Accessibility and structure of ribosomal RNA monitored by slow tritium exchange of ribosomes

J. Mol.

Hiol. (1981)

Accessibility

146, 241-257

and Structure of Ribosomal RNA Monitored Slow Tritium Exchange of Ribosomes XEAL

M. FARBEnt

Barth laboratory, (‘olltmbia l’niveraity. (Receiwd

ASD

('HARLEs

It.

by

('A~‘TOR

Depa~rtment of (‘hemistry Sew York. *V.Y. 10027, i’.S..3. 9 September

1980)

The structure of Escherichia coli 5 S ribosomal RNA within the ribosome was examined by comparing the slow tritium exchange rates of specific sites with those rates in the free 5 S rRNA. The pattern of tritium incorporation of 5 8 rRNA in 70 S and 50 S particles is nearly identical and differs significantly from that of 5 S rRSA free in solution. The exchange rate throughout most of the rRNA is markedly suppressed, predominantly due to steric effects by other ribosomal components. Only four short segments show facile exchange: G,,, A-CJ5, A-A-C, I0 and a G from one of the four C-C-U sequences, presumably G13. The presence of bound tightly to the P site of poly(U)-programmed ribosomes deacylated tRKAPh’ caused no significant difference in the exchange pattern of 5 S RNA. The fact that (C:)-A-A-C,7, postulated to base-pair with the T-Y-C-G sequence of tRNA, was not accessible in any of these ribosome or subunit samples seems to eliminate a direct functional role for these 5 S rRXA residues. The rates of tritium exchange into 16 S rRNA free in solution, 16 S rRNA in the 30 S subunit and 16 S rRNA in the intact 70s ribosome were also determined. The results reflect a loose 30 S particle structure which tightens upon 76 S ribosome formation.

1. Introduction Slow tritium exchange has proven to be a very powerful technique to study transfer RNA conformation (Gamble et al., 1976; Schoemaker et al., 1976) and tRNA protein interactions (Schimmel & Schoemaker, 1979: Farber B Cantor, 1980). In the preceding paper (Farber & Cantor, 19X1), we used this approach to investigate the structure of free Escherichia coli 5 S ribosomal RNA. Single-stranded and double-stranded regions were identified and possible tertiary interactions mapped. In Go. mature ribosomal RNA is not found free of proteins. Virtually all of it is bound as an integral component of the ribosome. Structure studies of RNA in the ribosome pose formidable challenges. However, this is the biologically more significant state. and it should be the ultimate goal of solution studies. For many t,echniques, this is not feasible due either to the conditions required or simply due t,o t’he complexity of the ribosome. However. the slow tritium exchange approach is well suited to study aspects of the situation of ribosome-bound RNA. t Present

address

tn22-2836/xl/Mo241-17

The Biologiml $02.00/0

Laborat~ories.

Harvard “41

I’niversity. (P 1981

(‘ambridge,

Avatiemic~

MA 02138.

Press Inc. (London)

ITS.;\ Ltd

212

s. 11. FAKKER

In the and

present

study,

slow

in

ribosome.

16 S rRNA

molecules

free

compared. t)est

in solution,

Studies

of precise

prrrrquisite txchange system

t’he of t,he

hypotheses to

do

all

not

incubat’ions

in

to probe

incorporation residues

of the

role

of thpse

residues

in 5 S rRIV4

is to

ribosomes. and

show

that

the

Henvr

we

must,

functjional

in the

of

sp4fic

studies

and

structure

into the

subunit

the rates

respective

structural 3H,0

is used

tritium

in the about

these

exchange The

exchange

damage

maintains

tritium

AIJI~ (‘. 12. (‘AXTOK

intact,

during

arr allow

function.

usrd

demonstratcx

rRSX

rihosome

5 S rRSA

conditions

integrity

of :5 S the

for first t’hr

a .4

tritium t,hat

t)he

length>

required.

2. Materials

and Methods

(a) Ribosomv and subct ttit /)rrparatiotl (i)

Salt-washed

ribosomes

Escherichia coli MRE 600 cells were grown in I”,, Bactotryptone. OK’j, yeast extract, O-.‘,q, NaCl to the early log phase (-+I,,, = @5), slow cooled to 15°C’. and harvest,ed on ice. The fresh cells were broken by grinding in twice the weight of alumina (Alcoa A30.5, Bacterial grade). brought to 100 ml in TMA I buffer (10 m&r-Tris. HCI. 10 mbvMg(Ac),, 60 rnw-NH,(‘I, 6 rn>v 2-mercaptoethanol, pH 7.4) and stirred in the presence of 025 mg electrophoretically pure DNAase I (Worthington) for 30 to 60 min at 4°C’. The extract was centrifuged at 12,000 revs/min for 30 min (SS34 Sorvall rotor) to remove the alumina and then centrifuged at) 45.000 revs/min for 0.33 h (FA 50.2Ti Spinco rot,or) to eliminate cell debris. Ribosomes were then pelleted from the resulting S30 supernatant by centrifugation at 45,000 rrrs/min for 2 h in the FA 50.2Ti rotor. These crude ribosomes were either directly used for subunit or RX,-\ preparation, or purified further by successively resuspending and pelleting first in TMA 1 buffer and then in TM,4 I buffer containing 1.5 M-NH,(‘I (at 45,000 revs/min for 2 h in the FA 50.2Ti rotor). The high-salt-washed ribosomes were resuspended in TMA I buffer, and clarified by low-speed centrifugation. (ii )

