Von Hippel, P.H., Revzin, A., Gross, C.A., and Wang, A.C. (1974) Symposium on Protein-Ligand Interaction, Konstanz, Germany. In press. Riggs, A.D., Lin, S., and Wells, R.D. (1972) Proc. Nat. Acad. Sci. USA 9, 761-764. Lin, S. and Riggs, A.D. (1974) Proc. Nat. Acad. Sci. USA 71;t;47,951. Files, J. G., and Weber, K. (1973) J. Biol. Chem., 248, ;lO-;21. Riggs, A.D., Suzuki, H., and Bourgeois, S. (1970) J. Mol. 48, 67-83. Biol., Riggs, A.D. and Bourgeois, S. (1968) J. Mol. Biol., 34, 361-364. Sobell, H.M., Jain, S.C., Sakore, T.D. and Nordman, C.E. (1971) Nature New Biol., 231, 200-205. Gilbert, W. and Maxam, A. (1973) Proc. Nat. Acad. Sci. USA 70, 3581-3584. Riggs, A.D., Newby, R.F., and Bourgeois, S. (1970) J. Mol. Biol. 51, 303-314. Pfahl, M. (1972) Genetics 72, 393-410. Lin, S. and Riggs, A.D. (1972) Proc. Nat. Acad. Sci. USA 2, 2574-2576. (1972) Biochem. Biophys. M., and Horiuchi,!C Oshima, Y., Matsuura, Res. conlm. 47, 1444-1449. Laiken, S.L., Gross, C.A., and von Hippel, P.H. (1972) J. Mol. Biol, 66, 143-155. Matthews, K.S., Matthews, H.R., Thielmann, H.W. and Jardetzky, 0. (1973) BIOCHIM. BIOPHYS. ACTA 295, 159-165.

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