Absence of thiolated nucleotides in ribosomal RNA of E. coli

Vol.41,No.5,

1970

BIOCHEMICAL

ABSENCE

W.

OF

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THIOLATED

IN

RIBOSOMAL

Scott

Champney

NUCLEOTIDES

RNA

and

College

of

University Irvine,

of California California

OF

Paul

E.

S.

COLI

Sypherd

Medicine at

Irvine

92664

Received November 2, 1970 Summary: associated fractionated ribosomal associated appearing ribosomal

Transfer RNA labeled with 35S was found to be reversibly with the 16~ and 23s ribosomal RNA species of --E. coli by sucrose gradient centrifugation. Recentrifugation of the RNAs resulted in a progressive decline in the amount of label with these molecules and a concurrent increase in the label in the 4s transfer RNA region of the gradient. Washed RNA contained no detectable thio-nucleotides. The

transfer

presence RNA

these

of --E.

compounds

(3).

in

Recently

are

also

Our

studies

molecule

several

present

as

on the

6)

(5,

species.

has

the

secondary

was

a contaminent successive

was

well

structure

and to

examination of

washes

ribosomal of the

suggested of --E.

the

revealed

that

16s

35S-labeled

the

for

described nucleotides RNA

(4).

ribosomal

RNA

thiol-bases

in

transfer

which through

role

thiolated

of the

preparations

in

been

ribosomal

function

RNAs

A

has

of the

examine

ribosomal

2).

that

coli

conformation

RNA

(1,

of t-RNA

which

constituents

nucleotides

documented

structure

made

us

of thiol-containing

been

minor

prompted

Our

types

coli

a report

this

by

of

could

RNA be

sucrose

removed

density

gradients. Materials

K-12 Cells

and

Methods

--E.

coli

strains

AT

722

(obtained

were

grown

MRE from at

37OC

in

600

(obtained

Dr.

A.

L.

Taylor)

ml

of

a minimal

200

from

1328

Dr.

G.

were salts

R. used

Craven) in

medium

and

these (7)

studies.

BIOCHEMICAL

Vol. 41, No. 5,197O

modified

to

Na2S04

was

Cell

contain present

at

suspensions

acetate

and

cell

at

RNA

in

10,000

was

Na

the

an

SW

27

25.

at

Samples

Em

in

a Zeiss

from

each

gradient

and

counted

in

Mark

fractions precipitated

Results

growth

resulted

ribosomal

and

bulk

RNA

L3-50

centrifuged

or

also

the

SW

collected

from

the

for

RNA

content

their PMQ

toluene

-M

EDTA.

layered

25,000

on

of --E. in

and

the

incorporation

RNA,

isolated

transfer

39

for of

for

using

rotor

by

the

5 hours

at

punctured

measuring

spotted

the

RNA

After

species

reanalyzed

on glass

counting

counter.

AT

fiber

fluid,

using

sampling,

were

pooled,

on fresh

sucrose

722

and

MRE

600

90%

these

strains,

was

sucrose

density

species

1329

by

of the

on

of about from

RNA

5

and

rpm

conducted

scintillation

a single

strains

0. 01 - M

above)

bottom

were

scintillation

coli

in

II Spectrophotometer.

fraction

and

was

ultracentrifuge at

was

the

RNA up

was

model

rpm

0. 1% and

0. 001

Spinco were

ethanol

to

(described

Mg

pressure

taken

NaCl

30pC/ml.

the gradients.

Discussion

Sulfur-containing into

II

containing with

and The

sulfate

260

analyzed

added

buffer

26,000

were

and

Chicago

gradient

at

a French

was

glucose.