Tight couplr ribosomw

The procedure of Wishnia et al. (1975) was used t,o prepare tight couple (A type) ribosomes. About 1 g of the solt-washed ribosomes was purified on a 10:; to 30% sucrose gradient in TM:2A III buffer (10 mM-Tris.HCl, 5 mM-Mg(Ac)B, 60 rnM-NH,(‘I. 6 mM-2-mercaptorthanol. pH 7.4) at 25,000 revs/min for 17 h in a Beckman Ti15 zonal rotor. The 70 S fractions were and concentrated by centrifugation (23,000 collected and brought to 10 mM-Mg(Ac)2, rers/min for 22 h in the FA 50.2 rotor). The pellets were gently resuspended overnight in TMA I buffer at a concentration of 20 mg/ml. and quick-frozen. (iii)

Ribosom,al subtcuits

Ribosomal subunits were prepared from crude 70 S ribosomes by zonal centrifugation according to Eikenberry rt al. (1970). The ribosomes were resuspended and dialyzed against TMA II buffer (10 mw-Tris.HCI, @3 mrv-Mg(Ac),. 30 mM-NH,CI, 6 mM-2-mercaptoethanol. pH 74) and centrifuged in a 7.4’& to 387; hyperbolic sucrose gradient in TMA II buffer at 26.000 revs/min for 14 h in the Ti15 zonal rot,or. The separated 30 S and 50 S subunit peaks were collected and brought to IO rnx-Mg(Ac),, pelleted at 30.000 rers/min for 16 h in the FA 50.2 rotor, resuspended in TMA I buffer, and quick-frozen.

(b) .4 nalytic sucrose

gradients of ribosomal particles

Small analytic gradients were used to ascertain the purity of the ribosomes and subunits. The following conditions were used in the SW 50.1 rotor at 45,000 revs/min with 3 to 5 -1,,, units of material : for 70 S, in a So/, to 20 “/;, sucrose gradient in TMA I buffer, 1.25 h

centrifugation or in a 10% to 30% gradient, 1.5 h; for subunits, in a 5% to 20% sucrose gradient in TMA I I buffer for 2 h or loo/, to 30% gradient for 2.25 h. The tubes were pierced. 3-drop fractions collected, 1 ml H,O added to each and the absorbance at 260 nm measured. The zonal preparations used produced extremely pure particles: the analytic sucrose gradients indicated no contaminating particles present. (c)

Protein

synth,eRis

assay

A modification of the Nirenberg & Matthaei (1961) polv(U)-dependent poly(phenylalanine) synthesis assay was used to assess the functional activity of the ribosomes and subunits. Ribosomes were in limiting quantities and the soluble enzymta fraction (SIOO) 1, as optimized. Subunits were assayed in a twofold excess of the complementary subunit. From @25 to 0.5 4,,, units of subunit or e-5 to 1 A,,, units of ribosomes were act,ivated (Zamir et al., 1974) and incubated for 30 min at 37°C in a 250.~1 assay mixture containing 5 to 10 pL1 of SlOO enzymes, 5 pg phosphoenolpyruvate kinase. 250 pg E. coli tRS.4 (Boehringer), 250 pg poly(U) (Sigma). The final concentrations of the reaction components were 50 mw-Tris.H(‘l (pH 75), 20 mM-Mg(Ac),, 50 mi%-KCI, 250 mMNH,(“L. IO mM-ATT-‘, @03 mM-GTP, 75 mM-phosphoenolypyruvate, @05 M of each amino acid except phenylalanine, 0005 mM-[‘4C]phenylalanine (100 mC’i/mmol, 0.125 &“I per rract,ion) and 6 rnhr-2-mercaptoethanol. (d)

RS.4

prrparatiorc.

The highest, purity rRNA can be obtained by growing cells, isolat,ing the crude ribosomrs and rxt,rarting with phenol all in one day. The crude ribosomes were resuspended in TMA 11 buffer and added to an equal volume of SC’E buffer (015 M-h’aCI, 0.015 M-Na citrate, 10 rn%lThe mixturtb EDTA. pH 7.0). WI vol. loo/, sodium dodecyl sulfate and 02 vol. 2* “/” bentonite. \ras kept on irck and intermittently vortexed with an equal volume of WE buffer-saturated phenol for 5 min. The layers were separat.ed by centrifugation at 10,000 revs/min for 5 min in t,hc SS34 rot,or. The procedure was repeat)ed 4 to 6 times. The final RNA solution was tsxtracted lvith ether several times and precipitated twice wit’h ethanol. The total ribosomal RNA was fractionated by zonal centrifugation. Up to 1500 A,,, units of rRSA in WE buffer, made 3 “/o in sucrose, was applied to a 5% to 20% sucrose gradient, (linear with volume) in WE buffer and centrifuged for 22 h at 30,000 revs/min in the Til5 zonal rotor. The a-l 260 of the pumped fractions was monitored; appropriate fractions pooled. dialysrd against SCE buffer, and precipitated with ethanol. ‘l’hra purity of the RNA was assayed by its ability to reconstitute into active particles (Traub pt nl., 1971) and by polyarr$lamide gel electrophorrsis. rsing the above protocol, pure 16 S rRh’;\, active in reconstltut,ion of 30 S subunits and showing no degradatiotr products on polvacrylamide gels. was reproducibly obtained. The 3 S rRNA. however. \vas rontaminat,ed iith tRSA present in the crude ribosomes. It) was purified on a preparative polyacrylamide gel and has been sho\l n to be active in rrconstitut,ion of 50 S subunits and ln~rcs by analytic l)olyacrylamide gel elec%rophorrsis (Farber 8r Cantor. 1980). (e)

