The helix-coil transition of E. coli s-RNA as measured by fluorescence polarization

Vol.

23, No.

5, 1966

BIOCHEMICAL

AND

TRE RRLIX-COIL

BIOPHYSICAL

TRANSITION

OF 9. m

AS MEASURRD BY FLUORESCENCE David

B. Hi liar

RESEARCH

COMMUNICATIONS

S-RR4

POMRIZATION*

and Maxime

MacKenzie

Laboratory of Physical Biochemistry Naval Medical Research Institute Bethesda, Maryland 20014

Received

April

29, 1966

In genera 1, the most soluble-RNA

(s-RM)

of danaturation

1965;

have

spectra

or of optical

rotatory

The sensitive

Here,

conjugate induced

results

in the acquisition

e.,

et al.,

1965).

the rotational

to a structureless,

random

measured

of a great

kinetic

unit

sophisticated

(Millar

Lamborg

and Zamecnik,

has only

taken are

been 1965). of an

through

the

interpreted

internucleotide

of freed-

of s-RNA at this

1964)

of fluorescence

The results

degree

analysis

and Steiner,

as s-RNA is

non-covalent

of

and Cantoni,

1965;

the polarization

50% of

transition

of fluorescence

transitions

transition.

of about

helix-coil

Felsenfeld

et al.,

in which

helix-coil

rupture

1963;

of polarization

of s-RNA is

that

of the

(Lamborg

Paeaan

experiments

showing

i.

et al.,

to polynucleotide

we report

thermally

1966;

studies

the use of either

dispersion

technique

applied

acriflavioe

involved (Fresco

Ray and Oikawa,

recently

rewarding

as bonds

of internal

temperature

rotation; is equivalent

coil. HBTHODS

S-RNA, 5. a, Chagrin

Falls,

phenol,

dialyzed

strain

Ohio,

freed

72 hours

B, was purchased of protein versus

from General

by repeated

frequent

changes

mechanical of doubly

Biochemicals, shaking distilled

with water

*The opinions or asserttons contaiued herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Ravy Department or the naval service at large. From the Bureau of Medicine and Surgery, Navy Department, Research task RR 005.06-0001.01.

724

Vol . 23 , No .*,5 1966

BIOCHEMICAL

and lyophi lized.

The preparation

ments of polarization performed solvent

AND

in detail

for 811 measurements

RESEARCH

of the acriflavine-s-RNA

of fluorescence,

as described

BIOPHYSICAL

and relaxation

elsewhere

(Millar

was 0.3 M KCI,

0.01

COMMUNICATIONS

conjugate,

measure-

time calculations and Steiner,

KAc,

pH 7.0.

were

1965).

The

The Szo,w of

the preparation was 3.96 (average of three determinations measured at temperatures of about 23'),

the intrinsic

viscosity

0.059 dL/g (25').

and the

weight average molecular weight (determined by the Yphantis meniscus depletion methtod, Yphantis,

1964), 26,500.

Assuming the extinction

coefficient

of the dye to be unaltered by conjugation there was only slightly one mole of dye combined with one mole of s-RNA in fair results of Churchich (1963). tlnction

coefficient

less than

agreement with the

A value of 21.4 was used for the specific

ex-

of s-RNA (Stephenson and Zamecnik, 1962). RESULTS

FLg. I shows the polarization

IOII1111

20

30

of fluorescence thermal profile

40I

60

I

50t

r

60

I

70

as

1

QQ

- 55

40-

-h -‘p

Fig.

I. Polarization of fluorescence thermal profile and absorbance 260 I+ thermal profile of 3. coli S-RNA. X excitation 436 q~, X exit via Corning 510 sq.bcutoff filter. 0, l/P + l/3; 0, 480.

725

Vol.

23, No.

5, 1966

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

f+' 3 versus Fig.

