Specific photoreactions between psoralens and yeast-tRNAPhe

BIOCHEMICAL

Vol. 106, No. 1, 1982

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 179-185

May 14, 1982

SPECIFIC

PHOTOREACTIONS BETWEEN PSORALENS AND YEAST-tRNA Peter

Eiqil

Nielsen

Phe

.

& Vaqn Leick

Department of Biochemistry B, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhaqen, Denmark Received

March

30,

1982

Phe and two psoralens, 8-metThe photoreactions between yeast-tRNA hoxypsoralen (8-MOP) and 4'-aminomethyl-4,5',8-trimethylpsoralen It is found that AMT reacts more (AMT) have been investigated. efficiently and with more sites on the tRNA than does 8-MOP, and that the AMT-photoreaction is inhibited by ethidium bromide and indicate that psoralens photobind enhanced by EDTA. The results to specific sites on the yeast-tRNAPhe molecule. INTRODUCTION Psoralens,

a class

of compounds belonging

have been used in ancient are presently

employed

compounds are also conformation interstrand

thereby

in the

of double

stranded

its

tRNAs constitute

a very

a well

tertiary

described

ded into

the L-shape

ded regions,

the

to be restricted fication

of

both

structure

1.

the

these

Abbrevations:

in the nucleic

sites of

acids,

since

(2).

It

class

capacity

cloverleaf

containing

both

single

of psoralens

could

lead

tRNA and the

interaction

AMT: 4'-aminomethyl-4,5' 8-MOP: 8-methoxypsoralen.

they

and

induce

Since

acids

having

which

is

and double

folstran-

be anticipated

In the hope that to a better

The

structure

(3).

of nucleic

the

could

(1).

to transfer-RNA

structure,

sites.

and

was recently

photobinds

specialized

the

of psoriasis of the

acceptor

specific

treatment

study

amino acid

(4),

furocoumarins,

disease

therapy

(8-MOP)1

photobinding to

skin

upon irradiation

8-methoxypsoralen

decreasing

for

light

used as tools

cross-links

shown that

medicine

to the

the

identi-

understanding

of

of psoralens

,8-trimethylpsoralen 0006-291X/82/090179-07$01.00/0

179

Copyrighf 0 1982 by Academrc Press, Inc. All rights of reproductron in anv form resw ,~d.

Vol. 106, No. 1, 1982

with

nucleic

BIOCHEMICAL

acids,

we investigated

AMT (4'-aminomethyl-4,5' The results for

AND BIOPHYSICAL

indicate

the

RESEARCH COMMUNICATIONS

photobinding

,8-trimethylpsoralen) the

presence

8-MOP and two or more sites

8-MOP and

to yeast-tRNA

of one major for

of

Phe .

photoreaction

site

AMT.

MATERIALS AND METHODS Phe Yeast-tRNA and nucleases Sl (EC 3.1.4-) and T were purchased from Boehrinqer Mannheim. [3H1-8-mO~E~a~.~;~~,~~d by heating 25 mq of 8-MOP (Fluka) dissolved in 3.3 ml dry dioxane containing 380 ~1 fuming H SO and 200 ~1 tritiated water (25 Ci/ was cooled to room ml, Amersham) at 60°C for 3 h? The mixture temperature, 10 ml of ice-water was added and the f3H]-8-MOP was extracted into 2 x 5 ml ethyl acetate. The organic phase was reduced to 500 ~1 and run through a silicaqel column elufed with toluene ethyl acteate; 1:l. The fractions containing [ HI-8-MOP were pooled and evaporated. The product (% 1.5 mCi) had a specific activity of s 40 mCi/mmole and was > 95% radiochemically pure by TLC-analysis. An analogous experiment done with D20 showed that the exchange took place (mass-spectral and NMR analysis, J.B. Hansen and P.E. Nielsen, unpublished results) at the 5-position. L3~I-~~ was a kind gift from Dr. John E. Hearst and the specific activity of the sample was ?r 10 mCi/mmole. Irradiations were performed with light from an Osram Sp 200 mercury lamplfiltered throyqh 5 mm acetone giving a light intensity of 2 x 10 qunata/s.cm (350-45Onm). The stirred samples were kept in an ice bath during the experiments. Before nuclease digestions the photoreacted tRNA was precipitated and washed with ethanol to remove excess radioactivity. Digestions with nuclease Tl (3 units/p9 tRNA) or nuclease Sl (30 units/u9 tRNA) were performed in 50 mM Na acetate, pH 6.5o 5 mM MqCl (and 2mM ZnS04 in the case of Sl) for 20 min at 37 C. The tRNA i! raqments were analyzed on 10% (S ) or 29% polyacrylamide slab-gels (5,6) which were scanned a 4 260 nm and subsequently sliced (2 mm) for scintillation counting (luma solve, lipo luma; Lumac). RESULTS AND DISCUSSION Both 8-MOP and AMT were found tRNAPhe in a light observed

that

dependent

AMT reacted

terms of photochemical tRNA (Table with

results

obtained

In order tRNAPhe,

reaction

(Fig.