Slow

trifiuvn

cxcharqP

ir~to

rihoaomal

particlrs

The genrral schtbmr of the tritiation protocol is shown in Fig. 4. To minimize, dilution of the 3H,0-specific activity. thr ribosome stock solutions had to br very concentrated. Ribosomes were first activated by incubation at 37°C’ for 20 min it1 20 rn>r-Tris. H(‘I (pH 7.,5). 200 mnl-NH,(‘I and 3 miv-2.mercaptoethanol. The solution was tht,n ~rntrifugrd for 4.1 h at 35.000 rrvs/min in the SW41 rotor and the supernatatrt discarded. It was found that, the tight -couple ribosomr pellet could be gently resuspended overnight, at l’(’ in (‘AC’ buffer (10 mu-Na cacodylate, 10 mM-Mg(Ac),; 50 mM-.NH,(‘I. 6 rnbl2-mc~rcaI)toethanol, pH 7.0) to yield perfectly clear solutions ranging from 160 to 170 mg/ml. Ribosomes and subunits were incubated at 40 to 60 mg/ml in 2 to 4.5 Ci 3H,0/ml at 37°C‘ for 24 t)o 28 h in (“.2(’ buffer. One reaction contained 376 $ of 2480 .4 260 units/ml ribosomes. 130~1 (‘.-I(’ I)uffer. 19 PI 10 x (‘A(’ buffer. and 175 PI of 12.5 Vi 3H20/ml. The reactions wer(l II

244

S.

M.

FAKRER

ASD

(‘.

K. (‘.-\N’l’OH

t,erminated by quenching on ice for 5 min and precipitating the particles with 2.0 vol. cold ethanol. After centrifugation, the supernatant containing the bulk of the radioactivity was carefully removed and discarded. The pellets were resuspended in 1 ml CAC buffer and exhaustively dialysed against cold buffer. Three to four rapid dialyses (20 to 30 min each) removed the free 3H,0 and very fast exchanging t)ritium from both the RNA and protein. The samples were additionally dialysed overnight, at 1°C. The samples were extracted with phenol several times and the RR‘A fractionated on So, to 20’S, sucrose gradients in SCE buffer (30,000 revs/min for 16 h in an SW41 rotor). The I6 S 13H]rRNA peak was pooled, redialysed overnight against Hz0 and precipitated with ethanol. The 5 S [3H]rRNA peak was similarly dialysed, concentrated, and further purified on a ;S($;, polyacrylamide gel at 100 T’ for 3.5 h. (III experiments in which tRiYA was present during the tritiation reaction, a ST/, polyacrylamide gel was found to resolve the bands adequately and yield the highest recovery after elution. (‘ontrol experiments showed: 357, recovery of .5 S RX.4 from a 157; polyacrylamide gel, 450 ./O recovery from a loo,,, gel and 55 to 60”~~ recovery from a 546 gel using the elution procedure described below.) The gel bands were eluted b;Y a modified crush and soak technique. The bands were located by ultraviolet shadowing. excised, and crushed by passing through a 3-ml syringe (without needle) directly into a lo-ml syringe. The IO-ml syringe, fitted with 2 layers of glass-fiber filters (Whatman GF), conveniently fits into a 15.ml Corex tube. The gel mat,erial M-as rovered with 05 M-NH&, 0.01 M-Mg(Ac),, O.l(‘,, sodium dodecyl sulfate, 01 mM-EDTA and left at 1°C’ (t,o prevent exchangr-out) for 24 to 18 h. The RN4 was recovered by brirfl) centrifuging t,he C’orex tube containing the syringe at 2OOOg and washing with the abovtl buffer. The eluant was extracted wit,h phenol and precipitated with ethanol. (f) The T, and was performed

Etlzymatic

digrstions

and

pancreatic RNAase digestions, as described in the preceding

mappi,lg

fragment paper,

oj .i S rK.Vd isolations

and

scintillation

count,iny

The tritium incorporat,ion rates were determined by a specific activity measurement and normalized as described previously (Farber & (‘antor. 1981). For the intact molecules, specific activity was measured as 3H cts/min per .-I 260 unit using 80 cts/min per A,,, unit for l6S rRSA and 1180 cts/min per -d,,, unit for ,5 S rRN4. For the enzymatic digestion products of 5 S rRXA. specific activities were determined from t,he 3H/32P ratio as described in the preceding paper.

3. Results (a) Ribosonws Ribosomes

incubated

or subunits;

and

subunits derived

nsni//tczirr from

the

activity E. coli.

a:ftpr strain

21 hours MRE

600.

cct 37”(’ RNr\ase

1 wcrc

at 37°C‘ under conditions identical to t,hose used for t)he tritiation reaction hut in non-radioactive water. Stringent nuclease-free vonditiona were used during the preparation and all subsequent, handling of these particles. At various irected poly(phenylalanine) synthesis times samples were wit,hdrawn for poly(V-d assays and polyacry!amide gel electrophoresis. The protein synthesis assay results for 70 S and 50 X particles are shown in Figure 1. As can he seen, the particles showed less activity in Tris buffer than in cacodylate buffer. This is presumably due either tjo t,he effects of the primary amine or to the pH since the cacodylate buffer maint’ained 80 to 1009, activity after 21 hours. It was used in all further experiments.

TRITIUM

EXCHANGE

OF

RIBOSOMAL

RNA

245

80

(a)

80 60 \ 40

\

‘\

(b)

20

0

10 Incubation

20 time (h)

FI(:. 1. Time wurse of inactivation of 50s subunits (a) and 70s ribosomes (b). Particles were Incubated at 37’C in Tris buffer (10mwTris.HCl, pH 7.4, 30mwNH,CI, lOmr+-Mg(Ac),, Bmw%mercaptoethanol) (- - - - ) or in (XC buffer (10 mM-Na cacodylate, pH 7.0, 10 mwMg(Ac),. 50 rnM SH,(‘I. 6 mw%mrr(*aptoethanol) (-). .4t roughlv 5-h int,ervals, samples were withdrawn ant1 assa,ved for remaining [‘Y”]Phe-incorporated polv(L-)-d”irected protein synthesis activity.