I

T//l;

also

shows

scale

is

Since

P (polarization)

in

at

about

cribed

in

detail

rotational

the

technique temperature sucrose

the

that

as 45’-50’.

the

presence

L+f P

(Millar

of

fluorescent

stranded

varying

solutions.

Qby Under

of

(1963), the

these

use

of

the for

e.

by

in

small. a selective to

case)

present

P at

As

des-

gain

in

the

adenine

wFth

the

constant of

freedom

P

may heavfly

possibility

rotational

T/q.

a fall

concentrations if

of

a decrease

measuring

various

conditions,

values

bound

this

temperature

rigidity,

(covalently in

i..

with

that

1965)

terminus

The

vanishingly

Steiner, label

viscosity.

lg~.

internal

I shows

We tested

Wahl

260

register

P becomes

CCA

solvent

at

in

Fig.

and

polarization. and

.

55O-60’

0 is

profile is

elsewhere

Gottlieb

and

(centigrade)

in

the

Kelvin

thermal

At

single

and

T

a rise

apparent of

degrees

registers

freedom of

T is absorbance

so

expressed

begins

weight

the

adjusted

P is

residue

where

buffered of

the

Ei-

7

--IF0 + -,a 6

, 5

4

200

f SC

400 T’T

Fig.

IT. Polarization solutions.

values X excitation,

at

25O and 4S”. A exit as in

726

R is Figure

varied I.

0,

by the use of 4S”; 0, 2S”.

sucrose

Vol.

23, No.

label

L+1; P 3 decreases

of TIQ,

experiment. and the both

No evidence intercepts

This

at 45',

I.

BIOPHYSICAL

T/Q is

at first

rapidly.

Fig.

of curvilinearity

at T/h

temperatures. Labtel

AND

L+L P 3 versus

is. present,

values

the

BIOCHEMICAL

5, 1966

= 0 are result

the

but

COMMUNICATIONS

with

decreasing

2 shows the result shown at 25'

same within

shows that

e. at about

Linear

is

the

RESEARCH

of this

nor

at 45'

experimental

no free

transition

error

rotation

is

for

acquired

by

initiation.

DISCUSSION The estimated good agreement

Tm of the absorbance

with

the

results

(1966),

who used only

ization

becomes vanishingly

the disrupted in internal acquire that

indica,ted this

by the

tional

label

rotational

forces

melting

in

this

freedom

is

of a conformation

freedom, region

that

the

organization

influences-and

label

idea

the

for

i.

that

freedom

would

not

freely

first

find

stabilizing in

label.

and not exposed

explanation

label

by the

of the

e. always

be

due to a frac-

happen

stranded

the

however,

the acquisition

is single

loss

expect,

in part

mobility

the

does not

We did

possibly

that

label

the structurally

could

CCA terminus

we feel

that

rotational

of a-RNA-

therefore

rotational

at 55O is

-et --al

the polar-

for

the

(1964).

in

indicates

responsible

One might

This

restrains

range

at 55'.

responsible

conceivable.

That

that

and Wahl

the are

are

57O-58'

and Henley

presupposes

45o)

of Gottlieb

(1966)

strengths.

region

freedom (circa

is about

temperature

Loss in polarization

which

indicates

in the helical

the

this

conclusion

technique If

ionic

in this

begins

to be so.

evideuce

in

rotational

transition

rotational

small

This

preferential as the

different

forces

rigidity.

curve

of Kay and Oikawa

slightly

secondary

melting

is

loss But,

involved

to solvent likely

to be

more correct. One further fluorescent Steiner, from the

residue. 1965)

20*to loss

possibility

about

Some

show about !iO".

It

in P shown in Fig.

is but

a change not

all

in the excited

lifetime

(r)

Labelled

polynucleotides

(Millar

a 20% to 50% decrease

in fluorescent

intensity

can be calculated I cannot

(Millar

be accounted

727

and Steiner, for

1965)

by such a change

of the and

that in r.

Vol.