more efficiently

1). than

with

yeast-

However,

it

8-MOP both

and number of psoralens

behaviour

of the compounds is

with

other the

types

nuclease

with

Sl or RNase Tl. 180

was in

bound per in full

accord

of RNA and DNA (7).

specificity

had been photoreacted

with

covalently

yield

to determine

which

was digested

This

1).

to react

of

the

reactions

yeast-

13H1-8-MOP or [3~1-~~~, The nucleotide

fraq-

Vol. 106, No. 1, 1982

BIOCHEMICAL

Ir

0

(Fig

these

results

indicate

specific

site

activity

is

digest

(Fig

found

of

with

the nucleotide

to

8-MOP binding,

for

nuclease

(Fig

two psoralens

2.

with of

of the is not

Phe . The problem

demonstrated

of the photoreactions

by ethidium

on DEAE cellulose

po-

A5*

pursued.

In are

by the experiments 5).

the binding by the bromide.

was consistent

181

the

suggesting

tRNA molecule

as indicated

between

on the

nucleotide

is being

(Fig

of

but

2 thereby

on the

3a) and RNase Tl difference

known,

Sl-

has not

psoralen

to be between

more sites

radioof the

nucleotide

an octanuleotide site

the

one oligonucleotide this

influence

The

to one major

fragment

the A35-A,6

was further

An analysis clusion.

gels.

with

AMT photoreaction Sl

slab

8-MOP binds

in the gel

indicates

the yeast-tRNA

The qualitative

tion

the

characterized

since

The identity

since

of a) 2.5 were witha polyacrylcounted.

of the molecule,

only

8-MOP photobinding

and G65 of

available

4).

yet

in the gel

contrast

3'-half

associated

(Fig

have been well

on polyacrylamide

3b) and apparently

been determined

the major

which

3,4 and 5) that

(Figs. in the

the Tl-digest

migration

digestions, were analyzed

2, 6,8,9),

sition

30

Na acetate, H 6.5, 5 mM MgC*l in the presence cpm or b) 10 3 cpm tritiated p t oralens. Aliquots at the times indicated. The samples were run on gel, from which the tRNA bands were cut out and

from

ments

RESEARCH COMMUNICATIONS

1 20 min

10

a

25 mM x 106 drawn amide

AND BIOPHYSICAL

pattern

sites of

for inhibi-

The experiments with

this

con-

the

Vol. 106, No. 1, 1982

BIOCHEMICAL

AND BIOPHYSICAL

Table I. Efficiency of psoralen photobinding The numbers were calculated from the results (30 min).

to yeast-tRNAPhe. shown in Figure

psoralen bound per tRNA ~-MOP

RESEARCH COMMUNICATIONS

yield (rel.to

0.07

of

1

photoreaction added psoralen)

0.15%

0

D\E D G %

/G\A

A F y

+*~

G/A

G/

PG-L --C-G _G-C’O 3-U 5A-U U-A A” U - A, c Umn GAtAC z I I I I I AU m5C U G/U G/ C

C= m:G /c-G\A

T

cLl

k

a I

I m 3-

‘t’

m’G Gz;.

2-

c-6 A-U j”G - Ch C,A+ A YW U .w Gm Art AI.,

02

I\

b

”

l-

...

0

03

Figure 3. S -digestion of tRNA reacted with r3Hl-psoralens. 30 vg of tRNA Y er Q irradiated for 15 min in the presence of a) 5 x 105 cpm of [ HI-AMT or b) lo6 cpm of [3HI-8-MOP. The tRNA was precipitated with ethanol and subsequently digested with S The products were analyzed on a polyacrylamide gel. The dot 4'ed line is the UV trace (260 nm) of the gel, showing intact tRNA, and the fragments of lengths 41-42 (fraction 36), 35 (fraction 38) and 33 (fraction 39) nucleotides.

BIOCHEMICAL

Vol. 106, No. 1, 1982

0

10

AND BIOPHYSICAL

20

0!i

30 40 Fraction

Fraction

RESEARCH COMMUNICATIONS

50 no

60

70

no

The tRNA Figure 4. T1-digestion of tRNA reacted with 13HI-8-MOP. was treated as described in the legend to Fig 3 except that nuclease T was used instead of S . The fragments (dotted line) are of 29, identified on the lengt ii s 20,19 (intermediates + 12 (fraction basis of the fluorescence from the yW base), 8 (fraction 36), 6, 5 and 4 nucleotides. Experiment Figure 5. T -digestion of tRNA reacted with L3~l-~~. in Fig 4. analogous t b the one described

showed dium ted

a drastic bromide, (Fig

binding site the duce

decrease while

6).

These

sites

are

latter.

