Veal heart ribonuclease P has an essential RNA component

Vol.

96, No.

September

BIOCHEMICAL

2, 1980 30,

AND

BIOPHYSICAL

RESEARCH

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Pages 831-837

1980

VEAL

HEART

Eiko

Akaboshi,

Department

Received

of

RIBONUCLEASE

P HAS

Cecilia

Biology,

AN ESSENTIAL

Guerrier-Takada

Yale

RNA

and

University,

New

COMPONENT

Sidney

Haven,

Altman

Ct.,

06520

USA

7,198O

August

Summary: The activity of RNase P (EC 3.1.26.5) from veal heart can be abolished by pretreatment of the enzyme preparation with micrococcal nuclease, pancreatic RNase A, or RNase Tl. This indicates that veal heart RNase P contains an RNA component essential for function of the enzyme as has also been shown for E. coli RNase P (l-3). 3Additionally, veal heart RNase P has buoyant density in Cs2S04 of 1.33 g/cm , which is intermediate between that protein and nucleic acid. RNase is

(4-61,

This

P,

necessary

enzyme

has

been

detected

for

the

biosynthesis

recognizes

substrates,

tRNA

sequences evidence

which

near have

(l-3).

In

also

requires

some

precursor its

an

molecules, cleavage.

that

E.

coli

RNA

we

show

component

(4).

RNase that for

an

the

structural rather

of

paper

procaryotic of

of

sites

shown

this

aspect

in

and 5’

eucaryotic

termini

of

than Both

P consists eucaryotic

of

specific

RNA

enzyme,

molecules. its

nucleotide

functional of

organisms

tRNA

conformation

a of

and

physical

and

protein

veal

heart

moieties RNase

P,

function. METHODS

Preuaration of RNase P: 426 g of fresh veal hearts, defatted and sliced, were homogenized in 300 ml of ice cold buffer TM (20 mM Tris.ACl, pll 7.6+/15 mM Mg(GAc)2 / 6 mM I-mercaptoethanol) containing 20 mM NH4C1 and 20gM PMSF . The buffer was then made 0.7% in Triton X-100 and 0.3% in sodium deoxycholate. The homogenate was left for 30 min on ice and then centrifuged at 16,300 x g The resulting supernatant (S16) was diluted two-fold with for 30 min at 4O. buffer TM containing 20 mM NH Cl and loaded onto a DEAE Sephadex A-50 column (2) (bed volume 1.5 1) equill -% rated with the same buffer. The loaded material was eluted stepwise with successive two liter washes of buffer TM containing 0.06 M NH4Cl, 0.2 M NH4Cl and 0.35 M NH4Cl. The RIfase P activity was elated in the last wash step. The pooled, active fractions were passed through a Biobeads (Biorad Corp.) column (18 ml bed volume) to remove the Triton X-100. The resulting active fractions were concentrated using (NH4)2S04. The lo-25% (0.1 g to 0.25 g/ ml original volume) (NH4)2S04 pellet was resuspended in buffer TM containing 0.2 N NH Cl and applied to a Sephadex G-100 column (1.3 1 bed volume). The RNase P act 4 vity elated just after the void volume Assav of RNase P activity: Radioactive substrate (E. coli tRNA br precursor) was reaction mixture 2-mercaptoethanol, substrate.

prepared and assayed (30 pl) contained 0.1 IIIN Na2EDTA,

as previously described 5 mM MgC12, 100 s&l NH4Cl, 50 mM Tris.HCl. pH 8 and

(5). The 1mM 5.000-7,000

standard cpm

of

0006-291X/80/180831-07$01.00/0 831

All

Copyright 0 1980 rights of reproduction

by Academic Press, Inc. in any form reserved.

Vol.

96,

No.