,L\ similar study with 30 S subunits and another 70 8 ribosome preparation is depicted in Figure 2. Again. in cacodylate buffer, over 900/6 of the original protein synthesis activity was maintained by the particles after 40 hours of incubation. The insert, shows the activity of 30 S subunits over a ten-day period. Significant activity is present, even after three to four days at 37°C. The incubated samples were also analysed on polyacrylamide gels to check for RXA degradation. Samples were first incubated in 05% sodium dodecyl sulfate for 30 minutes at 37°C to disrupt the Fjarticle structure. The gels were purposeI) overloaded to see low levels of degradation products. Figure 3(a) shows a typical 70 S incubation sample. Little rRNA degradation is seen until after 24 hours. A time course of a typical incubation of free 16 S rRNA, shown in Figure 3(b) indicat,es totally intact RNA after 50 hours. Figure 3(c) shows a gel of low molecular weight RNA from ribosomes that were tritiated for 24 hours. In this experiment tRN;Z was bound to the ribosomes during the incubation. Lane 3 is the tRNA/,‘, S rRXA peak of the sucrose gradient and shows the absence of any degradation products from the samples or from larger rR?;As. Lane 2 shows the 5 S [ 3H]rR,SA after further purification from the [3H]tRSA.

Incubation I IO

0

time (days) I

20 Incubation

I

30 time (h)

I

I

40

50

Flc:. 2. Kinetics of inactivation of 30 S subwith () and 70 S ribosornes ( - - - - ) incubatecl (‘A(’ buffer at 37°C”. See the lepencl to Fig. 1 for details. The insert shows the 114(‘JPhr-inc~or~,oration activity of 30 S subunits incubated at 37”(’ for up to 10 tlayh.

in

These results demonstrate t,hat by employing stringent nucleasc-free conditions. ribosomal RKA remains intact and ribosorm particles remain actjive during a 2-L hour incubation at 37°C’. Thus the tritium-exchange approach can he used as a probe of conformation without the risk of structural perturbations. (I))

(‘ontparisott

of thP atrd

16 A’ rRS,-l itt thP

it/tact

itt

soltrfiott.

itt thP 30 A’ srthtrtri/

rihosom~

To see the effect of ribosomc structure on 16 S rRNA conformation and accessibilit,y. 16 S rR)N.A. 30 S suhunit,s and 70 8 ribosomes were incubat’ed in 1..5 ( ‘i 3H20/ml at 37°C’ for 23 to 27 hours in cacodylate buffer cont’aining 10 rn>l-Mg(Ac),. The exchange react,ions were effectively quenched by cooling on ice and the free 3H,0 and rapidly exchanging triGurn removed by prwipitation and exhaustive dialysis at, 1°C’. All samples were extra&t4 with phenol. and the 16 S rRXA4 ma,s suhsequentjly purified on sucrose gradients. Only tritium in slow-lp exchanging sites remained. since the specific activit)y of all three samples remained corMant during additional dialysis (da.ta not’ shown). The rate of tritium incorporation into the (‘(8) position of a free guanosine or adenosinr residue is known ((:amhle 4 ~1.. 1976). The rate of tritium incorporation

TKITlVM

ES(‘tl.-\?r’GE

OF

RIKOSOMAL

“47

RN.4

Cc)

I

2

3

4

5

(b)

I~‘Ic: 3. l’olyac~rylamide gel electrophrmsis of’ rKNA after im~rrlmtion at 37 (‘. At the indicated times samy~les of i0 8 ribosomes (a). anti 16 S rKSA (b) were made (M”,, in sodium tlotircyl sulfate, maintained agarose composite gels. Length ot ;,t 37’ (’ for 30 min and electrophoreseti on %V,, ~)ol~ac~~~lamitlP/O~5”,~ mrnbation wtas~ (a) Lane 1.0 h : 2. 15 h ; 3,23 h : 1.1” h : 5.50 h. (b) Lane 1.0 h : 2. 15 h : 3, ‘3 h : 1.42 h : 5. JO h : ti. !)O II. (kl?r wvpre fixed with 1 M-C”H,(‘OOH, stained with met~hylenc blue anti destained in Hz0 overnight. (I.) .i I!“,, to 15” o gradient polya~~rylamitir ye1 of the extracted I
into a purine in a macromolecule, however. is sensitive to its microscopk wvironmrnt and is suppressed by a combination of inkand intermolecular int~eractions. This reduction in exchange rate is expressed in terms of the ret,ardation coefficient, R, where :

R=

calculated

exchange

experimentally

rate for an open structure observed

exchange

rate

-.

248

S.

M.

FARBER

ANI)

(‘.

K.

CANTOR

The observed exchange rate for the three 16 9 rRNA samples was determined from specific activity measurements (see Materials and Methods). The exchange rate for an open, unstructured 16 S rRNA molecule was calculated from the sum of the exchange rates of the individual nucleotides and the composition of 16 S rRNA as determined from the nucleotide sequence. Table 1 shows the results. The sixfold suppression in the exchange rat,r of free 16 S rRNA over a totally open, unstructured polynucleotide chain is due to the super-position of the effects of base-stacking. and secondary and tertiary basepairing interactions. The ret,ardation in the exchange rate of ribosome-bound 16 S rRNA may be due to additional intramolecular structure or to steric effects of protein-nucleic acid interactions (Schoemaker & Rchimmel, 1976: Schimmel, 1977 : Farber & Cantor, 1980). The difference in R values between free 16 S rRNA and 16 S rRNA isolated from tritium-exchanged 30 S subunits indicates t,hat. whichever mechanism is predominant,, the overall tritium incorporation is very similar. However, the results with I6 S rRNA isolated from tritium-exchange 70 S particles indicate that subunit association greatly affects the 16 S RNA structure. The large increase in the R value indicates a substantial reduction in the tritium labeling.

1

TABLE Effect

of

ribosome

structu,re

RAVE titration

on overall

exchange

rates

Exchange conditions

RNA

6.1 !W 15.1

5 s rRS.4 5 s rRNA 3 s rRN.4

50 5 io 5 iG-4

tHNAPhe t RNAPh’ t

Retardation

(c)

coefficient.

