Differences in physical and biological properties of 50S ribosomes and 23S RNAs derived from tight and loose couple 70S ribosomes

Vol. 124, No. 3, 1984

BIOCHEMICAL

November 14, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 970-978

DIFFEBENCES IN PHYSICAL AND BIOlOGICAL PBOPEBTIES OF SOS RIBOSOMES AND 23s BNAs DEBIVED FBOM TIGHT AND l0OSE COUPLE 705 BIBOSOMES D.P.Burma, D.S.Tewari,

A.K.Srivastava, S.Srivastava D. Dash and S.K. Sengupta+

Moleoular Biology Institute of Medical

Unit, Department of Bioohemistry Soienoes, Baoaras Hindu University and

*Department

of Chemistry, Banaras Hindu Varanasi-221005, India

University

Received September 28, 1984 Tight oouple (TC) 50s ribosomes on treatment with kethoxal lose their oapacify to associate with 30s ribosomes whereas loose oouple (IC) 50s ribosomes on auoh treatment fully retain their assofrom treated oiation capaoity . The same is true for 235 BNAs isolated a(# ribosomes or isolated 235 RNA8 direotly treated with kethoxal, so far as their oapaoity to aesooiate with 165 RNA is oonoerned. At oerTC 23s RNA is highly susoeptible to the tain 3416” ooaoentrations nuoleolytio aotioa of single-strand speoifio enzyme BNase I; LC 23s The b$g+*-dependenoies of the two speaiesT;; RNA is quite resistant. 23s RNAS for association with 16s RNA are also quite different. fluoresoenoe enhancement of ethidium bromide due to binding to TC 23s of I.C 23s RNA is slightly less than 7.C 23s RNA. The hyperohromicity RNA due to thermal denaturation is somewhat mole than TC 235 RNA. lC 235 RNA has slightly more elliptio CD speotrum than TC 23s RNA. These results olearly show that 235 RNA8 present in TC and l.C 50s It has been reoenfly ribosomes are distinot from each other. demonstrated in this laboratory that they oan be interooaverfed by the agents involved in translooatlon and thus appear to be 0 1984 Academic Press. Inc. oonf ormomers. It

is well the

population, oiated

at

higher

Kg++

tight

low Mg+*

active

former

are damaged

derived

and native

oouple

705 ribosomes oapaoities

been made from 0006491X/84 Copyright All rights

0

of

oouples

than

where

for the

the

subunits

ones. It

present

Inc. reserved.

of 705 ribosomal

It

ha8 been

970

the

require are

suggested

realised in

difference

of 70s rihoaomes

to understand

asso-

Loose oouples

was also

the

remain

whioh

respeotively for

$1.50 1984 by Academic Press, reproduction in any form

oouples

association.

are responsible

to time

two types

30s and 505 subunits

(i-4).

505 subunits

the

are

and loose

tight

ribosomes

of time

there

oonoentrations

oonoen trations

biologically

ciation

known that

that

the

the

tight in

and loose the

(5,6).

differences

that

less

asso-

Attempt in

the

has

BIOCHEMICAL

Vol. 124, No. 3, 1984

of 50s ribosomes

constituents

whioh

will

be henceforth

respeative will

ly.

derived

referred

No differenoe

be shown here

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

that

from

markedly

in physical

likely

involved

in

to the

P site.

the

and loose

couples

to as TC and Ix: 50s ribosomes

oould,

however,

23s RNA8 derived

differ

tight

from

and biological

traoslocation

be detected

(6).

It

TC and lC 50s rihosomes

properties

of peptidyl

and are most

tRNA from

the

A site

Materials

and Methods TC and LC 50s ribosomes were isolated by suorose density gradient centrifugation (in presence of 4mM kg*+) aocording to the method of Chapman and Noller (7). TC sod U: 235 RNAs were isolated from the oorresponding 505 ribosomes by the method of &nils et al.(S). The kethoxal treatment of 50s ribosomea were oarried out as described by Herr and Noller (9). In order to determine the association oapacity before or after kethoxal treatment TC or l.C 50s ribosomes were mixed with equivalent amount of 30s ribosomes and subjected to suorose gradient (5-30$) centrifugation in TMA oontaining 2OmM Tris-RCl, pH 7.5, 3omM NH4C1, iombi Mg(OAo)2 and 6mMP-mercaptoethanol at For measurement of the association capacities of l28,oooxg for 2.5 hr. TC or I& 23s BNAs eaah was separately mixed with equivalent amount of 16s RNA in 2OmM Tris-HCl, pH 7.5, 4OOmM KC1 and varying amount of Mg(OAo) and subjeoted to suorose gradient (5-20$) oeotrifugation at to the method of Burma et al. (10). 96,000x8 f or 6 hr aoaording The kinetios of degradation of 50s ribosomea (both TC and lC) were followed by measuring the hyperohromicity at 260 nm in PMQ II Zeiss speatrophotometer according to the method of Dutta and Burma 11). The thermal denaturation of TC and lC RNAS was followed in Gilfor d spectrophotometer attaohed with a thermoprogrammer. The binding of ethidium bromide to TC and Lx: 23s RNAs was studied by measuring the fluorescence produoed (exoitation at 540 nm, emission at 590 nm) in a Perkin-Elmer spectrof luorometer. The oircular diohroio measurements were oarried out in a Jasoo Mode 1 J-SOOA speotropolarimeter. Results Effeats an dI1:

of kethoxal Noller

treatment and his

aoworkers

50s ribosomes

on treatment

kethoxal

their

oapaoity

subunits

in

tight results

lose couple

presented

kethoxal iOmM ?dg*)

lose

their

whereas

on the

with

assooiation

showed that

guanine

base with

is

TC SOS ribosomes oapaoity

l.C 50s ribosomes

fully 971

with retain

of TC both

specifio

each It

experiments. that

oanaoities

(7,9,12)

to assooiate

their

in Fig.lA

the

association

30s and

reagent

other. evident

They from

on treatment

30s ribosomes their

used the

with (even

association

at

Vol. 124, No. 3, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

23s 4

l 6si23s

L

*

4

8

12

16 20 FRACTION

NUMBER

Effects of Kethoxal treatment on the association capacities Fig.1. of TC and lC 50s ribosomes and TC and II: 23s BNAs with 30s ribosomes and 16s RNA respectively ““P-labeled TC and IX 50s ribosomes (40 pmole each) were individually incubated with kethoxal in triethanolamine-IICI, pII 7.0 under the condition described by Herr and Noller (9). Following alcohol precipitation, solution and dialysis, either type of SOS ribosomes (7.8 pmol, 2x104 counts/min) was separately mixed with equivalent amount of 30s ribosomes in TMA containing IOmhf Mg++ and individually subjected to sucrose gradient (S-3&) centrifugation as described un@r *Experimental’. The gradient contains the same buffer (TM). P-labeled TC and IX 23s RNAs were isolated from kethoxal-treated TC and IC 50s ribosomes and 4.7 pm01 (2~10~ counts/min) of each were individually incubated with equivalent amount of 16s klNA in reconstitution buffer (2OmM Tris-HCl, pH 7.5, 4001&I KC1 and 2OmM Mg++) aud subjected to sucrose gradient (S-2&) centrifugation for 6 hr at 96,oOOxg as described under 8Experimen+fa11. The gradient contained the same reconstitution buffer. B. -A--

A.

-O--32P-labeled -o-

TC 50s ribosomes

32P-labeled

I.C SOS ribosomes

The same is

.

treated

TC and II: 50s ribosomes with

23s RNAs are the

behaviour

reflected

found

16s RNA is treated

with

-A-

to be true

capaoity

capacity

32P-labeled 32

(Fig.lB)

oonoerned. kethoxal

of 50s ribosomes

for

(results

towards

in 23s RNAs. 972

P-labeled

LC 23s RNA

23s RNAs from

so far This

TC 23s RNA

is true not

kethoxal

as their

kethoxalassociation

even when naked presented). treatment

Thus is

Vol. 124, No. 3, 1984

Kinetics

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of degradation

of TC and LC 50s ribosomes

RNase I has been studies

on the

ribosomes

behave

concentration resistant at

a rapid

struoture

rate

At

enzyme

h$g++

However,

and at high

Mg*‘+ concentration

The results

obtained

at 2.5,

(13-16).

are

both

also

quite

are degraded observed

are degraded

are

indicative

4

6

8

10

12

to RNase I. of

14

MINUTES

Fig.2.

Kinetics of degradation of TC and LC 50s ribosomes by RNase I at different Mg++ concentration 1 AzCo unit of each type of 50s ribosomes (LC or TC) was incubated with 4 units of RNase I in a total volume of 1 ml containing 100 pmoles Trig-XI, pH 7.0 and varying amounts of t&+. The increase in A260 was followed ot 260 nm, as mentioned Under 'Experimental'. Mg++ (mx) 10.0 0.1 2.5 5.0 20.0 ?..I2 cl A V 0 0 TC 0 A v n + 973

at

rapidly

,

2

the

on the

are

are resistant

5 and 10 mbI I@“‘*

in

TC and LC 50s

TC 50s ribosomes

Mg++ both

(2omM)

laboratory

FlNase I depending

differences

at O.imM

this

II: 50s ribosomes

whereas

Similar

in

subunits toward

2.5m?d

action

(Fig.2).