2, 1980

BIOCHEMICAL

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Ribonuolease inactivation of RNase P: Micrococcal nuolease: 10 81 of RNase P purified through the Sephadex G-100 step (about 20 pg of protein) was mixed with 1 pl of 50 mM Ca (OAC)~, 3 ~1 of 50 gbf PMSF and 1 ~1 of MNe (0.17 units or 1.7 units per 15 pl pretreatment mixture as desired) a kind gift of The mixture was incubated at 37O for Dr. M. Laskowski, Sr., in 1 j!M NaCl. 40 min. 10 gl of 10 mM RGTA was added to inactivate the MN by chelating Ca2+ . The RNase P activity was then assayed in standard reaction mixtures containing 1 mM PITA and 10 nbl PMSF. RNase Tl and RNase Pretreatment with these enzymes (separately) was A: carried out in buffer TM containing 0.2 M N’R Cl and 10 uM PMSF. 1.5 units of RNase T1 or 1 pl of RNase A (1 mg/ml) was ad % ed to 55 pl of RNase P (as The sample was incubated for 40 min at 37O. In some cases the above). amount of RNase A and RNase P were doubled to provide larger sample volumes. After incubation (NR,) SO4 (0.49 mg/ml) was added in the ratio of 90 pl of After centrifugation the pellet was (NR4)2S04 solution to 1 10 1.11 of sample. redissolved in buffer TM containing 0.2 M NR4Cl. This procedure effectively removes most of the RNase T1 and RNase A from mixtures containing these enzymes and RNase P. The resuspended pellet fraction was used directly for assay of RNase P or further purified by applying to a sucrose gradient. Gradients were formed by layering steps of buffer TM with 0.2 M NR4Cl Each step was 150 ~1. containing 20%. 15%. lok or 7.5% sucrose. Centrifugation was carried out in tubes of 0.65 ml capacity in the Beckman SW65 rotor with special bucket adaptors for 4 hr at 59,000 rpm, or for 14 hr This gradient purification procedure was used because at 31,000 rpm at 4O. the molecular weight of RNase P is much larger than that of either RNase T1 or BNase A. The tubes were punctured with a 27 gauge hypodermic needle and one drop fractions were collected. &2@+ densitv aradient centrifuaation: Preformed step density gradients were by layering 0.19 ml each of 45W, 37% and 31% Cs2S04 solutions made in TM buffer containing 0.2 M NR4Cl. The sample, 0.05 ml, was then layered on the gradient made in cellulose nitrate tubes of 0.65 ml capacity and the tube were centrifuged in the Beckman SW 65 rotor equipped with tube adaptors. Centrifngation was carried out for at least 6 hr at 3O and 59,000 rpm. Longer run times did not alter the results. Two drop fractions were collected after puncturing the bottom of the tubes with a 27 gauge hypodermic needle. Aliquots of each fraction were then assayed for RNase P activity in standard fashion.

MN uretreatment: Methods, 1.2

and

When

little 7.8)

or as

autoradiograph

absence intactness *Abbreviations:

can of

contrast,

control of

residual

be the

P is

seen

show the

by

MN -

the

RNase

with

P activity lack

polyacrylamide

precursor PMSF RGTA

pretreated

RNase

incubations,

Ca2+, of

no

RNase

of

gel carried

P is substrate

still

MN as is

RNase analysis

out

in

active as

seen

- phenylmethylsulfonylfluoride - ethylene glycol-bis-(8-aminoethyl N - N’tetraacetic acid micrococcal nuclease

832

observed

the

the

absence

(Fig.

lA, Fig.

the

lA,

products reaction of

shows

ether)

lanes in

the

mixture.

RN or

lanes lA,

in

(Fig.

P cleavage of

in

described

3-6). that

in

By the

The the

MN

is

Vol.

96, No.

EIOCHEMICAL

2, 1980

AND

EIOPHYSICAL

RESEARCH

COMMUNICATIONS

Origin-

pt RNAtRNA(,” 5:

A. Figure 1: Inactivation of a veal heart RRaso P by pretreatment rith micrococcal nucloaso or RRaso T 1. A. Lanes 1 and 2: Pretreatment rith 0.17 units of RN, 0.5 nl or 1 pl in assay mixture respectively. Lanes 3 and 4: Pretreatment with RN as in lanes 1 and 2 but no Ca2+ present. 0.5 pl and 1 ul assayed respectively. Lanes 5 and 6: Mock pretreatment, Ca2+ present but RR absent, 0.5 pl and 1 ul assayed respectively. Lanes 7 and 8: as in lanes 1 and 2 but 1.7 units of RN used. B. MN pretreatment followed by (N84)2S04 fractionation as described in the methods. Experimental conditions as in Fig. 1A. lanes 2.4.6.8 respectively. C. Pretreatment with RRaso T1 follovod by (Ml )2SO4 fractionation as described in the Rethods. Lane 1, Control (no Ware T1); 1 al assayed. pro 4 roatmont Lane 2: Pretreated rith INare T1; 1 The position of the precursor of tRRA substrate and the RRaso P pl assayed. cleavage fragments containing the mature tRRA sequence with the ‘extra’ 5’ are indicated in the figure. proximal fragment,

completely

inactivated

activity the

must active

veal

as

the

true

of

the

mature

left the

at

due

products

the

tRNATyr origin

in

fractionation

preparation

is

RNase P have

expected

by

of

the

gel

of

our

results

Methods.

used

at

the in

origin these

P.

The

by

of

lanes

Since

to

lB,

experiments

RNaso RNase

in

which

the

same

5’

as

that

terminus

radioactivity

was

complicating the

P with

(hlR4)2S04,

P after little The

eleotrophoresis. was

833

l-4,

analysis

further

pretreated

lanes gel

some

the

possibly

the

by

fingerprint

fractionate of

the

generated

at

1,2,7,8),

decided

ribonuclease

products by

shown).

of

the

residual

P cleavage

precipitation

Pig.

any

cleavage

RNase

lA, we

and

characterized

not

(Fig.

in

Ca2+

accurate

Reassay

illustrated

of

been

(data

mixture

seen we

active

sequence

the is

removal

RNase

pretreatment

described

substrate

to

heart

interpretation

completed as

be

after

used

such or

MN for

no

Vol.