(‘omparison

70 s + poly( defined

of 5 S and

1:)

12.7

in t,he test

rR,VA in

the

in solution, intact

in

the

50 S subr~nit

ribosonle

A comparison of the tritium exchange into 5 S RNA free in solution, in the 50 H subunit and in bhe ribosome was also performed. The samples were incubated in 4.5 Ci 3H20/ml at 37°C for 24 hours. In the cacodylate buffer used, the 5 S rRNA assumes the R conformation. The reaction was terminated and the RNA isolated as described for 16 S RNA. The 5 S rRNA was additionally purified on a ri”,b polyacrylamide preparative gel and the 3H cts/min per .-I 260 unit were measured.

TKITIUM

EN(‘H.-\SGE

OF

RIROSOMAL

249

KSA

The results are given in Table 1. Compared to 5 S rRNA in solution, 5 S rRNA in the 50 S subunits, which we will call 5 S I-RNA (50 S), shows a marked reduction in exchange. 5 S rRNA in the 70 S ribosome, called 5 S rRNA (70 S), labels at an identical rate to .5 S rRNA (50 S). This suggests that the 5 S rRNA in 50 S and 70 S particles is in similar, if not identical, environments and no discernible rearrangement in the 50 S subunit upon association occurs in the neighborhood of the 5 S rRS=\ molecule. This is in marked contrast to effects of t’he 50 S particle on t)hr state of the 16 S rRNA in 30 8 subunits. (d) !!Mium-exchangr

rates

of specijk

sequences

in

5 S rRNI1 (50 S) an,d 5 S rRNA (70 8) To confirm that these molecule-averaged R values of 5 S rRNA truly reflect identical environments of the RR’X in 50 S and 70 S particles (and not the simultaneous rate enhancement in one region of the molecule and decrease in another). a T, RYAase analysis, as outlined in Figure 4, was performed. The three isolated 5 S rRNA samples were enzymatically digested, the products isolated and the labeling rat,e of each fragment determined. To facilitate autoradiographic location of the T, fragments and measurement of their specific activities, uniformly labeled 5 S [ 32P]rRNA was added to each of the tritiated 5 S rRNA samples. The observed labeling rates are converted to R values and plotted in Figure 5. As can be seen from Figure 5, most of the T, fragments of ribosome-bound 5S rRXA show greatly retarded exchange rates. These values are consistent with strong protein nucleic acid interactions and indicate that these regions are embedded in the ribosome structure. This is in agreement with the high R values of the intact) molecule. The pattern of tritium exchange into T, RKAase fragments of 5 S rRNA exchanged in 50 S or 70 S particles appears to be the same within

5SRNA 50 S subunit 70 S ribosome

Incubate 3He0

in

(a) Ethanol ppt f b) Exhaustive dialysis

37°C

40 c

5 S[3H]RNA 5 s [3ktI~~~ (50 s) + 5 s [32P] 5 S [3H] RNA (70 S)

(a ) Phenol extract (b I Sucrose gradient (c) Preparative gel

RNA

40 c

5 S RNA Pz;z;tic

(a ) Autoradiograph ( b) Isolate pancreatic fragments

T, fragment each

into

sample

RNase

( b) DEAE poper electrophoresis 32P]RNA

rate of each

(a ) Autorodiogroph ( b) lsolote T, fragments

(a 1 Tt digest (b) 2-D map -

(a)

5S[3H,

Labeling

FIG. 4. F‘low chart outlining specifir iwi ot’ 5 S rRNA.

the experimental 2-D. 2.dimensional.

protovol

used

to analgse

the slow

5 S RNA

tritium

sample

exchange

ratr

24 k

20 16 12 8 4

p:

II-I I

24 20 16 12 8 4

20 4:

16

LJl

?m-ldLl 123456

9

ion

Fragment

14

16

17 18 19 20

no.

experimental error. Note that T, fragment 5. (:-(‘-,4-&,. the complement, to t,he t,RNA sequence T-Y-(‘-(:. is not, accessible. Only three T, fragments, numbers 1. 6 and 17, show substantial tritium incorporation indicating t,hat only these regions are exposed. Fragment 6 of ribosonlc-houlltl 5 S rRSA appears to incorporate t’ritium faster than the corresponding sequent of 5 S RX;1 free in solution (set below). The effect of 30 S to 50 S subunit association on thew exposed regions can be seen by calculating R,,,/R,,,. This rat,io is equal to k,,,/k,,,. the relative rate of t’ritium incorporation into a fragment of 5 H rRXA in the 70 8 particle wmpared with that in the 50 8 particle. As indicated in Table 2. at the T, fragment level of resolution, there is clearly no difference in fragments 1 and ti. Fragment 17 shows a slight subunit effect : unfortunately, this fragment is 3.5fold redundant in the 5 S RNA sequence. However, according to experiments with ket,hoxal (Delihas rt al.. 1975; h’oller $ Herr, 1971), where the specific (‘Y-C sequence can be determined, only one copy, P-CC, 3. is surface-reactive in the intact, subunit or ribosome. If we

assume that (‘-(‘-(i,, is the only C’-(‘-G fragment exchanging as an exposed region and that the other C-C& are retarded 20 to 30.fold, the labeling rate of (‘-CC: 13 can be estimated. Using these corrected rates, the k70s/k50S ratio for (‘-(‘-(:,, is 0.79. This is just outside our experimental error and indicates that a slight protection of (iI occurs upon subunit association (Herr Kr Soller. 1979). More information about’ the pattern of tritium exchange is available from pancreatic RX‘Xase digestion ofthe T 1 fragments of tritiated 5 S rR?iX. The results of such experiments are summarized in Table 3. The low K values of individual purine residues in T, fragments 4. ti and 17 confirm that these residues art’ partially 01’ fully awwsible in t,he 70 K subunit.