8-MOP

close

A recent

a conformational

AMT

binding

photobinding

results

while

(10,11,12),

in

was

indicate to the

study change

or

in

that

overlapping

8-MOP has to

site(s) also

shown

yeast-tRNA 183

the

presence

only

one

of

moderately

or

more

the

ethidium

seems

little

that

AMT

Phe , not

ethiaffec-

of

the

AMT

bromide related is very

able

to to

different

in-

Vol. 106, No. 1, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I1 12

I I, I, 3 4 5 6 etbr/t,wA Figuw. Effect of ethidium bromide on the photoreactions. YeasttRNA was photoreacted with psoralens as in Fig 1 but in the presence of ethidium bromide. At the higher levels of ethidium bromide the results have been divided by the transmission of the solution at 350 nm (40-90%) thereby taken the inner filter effect into account. 8-MOP: e-e-0, AMT.: o-o-o.

than

one induced

such an effect

by ethidium

to the presence of

sults

this

in behaviour

II

the

of

availability

was inhibited.

8-MOP did not

exhibit

backbone

In the

This

of

presence

and the

of EDTA, for

enhanced while

(6,9,13)

re-

example, the 8-MOP

a general

"open-

or to an increased

absence of Mq2+ ions.

in the

interac-

the tRNA as the

may be due to either

structure

of phosphates

8-MOP and AMT may be due

in the latter

was significantly

the tRNA tertiary

been shown that binding

phosphate

suggest.

the AMT photoreaction

ing"

between

of the amino group

with

in Table

reaction

while

(6).

The difference

tion

bromide,

It

has

more sites

are available for ethidium bromide 2+ (14). Conversely, spermine, which in the absence of Mq

Table II. luence of EDTA and spermine on psoralen photobindinq. Yeast-tFWAU was photoreacted with the psoralens as described in Figure 1 (15 min) except for the addition of EDTA or spermine, respectively. 8-MOP Addition

AMT

cpm

%

cpm

%

none (control)

1000

100

1400

100

EDTA (10 mM)

680

68

2800

200

400

29

spermine (5 mM)

184

BIOCHEMICAL

Vol. 106, No. 1, 1982

bind

to the

phosphate

on the AMT binding We did

not

this

may explain

here

and those

molecules

they

show that

results

the

thermore,

the

influence

on the photoreactions

of

two psoralens

the results

and

presented

that

four

implications. yeast-tRNA Phe

First

sites,

that

ethidium it

in this bromide,

may be possible

more thoroughly

tRNA conformation

e.g.

with

and these

investigated

of both infer

psoralens

8-MOP

per tRNA.

specific

have been characterized for

tRNA with

between

have several

involve

for

as probes

the

the photoreactions

identical

sites

to saturate

Ou and Song (3) who reported

are not random but

effect

II).

be photoreacted

The present

RESEARCH COMMUNICATIONS

had an inhibitory

(15),

the discrepancy of

could

backbone

(Table

intend

AND BIOPHYSICAL

during

of all psoralens

sites

are not

study.

Fur-

EDTA and spermine - when the

- to use psoralens protein

synthesis.

ACKNOWLEDGEMENTS The support of the Danish gratefully acknowledged.

Natural

Science

Research

Council

is

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. lo. 11. 12. 13. 14. 15.

Anderson, T.F. and Voorhees, J.J. (1980) Ann. Rev. Pharmacol. Toxic01 20, 235-257. K-J-Jr. (1979) Photochem. Photobiol Song, P.-S. and Tapley, 2, 1177-1197. (1978) Biochemistry 17, 1054-1059. our C.-H. and Song, P.-S. Rich, A. (1977) Act. Chem. Res. 9, 388-396. Donis-Keller, H., Maxam, A. and Gilbert, W. (1977) Nucl. Acids Res. 4, 2527-2538. Nielsen, P.E. (1981) Biochim. Biophys. Acta 655, 89-95. Isaacs, S.T., Shen, C.-K.J., Hearst, J.E. and Rapoport, H. (1977) Biochemistry 16, 1058-1064. RajBhandary, U.L., Stuart, A. and Chang, S.H. (1968) J. Biol. Chem. 243, 584-591. Wrede, P. Wurst, R., Vournakis, J. and Rich, A. (1979) J. Biol. Chem. 3, 9608-9616. Jones, C.R. and Kearns, D.R. (1975) Biochemistry 14, 26602665. Wells, B.D. and Cantor, C.R. (1977) Nucl. Acids. Res. 4, 1667-1680. Liebman, M., Rubin, J. and Sundralingham, M. (1977) Proc. Natl. Acad. Sci USA 3, 4821-4825. Rich, A. and RajBhandary, U.L. (1976) Ann. Rev. Biochem. 5, 805-860. Urbanke, C., Rijmer, R. and Mass, G. (1973) Eur. J. Biochem. 33, 511-516. Quigley, G.J., Teeter, M.M. and Rich, A. (1978) Proc. Natl. Acad. Sci USA -'75 64-68. 185