96, No.

pretreatment cell

of

RNase

protease

2).

where

precipitation

residual

BNase lane

is

BIOPHYSICAL

and

shown

that

to

included

RESEARCH

which

be

in

P does

prior

to

free

all

Koski of

our

COMMUNICATIONS

used

to

proteases.

inactivate

In

pretreatment

of

BNase (NB4)2SO4 Methods.

assays

of

some

the

KB

addition

mixtures

to

BNase

then

further

Even

after

relevant 2A,

fractions 2-4).

soluble

the

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(see

To

digested

by we

A pretreated

apparent

with

exclude

BNase

of

gradient

where

were

BNase

A in

reconstruction

to

reaction

RNase

834

BNase mixtures containing

P activity.

the

fractionated

by

described

evident

in in

there

gradient,

presumably

presence control is

A pretreated with

from

particular,

activity

polyacrylamide that

mixtures, for

out

Tl

2.

enzyme

the

RNase

Fig.

the is

(N84)2S04

the

such

carried

A bound possibility

no

the the

BNase to

lane

in

sucrose

of

A was

this

containing

the

by

as

In

of

products

were

RNase

in

whereas

lC,

2B).

fractionated amount

sedimentation

residual

gradient

were

respect

mixtures

Nevertheless,

the

samples,

Figure

precipitated the

under

residue with

fraction

containing

experiments

in

(Fig.

size. the

last

velocity

bottom

of

small

is

residual

assayed

top

from

A and

result.

RNase

its

the

(NB4)2S04

mixtures

observed

some

activity

breakdown

the

shown

loses

a slight

pretreatment

fractions

gradient

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by

so,

accumulated

A pretreated

A which

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2-4)

BNase

samples,

at

as

steps

in

fractions

from

same

in

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result

Tl

mixtures

removed

end

these

precipitate

P.

the

Methods)

gradient

of

lanes

by

purified

BNase

because

evident

the

activity

and

RNase

RNase

unresuspended

sediment

same

P is

sucrose

fractionation,

for

The

BNase

pretreatment

of

shown).

with

not

the

purification (see

degradative

does

(8),

is

A oretreatment: and

P pretreated

Tl

assay

activity

(not

inactivation

lanes

RNaSe

RNase

Further

mixtures

(Fig.

RNase

centrifugation

these

the

been

Since

Tl

2).

gradient

due

has

PMSF

lane

conditions

and

and

P (1)

uretreatment:

lC,

the

BNase

AND

.

(Fig.

1C.

coli

P (7).

RNasel

be

IL

inhibitor

Methods)

by

BIOCHEMICAL

2, 1980

is

expected

of

BNase

treated seen

P in sample

in

the

sample

various

to

same

(Fig.

2B,

concentrations beads

and

P cleavage

both

yielded

products

containing

the

the might

pretreated

control

We detected

of

treated both

BNase

Vol.

96, No.

8lOCHEMlCAL

2, 1980

12 34 k-

Origin

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

123468101214CR

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Figure 2: Sucrose gradient fractionation of RNase P after pretreatment with RNase P vas pretreated rith RNase A as described in the Methods. RNase A. The mixtures (with RNase A and control lacking RNase A) were then fractionated by ammonium sulfate precipitation and the relevant resuspended pellet layered on sucrose gradient as described in Methods. Gradient fractions (1 pl aliquots) vere then assayed for RNase P activity. The figure shows an autoradiogram of the polyacrylamide gel analysis of the RNase P assay. The numbered lanes indicate relevant gradient fractions with number 1 being the bottom of the gradient. Fractions not shown in the autoradiogram had no activity. A. Control pretreatment (no RNase A). The lane labeled ‘C’ was a control incubation with no added material from the sucrose gradient. The lane labeled ‘R’ vas a reconstruction (see Text) with equal amounts of material from Fig. 2A lane 4 and Fig. 2B lane 4. The position of the RNase P cleavage products are indicated in the figure. Small amounts of spontaneous breakdown products or RNase A break down products (Fig. 2B) are visible in the autoradiogram. They do not have the same mobility as the RNase P cleavage fragments (see Fig. 2B lane 1 especially).

P and

RNase

mixing 2A,

for lane

RNase in

A activities the

4 and

Fig.

2B, is

subsequent Baovant

RNase Methods) preformed

P preparation

Cs2S04

indicate

that

recovery

of

activity,

level

as

RNase

of

crude

was

further buffer

is

of very

this

poor.