(i 4 Ii

I3 .i 14 IX 2 Ifi 4 I .> IO 3

A:\(’ t: (: .I( * (: .\.I(’ ;\;\.I\(’ .\( : .I( ’ .I‘\(: .Al. Al’ (: .I( : XC:

“$1 2.4 +x ti.3 23 > 10 >“5 “7 (13) 3I I7 zI 27 “4 31

2.7 1 .n 7%

‘i.(i

252

S.

M.

FARBER

AND

(‘.

I(

(‘ANT011

The R values from pancreatic RSAase analysis of the other T, fragments range from 19 to 70 and largely confirm the conclusions from the T, analysis that most of the 5 S rRNA is not exposed. (e) Effect of P-site

bound

deacylated

tRS.4

OH 5 AS rRS=2

cxccrssihility

The results in Figure 5 and Table 3 indicate that, the A-A-C,, region of 5 S rRNA. postulated to interact with the T-Y-C-G sequence of tRNA, is not accessible in either the 70 S ribosomes or the 50 S subunit. As a result of experiments with kethoxal, from which they drew the same conclusion, Delihas rt al. (1975) argued for an allosteric effect, suggesting that the (:-L4-A46 sequence of 5 S rRNA might become exposed when the ribosomal P site is occupied in the 70 S ribosome. To t,est this hypothesis, we bound yeast tRNA”h’ to the P site of the ribosome in 20 rn>IMg(Ac), and incubated the sample in 3H,0. Evidence shown elsewhere indicates that the tRNAPh” is stoichiometrically bound and remains bound for the duration of the experiment (Farber & Cantor. 1980). Th e experiment was done both in t,he presence and absence of a poly(U) message. 70 S ribosomes in cacodylate buffer containing 20 mal-Mg(Ac), were incubated for 15 minutes at 37°C with poly(U) at a concentra,tion of 5 pg per .jzbO unit of ribosomes. Yeast tRNAt’h” (Boehringer, 1452 pmol/.il 260) was added and incubated for an additional 15 minutes. Finally, a sample of 3H,0 (125 to 155 G/ml. final

FIG:. 6. K values ofpancreatic ribonucleasedigestion poly(U) ribosome extracted from a tritiated tRNAPh’. (1975) as modified by Garrett, & Noller (1979). The data for each measured boxed fragment are indicated for clarkg.

produets of’the unique T, fragments of5 S rliSA complex. The model shown is t,hat of Fox 8r Woese sequence is numbered by subscripts. The exchange alongside by larger numerals. Hyphens omitted

‘I’ItI1’11TM

EXC’HASGE

OP

lITBOSOM.41~

KS.4

353

activity 2 to 3 (‘i/ml) and an appropriate volume of a tenfold concentrated cacodylate buffer (20 mM-Mg(L4c)2 final concn) was added, the tube was gently mixed and incubated at 37°C for 27 hours. Ribosomes and ribosomes with tRNAPh’ but without poly(U) were similarly incubated. The reactions were terminated and t,he RX&4 extracted as described previously. The 5 S rRNA and tRXB were separated on a 5?& polyacrylamide gel and eluted (Fig. 3(c)). 5 8 [32P]rRNA was added to the tritiated 5 S rRXA samples and the mixture was analysed by T, and pancreatic RNAase digestions. The csalculated R values for specific sites in 5 S rRXA in the ribosome, in t’he l)resence of tRSXPh’ and in the presence of tRSAPh’ and poly(U) are listed in Table 3. Ten fragments from 5 S rRNA in the ribosome or in tRP\‘A-ribosome complexes had R values ranging from 10 to 70. The suppression in exchange rate for these sites must certainly derive from strong protein-nucleic acid interactions. The results indicate that ribosome-bound t’RNAPh’. either with or without p01y(t~) does not significantly affect the 5 S rRS.4 tritiation pattern and. in particular, the X-=\-C’,, region (of fragment 5) is not exposed. A complete set of R values for pancreatic RNL4ase digestion products of 5 S RNA in the ribosometRSL4 poI\-(I-) complex is projected on the 5 S RSA sequence in Figure 6. A projection for R values for 50 S or 70 S bound 5 S rRNL4 in t)he absence of t,RNA would be nearly identical. It is interesting that the two regions of the .5 S rRN.4 showing appreciable exchange in riboxomes lie at opposite ends of the Fox & Woesr ( 197.5) st~rurture.

4. Discussion Slow tritium txxchange provides quantitative data on RSA structure in the ribosome. Since the incorporation rates are a function of electronic and steric rffects. measured R values can reflect base-stacking, base-pairing, tertiary RSA c*onta& and protein-RNA interact,ions. The overall exchange into a molecule provides gross information about thcb molecular environment. The dramatic reduction in labeling rate of 5 S rRNA in the 50 S subunit compared to 5 S RNA free in solution cannot be account’ed for 1)) intramolecular interactions alone and suggests that much of the molecule is tightly tarnbedded in t)he subunit structure. (For comparison note the R values for free and ribosome-bound tRNA listed in Table 1.) The nuclease digestion analyses confirm t,his interpretation: only a few sites are exposed: A-A-C’,,,, (G41, A-C’,, and most probably (i13. Formation of the TO S particle does not expose any of the extensivel? buried 6 S rR?;-4 regions nor strongly shield the few exposed sequences. These results imply that 5 S RXA is not at, the subunit interface. Tritiurn exchange indicates a number of differences between the structure of 5 S rRNA in solution and in the ribosome. Two sites. L4-A-C, 1O and Gdl, exchange faster when 5 S rRNA is in the ribosome than when free in solution. A plausible flxplanation for this enhancement is that the 5 S RNA structure in the ribosome is (*onstrained so that the phosphodiester backbone is kinked near these exposed residues. unstacking these bases and thereby increasing the exchange rate.