5 and lOmbI Mg++.

used

of ribosomal differently

quite

of I&++. to the

extensively

by RNase I

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 124, No. 3, 1984 was added

to all

were

rinsed

with

0.2

bound

rapidly

N NaOH.

L3H]-NTP

were

for

4 times

were

was determined done on 5,8,23

development

displacement TTX displacement statistically

regard

of all

in young

not

of the

ages

showed

than greater

cultures

(day

in older

cultures

1.

SC-DRG development determinations.

than

of

variance

means

indicates

with

error

is

3 = day

the

8 > day

981

that with

younger

cultures,

and cultures

(Days)

(1 nM) binding

Student-Newman-Kuels day

A one

on day 27 (p<.O5).

Each value

bar

27).

showed

(p<.O5)

was no

j’, 2327

of C3H]-NTP

in culture.

The

assays

in day 23 cultures

TTX (1 $I) displacement

(day

In these

displacement

cultures.

5) and there

from each other

binding.

in

1 shows TTX (1 PM)

on 5, 8, 23 and 27 day old

AGE OF CULTURES

Figure

TTX was observed Figure

different

of [3H]-NTP

content

1951 (14).

et al.,

with

developmental

significantly

more displacement

three

four

for

cultures.

maturation.

(1 nM) binding

to TTX displacement

TTX showed

with

dissolved

The protein

of Lowry

of C3H1-NTP binding

displacement

of variance

day 5 and day 8 were

method

were

Cultures

HCl and assayed

spectrometry.

by the

was greatest

incubation.

and cells

with

and 27 day old

significant

way analysis

neutralized

and disappeared

of C3~l-~~p

30 minute

PBS (10°C)

scintillation

RESULTS : The displacement early

an additional in cold

Aliquots

by liquid

of the culture Assays

cultures

during

is the mean of 3

S.E.M.

One way

multiple 23 > day

analysis

comparison 27.

of

BIOCHEMICAL

Vol. 124, No. 3, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

r x lo2

Fig.4.

Binding

of ethidium

bromide to TC and u 23s RNAs

The binding of the dye was studied type of 23s RNA in 2.5 ml of reconstitution measurement wns made at every step following increasing amount of ethidium bromide up to method was followed in drawing the straight plots (18) A

Binding

lC

RNA

A TC 23s RNA

bromide

to TC and IC 23s RNA8

23s

of ethidium

The fluoresoenoe to rENAs

(17), somewhat

binding

of EtBr

23s RNA binds indioates

with

less

than

aase

DNA.

bromide

of DNAs.

From the

somewhat

This

more ethidium

ordered

The binding

Soatahard

is

bromide

structure

in agreement

in

with

the

enhanced

is

to TC and L1: 23s RNAs (Fig.4)

some more

TC 23s RNA.

of ethidium

suah as in the

rENA is

with 42 pm01 of each buffer. The fluorescence the stepwise addition of O.C45mM. Least square lines for the Scatchard

it

on binding of the

plot is

that

(18)

evident

dye to

of the that

TC 23s RNA.

LC This

LC 23s RNA in oomparison

data

obtained

to

by treatment

BNase I.

Thermal

denaturation

profiles

The thermal oompared

starting

with

melting equivalent

of 23s RNAs profiles

of TC and IC 23s IWAs were

amounts 975

(Calculated

from

‘Lzso)

of

BIOCHEMICAL

Vol. 124, No. 3, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

r

0.60 1 t 0.58 aP

o 0.56% a 0.54-

9‘ 0 x m

0.52!

05 0.50.35

45

55

65

75

06

85

OC

I 1 I t 1 J 220 240 260 280 300 320

X(nm)

Fig.5.

Thermal denaturation of TC and LC 23s RNAs The melting profile of each type of 23s RNA (0.15 A260unit) in 0.3 ml of 20 mMTris-HCl, pH 7.6 and 400 mMKC1 was followed in Gilford spectrophotometer attached with thermoprogrammer, as mentioned under Materials and Methods. Fig.6.

CD spectra

of L.Cand TC 23s

RNAs

The circular dichroic measurements were carried out in a Jasco spectropolarimeter as mentioned under i\iaterials and Methods. e

me- LC 23s iNA

the

two.

mately

On complete

melting,

2%) hyperchromicity

may be assumed

that

data

also

indicate

than

TC 235 RNA (Fig.

CD spectra

that

WA

IC 23s RNA has slightly than

the

TC 23s

TC 23s

more

To aocount

RNA.

for

two R3iAs have different

conformations.