835

2A,

lane

RNase conditions

P:

The

S16

by

(N84)2S04

0.2M

m4C1

Fig.

is RNase

that is

not

we

from

an (see

centrifuged

in

in

3.

about P has

Figure

1.33

lost

used.

precipitaion and

before

R with

fraction

shown

results,

coli

alone

A and

the

material E.

samples

under RNase

The

the

demonstrates

with

‘IN with

Methods).

density

Fig.

pretreatment

heart

in

experiment

fractionated

(see

3-S%

this

P activity

veal

versus

buoyant

the

seen

(compare

Thus,

by

for

g radient the

4).

abolished

dialysed

same experiment

lane

assay

densitv

and

the

reconstruction

P activity

the

at

g/cm3.

a buoyant

a

The

Vol.

96, No.

BIOCHEMICAL

2, 1980

I

AND

3

5

BIOPHYSICAL

7

FRACTION

9

II

RESEARCH

COMMUNICATIONS

13

NUMBER

Figure 3: Cs2SO4 buoyant density determination of veal heart Wase P. Centrifugation was carried out as described in the Methods. (0) BNase P activity quantitated by measuring radioactivity in gel slices from analyses assay mixtures for RNase P activity in gradient fractions. (Xl Protein concentration as determined by the method of Bradford (10). The two arrows indicate the position of DNA and hemoglobin markers as determined in separate experiments (see Text).

density

of

buoyant

densities

1.43

g/&

complex due

and is

to

1.55

g/cm3

under

of

various

E.

coli

denser

the

conditions

markers

tRNA Tyr:

than

presence

similar

protein

of

(hemoglobin: 1.38

but

a nucleic

(unpublished

g/cm31

less acid

show

dense

of

experiments).

1.25

g/cm’;

that

the

than

RNA or

be

inactivated

calf veal

DNA

The thymus

heart (91,

DNA

RNase presumably

component.

DISCDSSION We have with

any

of

pancreatic the

shown the

three

RNase

between

inactivated

by

characteristic evidence

that

been

heart

RNase

with

the

buoyant

that

of

presented

with

to

the

and

of protein.

veal

Cs2S04

cou

RNase

P is

also

complex

(1).

In

836

1 or

studies P in

A and

enzyme

T

RNase

E.

the

pretreatment

heart

RNase

that

RNase

inactivation

MN and

show

by

nuolease,

functional

density RNA

an RNA-protein

P can

micrococcal

Consistent

pretreatment of

has

veal

ribonucleases,

A.

observation

intermediate

that

also this

has latter

requires

a buoyant case an RNA

is is

density further component

P

Vol.

96, No.

2, 1980

vivo.

The

in veal

heart

reason

and

explanations

E.

be

with modified

centrifugation

for coli

can

associated highly

BIOCHEMICAL

RNase put

or

have

more

secondary

smaller

than

together

with

data

This

The the

somewhat

Acknowlednements: assistance. S.A.

is

does

these

P from

P complexes

than

All

RNase

between

forward.

conditions.

that

regarding any

source

observed

contribute

coli

will

We are indebted work was supported

be

and consist

to

E.

complexed

Isabel by grants

have it

could

RNase

P,

Our

for excellent the USPBS

RNA

more our

membrane buoyant

density

results,

suggest

an RNA-protein

Pinto from

be

under

the P.

the

a smaller

with

making

RNase

KB cell of

or

of

several

structure

to coli

may

P,

COMMUNICATIONS

density but

enzyme

tertiary

could

for

buoyant

apparent

RNase and

it

could

E.

not

procaryotic

RESEARCH

the

mammalian

Lastly, factors

BIOPHYSICAL

discrepancy

it

fragments.

that

the

AND

taken strongly

complex.

and

technical the NSF

to

Referenoes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Stark, B.C., Kale, R., Bowman, E. J.. and Altman. S. (1978). Acad. Sci. (USA). 15. 3717-3721. Kole. R., and Altman, S. (1979) ibid. 76. 3195-3199. Kale, R., Baer, M.F., Stark, B.C. and Altman, S. (1980) Cell 881-887. (1978) International Review of Biochemistry, 17, Altman. S. Clark, ed. (Baltimore: University Park Press). pp. 19-44. (1976) Cell 9, Koski, R.A., Bothwell, A.L.M. and Altman, S. (1979) Cell 17, 389-397. Garber, R.L. and Al tman, S. Koski, R.A., (1978) Ph.D. Thesis, (New Haven, Yale Universitv). Uchida, T. and Egami, F. (1971) The Enzymes, 4, P.D. Bayer, York, Academic Press) pp. 205-250. (1968) Methods in Enzymology, XII Part B.. L. Szybalski. W. K. Moldave, eds., (New York: Academic Press). pp. 330-361. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254.

837

Proc.

Nat.

19, B.F.C. 101-116.

ed. Grossman

(New and