254

S. MI. F.iKHER

ASI)

('. I<. ('AN'I‘Ol<

rllternatively, catalysis by a charged protein residue, by increasing the N(7) pK, or lowering the (‘(8) pK, of a purine residue, could enhance the exchange rate. (ia1 is believed to be in the center of a looped structure (see Fig. 6). It’ is t,he major site of chemical reactivity of ribosome-bound 5 S rRXX (Erdmann. 1976). In at least one study it was found t,o be more reac%ive to kcthoxal modification itt t,itc ribosomal subunit than 5 S rRSA4 free in solution (Gray rt crl., 1973). The tritiutn exchange data of fwe 5 S rRS.4 indicate (’14t is in a stacked, single-stranded region (I? = 1.9). In the r,iltosome-bortnct state. t~hc~ excshange rate for (ihI is tnortb consistent nit’h an unst,acked. single-stranded base (K = 2 to 3). We suggest, that t,hr ribosotnr structure constrains the (i4t lool) such that a bend owitrs bet\vcwt and (‘ ant. It should be noted t,hat in all 5 S rRX,4 sequenced to date. although r4tl I)osit,ioti -Cl is variant, tThO is uttiversallqc~)ttserved (Hot-i c’+ Osawa. 1979). This is rc~mittiscettt of the ‘.I.1 t)urn” in t’hr tRN.4 antiwdon loop (Vj3) and may be a common ft~at,urr in all RX&. The enhancement, in the exchange ittt,o A-A-C’, to can also be interpreted as the ribosome altering the 5 S RNA structure. The R value for 5 for A-A4-(‘,,o free in solution indicates it is in a stacked, single-stranded conformation. When the 5 S rRNA is bound to the ribosome, R is 2 to 3 for ;\-A-(!, to, suggesting an unstacking of the bases. In models of 5 S rRNA structure. the long duplex formed from the 3’ and 3’ ends of the molecule can be furt,her base-paired to form an imperfect duplex (“extended feature” , see Farber Kr (!ant,or. 1981). This imperfect helix places the two adenosines of A-A-C’, to in a bulge loop where their potential to stack is probably diminished. The tritium exchange data imply that this bulge loop ma) still retain some stacking in the free rRNA. but not in the ribosome. Ttt(~re is an increased rat)t. of trit,iutti rlxcttatige into .I-(‘,, and a decreaw exctia~trge in ?-lT,, in all three t~it~oson~c~-~~o~ttt~l 5 S rKSL4 st,ates wtwtt winpared wit,11 t’he solution sttwture. The sulq)ressiott in .4-I-4O could I)e ducb t,o ;I stabilization of the (‘-(‘-A-L 40. X-L-(X:,, duplex proposed in t,tte preceding paper. alt’hough a purel,v st)eric retardat,ion cannot Iw ruled out. l,ikcw+w t’he enhancement in 1\-(‘35 may imply an altered bast,-ljairittg scheme for this ttrllix. Some of these conclusions have been wnfirmed indel)ettdently. Vnfort~ttttately. of the many physical and biochemical approaches used to study 5 S rRSX in solution. wry few have been applicable to 5 S rRXA4 in t,he ribosome (Erdmann. 1976). Enzymatic hydrolysis of ribosotnal particles indicates that substrate size stretches of 5 S rRSA are not accessible (Forget & Rr,vniw. 1970). Indeed. even after trypsin digestion of the ribosome, 5 S rRN;\ is protected from nurlcase digestion (Feunteun cY-Monier. 1971 ). (‘hemiral modification. howe\-et., indicatrs that certain nnclrotidt~s reactive). Kethox,vlat,ion experitnettts ttavc demonstrated t.he arv surface awessibilit,v of both ( i 13 and (i4t in bot’h the 50 S (Noller & Herr. 1974) and 70 S partic~lrs (Delihas rt trl.. 197.5) in agreement wit’h these tritirtrn exchange results. Treatment of 50 S subunits with tnonoF)(‘t’f)hthali(~ acid allows the modification of trio adenines which were originally assigned to either Ab5 -& or L157-:~58 based on competition for t,hc tRSA fragment ‘I‘-Y-(‘-(:I) (Erdmann pt r/l.. 1973: Erdmann. 1976). Their subsequent ident,ificatiotr through srquenw analysis (Silberklang. 1976) has relocated the reactions t)o A 73 atrd Ag9. These results art difficult to correlat,e 1rit.h the tritium-exchange dat,a and with T-Y-(‘Yip binding to 5 S rRS.4.