23, No. 5, 1966

BIOCHEMICAL

From our data,

we cannot

of bonds

located

a marked

conformational

in internal

rigidity.

cluded

yeast

that

between

20’

coiled

near

change

and that

loss

of bonds

in rigidity From

time

axial

ratio

The axial viscosity, equation

for

of the ratio

(1953)

any large It

is clear

perturbations by

equivalent

system

in this weight

isxnediately

distant

the polarization

of equal

the

loss

in asyxkaetry like

randomly

between

yeast

a similar

change

and

conformation

the melting

of a critically

to explain

the

of about

size.

adjacent independent

to the terminus

5-6.

Thus

since

kinetic

they

would

at 25’

does not have

rotation

localized

units

that

terminus

of the over-all

area

at 25O and under

indicates

labelled

does not preclude

1964).

by the Scheraga-Mandelkirn

result

to the

the

sedimentation,

and rotational

This

the

compute

et al.,

From our

we may compute

with

weight

(Borisova 6-7.

the translational

of the relaxation

by comparison

molecular

is

ratio

suggestion

a value

ellipsoid

data

axial

freedom this

then

and then

manner

to be of similar

rotational

does,

conformational

prolate

conditions

area

s-RNA behaves

that

in

have con-

loss

mechanism

results for

(1966)

a marked

ts possible it

loss

et al.

we may calculate

sphere

an apparent

that

Henley

of a group

at 55’.

a rigid

and molecular

experimental

helical

the

solvent

estimated

of s-RNA appear the

follow

of P at 25’

((3 of s-RNA in this calculated

If

we observe value

the

value

our

must

s-RNA.

melting

the

responsible

melting it

COMMUNICATIONS

of bonds whose

yeast

If a parallel

j$. coli

is

is primarily

above 40’

then

RESEARCH

it

s-RNA in 0.2 M NaCl undergoes

vith

set

or a set

connection,

s-RNA may be assumed,

located

whether

which

In this

and 40°,

occurs

BIOPHYSICAL

determine

the terminus

polynucleotides.

event

AND

of s-RNA.

conformational not

be detected

technique. REFERENCES

Borisova, 0. F., Kiselev, L. L., Frolova, L. JSJ., Dokl. Akad. Churchich, Jr., Biochim. Biophys. Fasman, G. D., Lindblow, C., and Felsenfeld, G. and Cantoni, G. L. (1964). Fresco, J. R., Klotz, L. C., and on Quantitative Biology 28,

Surovaia, A. I., Turner-man, L. A., and Science (NAIJK), USSR 159, 1154 (1964). Acta 75, 274 (1963). Seaman, E., J. Mol. Biol. l2, 630 (1965). Proc. Natl. Acad. Sci. U. S. 5l, 818 Richards, 83 (1963).

728

E. G.,

Cold

Spring

Harbor

Symposia

Vol.

23, No.

5, 1966

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Gottlieb, Y. and Wahl, P., J. Chim. Phya. 60, 849 (1963). Henley, D. D., Lindahl, T., and Fresco, J. R., Proc. Natl. Acad. Sci. U. S. 55, 191 (1966). Kay, C. M. and Oikawa, K., Biochemistry 2, 213 (1966). Lamborg, M. R., Zamecnik, P. C., Ting-Kai, Li, tigi, Jr., and Vallee, B. L., Biochemistry 5, 63 (1965). Lamborg, M. R. and Zamecnik, P. C., Biochem. Biophys. Res. Cormrmn. &I, 328 (1'965). Millor, D. B. and Steiner, R. F., Biochim. Biophys. Acta 102, 571 (1965). Scheraga, 8. A. and Mandelkirn, L., J. Am. Chem. Sot. 75, 179 (1953). Stephenson, M. L. and Zamecnik, P. C., Biochem. Biophys. Res. Common. 1, 91 (1962). Yphantis, D. A,, Biochemistry 2, 297 (1964).

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