I.C 235 RNA has slightly

more

ordered

(approxithis,

it These

structure

5).

of 23s RNA8 The analysis

has

slightly

that

the

more former

of CD spectra

ellipticlty

has somewhat

(Fig.6)

than more

shows that

TC 23s RNA indioating ordered

structure

I.C 23s RNA thereby

than

the

latter. Discussion It between

is

clear

from

the

TC and LC 50s ribosomes

present lies 976

studies

that

in the constituent

the

difference 235 IWAs.

The

BIOCHEMICAL

Vol. 124, No. 3, 1984

TC 23s RNA isolated RNA treated

from

directly

kethoxal-treated

with

with

association like

those

of the

parent

towards

degraded

RNase

rapidly.

indicated

by our also

as thermal

however,

must most

been

studies

shown that

by treatment

can be converted GDPCH2P. with

EF-G, to

Further,

the

progress

to throw

proposed

the

binding

two RNAs.

50s are

conformations

region

The difference

as

in conformastudies

The best

acid (less

as well evidence

whereas

on the

mechanism It is

by EF-G and TC or lC 50s ribosomes

two.

These

of the

of the

results

conformational

in change

observed

to TC 23s RNA. already

observations

Work is

has been recently

converted

TC 50s ribosolnes

efficiently)

of the

laboratory

to TC 505 ribosomes

of either

a mixture

in this

out

of 23s RNA in translocation.

basis

interesting

of E

stalk

can he converted

translocation.

on the

different

TC ribosomes

LT/Ll2

carried

treatment in

treatment

quite

by CD measurements.

individual

LC 23s RNA on heat

the

GTP and fusidic

involvement

of 23s RNA during

been

of

LC 50s ribosomes

light

also

in

bromide

experiments

EF-G and GTP results

indioate

the

ethidium

Ix: 50s ribosomes

with

where

(13,14).

profiles

Some preliminary have

in

of the

the resistance

conditions

likely

obtained

is

with to

The most

be some differences

by the

deaaturation

curves

16s RNA are

studies

I under

earlier

suggested

have no capacity

The Mg++ -dependency

these

(or TC 23s

to associate

TC and lC 50s ribosomes.

There

of TC and lC 23s RNAs,

has,

TC 50s ribosomes

30s ribosomes.

made during

ribosomes

is

has no capacity

of TC and LC 23s RNAs with

observation

tion

TC 50s ribosomes

kethoxal)

16s RNA as kethoxal-treated associate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

obtained

that

A model

has

(12,20).

Acknowledgements Thanks are due to the University Grants Commission, New Delhi, the Department of Science & Technology, Government of India, The Council of Scientific and Industrial Research, New Delhi and the Indian Council of Medical Research for financial assistance. Thanks are also due to Pr0f.M.V.R. Rao of the Department of Chemistry, University of Delhi, Delhi for the help in CD measurements.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 124, No. 3, 1984

Referenaes Noll,R., 1. 2.

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

Noll,

M.,

Hapke, B. and Van Diggelen, G. (1973) in of transoription and traaslocation in eukaryotes' (Bautz, E., ed.), p.257. Noll, M., Hapke, B. and Noll, H., J.Mol.Biol., 80, 519(1973) Noll, M. and Nell, II., J,Mol.Biol., 90, 237(1974) Hapka, B. and Noll, H., J.Mol.Biol,, 105, 97(1976) Van Diggelen, O.P., Heinsius, H.L., Kalousek, F. and Bosch, L., J.Mol.Biol., 55, 277(1971) Van Diggelen, 0.~. , Oostrom, H. and Bosoh, L., Europ.J.Bioohem., 39, 811(1973) Chapman, N.M. and Noller, H.F., J.Mol.Biol., 109, 131(1977) F.A. and Cantor, C.R., Nuclei0 Acids Res., Amils, R., Mathews 5, 2486(1978) Herr, W. and Noller, H.F., J.Mol.Biol., 130, 421(1979) Burma, D.P., Nag, B. and Tewari, D.S., Proc.Natl.Acad.Soi., 80, 4875(1983) U.S.A., Dutta, A.K. and Burma, D.P., J.Biol.Chem., 247, 6795(1972) Herr,W., Chapman, N.M. and Noller, H.F., J.Mol.Biol., 130, 433( 1979) Raziuddin, Chatterjee, D., Ghosh, S. and Burma, D.P., J.Biol.Chem., 254, 10675(1979) Byasmuni and Burma, D.P., Bioohem.Biophys.Res.Com.,io4, QQ(lQ82) Burma, D.P., J.Sc.Ind.Res.(India), 38, 31(1979) Current Science (India), 51, 723(1982) Burma, D.P., Stevens, L. and Pasooe, G., Bioohem.J., 128, 279(1972) Saatchard, G., Ann.N.Y.Aoad.Sai., 51, 660(1949) Burma, D.P., J.Sc.Ind.Res.(India), in press Burma, D.P., J.Bioscienc@s (India), in press Regulation

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