0. 01 M

acetate

centrifugation

16 hours

dried,

a Nuclear

Samples

Fractions

absorbance

in

Beckman

gradient

tubes

filters,

the

1 rotor.

for

rpm.

centrifuge

was

in

Sucrose rotor

39,000

4OC

species,

at

phenol.

pellet

0. 1 - M

of RNA made

in

0. 5%

04

containing

saturated

the

containing

gradients at

and

35

H2S

was

water

ethanol 5.3)

7. 4)

sulfate

with

with

free

disrupted

dodecyl

fractionation

sucrose

19 hours. SW

(pH

(pH

were

times

supplemented

carrier

buffer

Sodium

absolute

and and

of DNAase

with

centrifuged using

tris

four

For 20%

0. 01 - M

p. s. i.

buffer

NH4Cl

lo-“E

extracted

acetate

and 5 x

20 pg/ml

precipitated

to

MgC12

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

added

limiting isotope.

fractionated gradient

RNA

Vol.41,No.5,

BIOCHEMICAL

1970

23

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

16 S

S

4s

3.c I

i I\ I \ I \ I ’

- 12 I - IO

1.0

-4

0.5

-2

FRACTION

Figure from hours

1: --E. in

Sucrose coli AT a Spinco

density gradient centrifugation of total RNA isolated 722. The RNA was sedimented at 25,000 rpm for 19 SW 25. 1 rotor. Samples of 0. 9 ml were collected, ( ------I at 260 cm measured were t and 0. 09 ml aliquots 35S radioactivity (-----). Brackets indicate fractions pooled analysis.

the absorbance counted for for subsequent

Figure

centrifugation. from

cells

gradient

of AT was

purification 23s

ribosomal

sample

RNA

each

gradient

4s

of the

major

pooled

and

RNA

the

type

was

90%

of the RNA

sulfur

label

the RNA the

RNA

radioactivity

was

extracted

found At

region.

species

RNA

then

of

of the

detected

this

in

stage

the of the

in

the

16s

and

by

the

brackets

gradient.

2 indicates position

separation

transfer

of

each

Figure and

the

the

than

regions

of

centrifugation. the

in 10%

1) were of

More

722.

about

Figure

1 illustrates

located

Fractions in

NUMBER

was

precipitated

separately the

the

(indicated

sulfur

1330

with

analyzed

location label.

of

each Transfer

by RNA

A

ethanol. sucrose

gradient

species RNA

(Figure

in 2A)

BIOCHEMICAL

Vol. 41, No. 5,197O

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 2A: Sucrose density Sedimentation at 39,000 rpm and 35S CPM/ml (-----).

gradient centrifugation for 5 hours. Absorbance

of MRE 600 4s RNA. at 260 nrn (-

Figure 2B: Absorbance

Sucrose at 260

density (-

gradient ) and

MRE

600

16s

RNA.

35S

centrifugation CPM/ml

of

Em

(-----).

Figure 2C: Absorbance

Sucrose at 260

density (-

gradient ) and

MRE

600

23s

RNA.

35S

centrifugation CPM/ml

of

nrn

(-----).

1331

)

BIOCHEMICAL

Vol. 41, No. 5, 1970

contained

essentially

However,

both

the

gradient

RNA of

ribosomal

RNA

substantiate

remaining

from

second in

the

gradient

were

again

RNA

and

260

-nm

the

number

with

of

of

4s

The

sedimented

which

remained results

transfer

location

in

of the

the

4s

transfer

a contamination

RNA

RNA

and

fractions through

for

16 hours

and

radioactivity

containing

sucrose

through

recentrifuged

conducted

Fractions

a third

further

the each

precipitated,

gradient.

RNA.

were was

that

suggested

suggestion,

run

The

in

RNAs

These

separation

each

at

The

gradient.

The

26,000 in

RNA

collected.

a

species RNAs

location

were of the

determined. gradient

profile,

calculated.

the

The

species

is

remained RNAs

radioactivity

specific in

continually

constant,

RNA

the

the

with

per

A

a function

specific

This

from

I as

the

declined.

transfer

(CPM

Table

Whereas

essentially

associated

activity

presented

centrifugations.

contaminating

of

activity

label

result

associated indicates

ribosomal

RNA

the molecules

centrifugations.

From

the

calculations

specific

were

nucleotides

2 x lo5

2 thiobases

per

activity

activity

found

performed

associated of

specific

preceeding

gradient RNA

repeated

activity

the

RNA

ribosomal

removal

adhering

again

each

transfer the

by

was

of

2C).

ethanol

species unit)

of the

and

sucrose

detected

label,

determined.

were

each

RNA

2B

through

label

For

sulfur

rotor.

pooled,

sedimented

each

27

radioisotope

ribosomal

the

initial

was

then

23s

gradient.

SW

each

of

the

sucrose

rpm

by

did

(Figures

To

of the

16s and than

region the

all

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to

with CPM/A

indicate

each 260

be

given

the

the

ribosomal unit

75 nucleotides would

after

by

I) is

transfer about

1332

wash,

number

of thiolated

If

a specific

equivalent

RNA 40

centrifugal

maximum species.

(Table in

third

thiobases

(8),

to then of the

an this 1500

average same nucleotides

Vol.41,No.5,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1970

TABLE Loss After

of

I

Radioactivity Repeated

from Centrifugation

Centrifugal Washes

RNA

rRNA

Specific

Activity

4s 105

1.5

x

lo4

3.4

2x

3.0

x

105

3.5

x

lo3

3.

3x

1.8

X IO5

1.4

x

lo2

7.3

of

third per

16s

RNA

molecule.

a specific

1. 4 x lo2

CPM/A

260

the

presence

indicating

molecule.

For

the

and

conclusions

results

strain

MRE

experiments as

in

the

binding

RNA

to

and

Monier

partial support ribosomal

was

species, have

the

suggest

constituents

of

transfer

RNA

contaminating used

23s

of

thiobase

per

5 x lo3

CPM/A

observed

for

of 0. 03

thiolated

16~

lo3

6x

10~

x

IO1

RNA

260

the

x

16~

would

unit.

A

RNA

after

the

(or

less)

nucleotides

a comparable

number

been

with

obtained

thus

was both

specific

computed.

AT

722

and

600.

These reported

One

activity

wash,

Similar

235

x

molecule

activity

16s

2.0

the

the

260)

1x

comprising give

(CPM/A

RNA

extraction

sequence our

RNA Current

RNA

ofs

thiolated

RNA

(4)

(9)

has

also

of both that

no

9

1333

in

the

would

be

indicated on

thiolated

are

the

recently contained

salt

expected The by

nucleotide

RNA nucleotides

fact high

RNA.

been

ribosomal

nucleotides

Indeed

ribosomal

experiments

analysis

conclusion

the

molecules.

to the

ribosomal (10).

ribosomal

procedure

of contaminants

16s

that

in

concentration

to enhance

binding

of transfer

Marcot-Queiroz composition

molecules are

and

(11, present

12, in

the

13)

Vol. 41, No. 5, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Acknowledgements These of General (GB8402).

studies Medical

were Sciences

supported (GM12686)

by

grants and

the

from the National

National Science

Institutes Foundation

References

8. 9. 10.

N., J. Biol. Chem. 240:3975 (1965). David, H., Studier, M. H., Science 161:1146 (1968). Hung, L., Jones, D., Proc. Natl. AZ. Sci. U. S. 53:979 (1965). Cotter, R. I., Gratzer, W. B., Biochem. Biophys. Res. Commun., 39:766 (1970). Syphzd, P. S., J. Mol. Biol., --In Press. Brownstein, B. H. and Sypherd, P. S., Science, In Press. Sypherd, P. S. , Strauss, N. , Proc. Nati. Acad. %i.. , 49:400 (1963). J. Biol. Chem. 242:4072 (1967). Lips=, M. , Doctor, B. P., Robinson, H. K., Wade, H. E., Biochem. J., 106:897 (1968). Marcot-Queiroz, J., Monier, R., Bull. Sot. Chim. Biol., -47:1627

11. 12. 13.

Fellner, Fellner, Fellner,

1.

2. 3. 4. 5. 6. 7.

Lipsett, Carbon, Carbon,

M. J., J.,

(1965). P., Sanger, P. , Ehresmann, P. , Ehresmann, (1970). -13:583

F.,

Nature 219~236 C. , Ebel, J. C. , Ebel, J.

1334

P. P.

(1968). , Nature , Eur.

J.

225:26 (1970). Biochem.