'I'ltI'I'1I'M

ES('HAS(:E

OF KIKOSOhlAI,

(a) 5 S RSd-tRSA

IiS,\

2.55

interactiorl

The postulated 5 S rRNA-tRSA interaction is so appealing that, despite t,rnuous experimental evidence. it has been widely accepted. It has been sufficiently demonstrated that the T-Y-C-G sequence is involved in tRNA binding to the 70 S et al., 1970). In addition, the most ribosomr (Ofengand & Henes, 1969: Shimizo recent studies agree that the interaction occurs with non-init,iator tRNAs in t’he incoming X site (Richter et ~1.. 1973: Crummt d al., 1974: Sprinzl e:t ~1.. 1976). Howcvrr. the evidence that the interaction occurs at the 5 S rRSA (i-C’-X-A,, secluen~ must be closely re-examined in the light of our results. The T-Y-C-(: tRNA interaction with 5 S rRNL4 is based on three argument’s : (1 ) the coml)lementary (‘-G-A-A sequence is conserved in all 21 species (totaling 33 strains) of procaryote 5 S rRNA that have been sequenced (Hori & Osana, 1979) : (2) binding of the tRSA fragrnent T-Y-(‘-(ill to 5 S rRXL4 is increased nearI> tenfold in the 5 S rRN&protein cot%plex (Erdmann et al., 1973); (3) the chemical modification of two adenines exposed on ribosome-bound 5 S rRNA abolishes t’he T-Y-(‘-(:l) binding to the 5 S rRNA-protein complex and reduces by half the protein synthesis activity in reconstituted particles containing this modified 5 S rRN.4 (Erdmann et ~1.. 1973). The trit,iun-exchange results cast serious doubt on this interact’ion. The 14-A-(‘a, sPqlltw(‘t~ has hccn shown not to be accessible in t’he 50 S nor the 70 S particle. Mrc~hanisms have been proposed (Delihas Pt trl., 1975) invoking a P-site bound tICS.4 to induce a ribosomal conformational change exposing the required sequence in the A site. The experiment,s done in the presence of P-site bound tRNA eliminate this hypotjhcsis. More complex, factor-mcdiat,ed mechanisms, however, have not heen eliminated. (1)) .4 t/ altemativr

explanation

atld chemical

for the t&vr~trclrotide ‘mod
bitrditzg

The apparentj binding constant of T-Y-C-Gp to 5 S rRSApprotjein complex is 72.000 \I ’ . and that of U-U-(‘4 is 22.000 v- ’ (Erdmann Pt al., 1973). Recent dat,a (Wrrde cd nl., 1978) indicate t’hat this binding constant, of T-Y-C-C:l) to t’he 5 S rRSA complex is two t’o three t’imes larger than that of any tct,ranucleotide t,estrd to free 3 S rRSA. The Watson&rick base-pairing of T-Y-C-G and U-I!-(‘X is identical a,nd their stacking interactions should be similar. The enhanced binding affinity of T-Y-(‘-(:p by such a small structural change seerns more reminiscent of l)roteill active site discrimination. Experiments by Ofengand & Henes (1969) demonstrating that cyanoethylation of N(1) of Y in T-Y-(‘41) reduces its inhibitory effect of tRNA binding support’s t’his notion. Knowledge of the exact location of the two monoperphthalic acid-moditied adrnines (A 73 and .&) eliminates the direct involvement of the 5 S rRSA (‘-(:-,1-A sec~uentY’. and helps support the argument implicat’ing protein in the trtratluc‘leotitle binding. There are no c*ompfimentjary 5 S rRSA sequences to T-Y(‘-(if) near AT3 and A,,. However. the nucleot’ide sequences involved in the interact’ion 1)rtwren 5 S rRNA and specific 50 S subunits proteins hare been idcnt,itied ((iray r>t al.. 1973) and these sites involve residues adjacent to L4,3 and

“56

N.

M.

F’ARBER

i\SI)

(‘.

Ii.

(‘ASTOR

A,,. Hence, chemical modifications that adversely affect T-Y-(‘Gp protein synthesis activity are near or at protein recognition sites.

binding

and

The exchange rate of 16 8 rRNA in solution is between that of the generally relaxed 5 S rRNA structure and the compact t,RNA structure urlder comparable buffer conditions. The percentage of the molecule base-paired can be rst,imated using the two-state model of Gamble d nl. (1976). They showed that a perfect duplex viral RSA had an R \.alue of about 10. If R for the unbourld state is estimated to be 2 to 3. then the amount of base-pairing in 16 S free in solution agrees well with the 60 to 70% estimated from spectroscopic studies (Allen blr Wong. 197% and references cited therein). When tritiated under identical ionic conditions, the overall exchange into 16 S rRNA in t)he 30 S subunit is only slightly decreased from that of 16 8 rRNA free in solution. This suggests a similar conformation. but, unambiguous interpretation is difficult due to possible simultaneous enhancement in some regions and decreases it1 ot,hers. The spectral studies cited indicate only subtle differences betw,een the conformation of 16 S rRNA free in solution and in the 30 S particle when properly compared in magnesium-containing buffers. The tritium-exchange data then suggest that in the 30 S particle ribosomal prot,eins are only minimally retarding the tritium exchange. This particle must have a relabively open or “loose” structure. Format,ion of 70 8 particles leads to a dramatic decrease in the 16 S rR?\‘=\ exchange rate. This could be due to increased ordering of the 16 S rRNA structure ajnd/or 16 S rRSAprotein (both 30 S and 50 S) interactions. Since optical data (Mcl’hie & Gratzer, 1966; Sarkar et al.. 1967: Rush 8 Xcheraga: 1966: Kabasheva ef rxl.. 1971) indicate that the RNA conformatiorr is not’ drastically altered by t,h(i subunit association, we attribut,e the large R value to a substantial tightening of the ribosomal proteins about the 16 S rRSA. The picture that emerges is one of a loose 30 S subunit tightening up upon 70 S ribosome formation. This loose 30 S st,ruct,ure rnay facititam initiation complex formation. The existence of a conformational change in the 30 S subunit upon 70 S formation has been well documented (e.g. \-an Holde k Hill, 1974). A specifit conformational tightening has been conrluded from prot’ein chemical modification and fluorescence experiments ((‘hang. 1973 : H uang & (‘antor, 1975 and references cited therein) and fast proton exchange studies (Sherman & Simpson. 1969). Experiments comparing the 16 S rRNA i)y kethoxylation ((‘hapman & Noller. 1977) also found generally less 1977) or RNAase digestion (Santer LQ Sham. ac:cessibility in the 70 S particle than t,he 30 S subunit. This

work

(:Ml9843.

was

support,4

We thank Alice

hy grants from the, ITS. Public Health Beekman for expert technical assistance.

KEFFREY\‘(‘FS J _ Allrn, Bush,

8. H. & Wong. K-l’. (1978). J. (‘. A. & Scheraga, H. A. (1966).

Hid. Phrm. Riochpmisfry.

1* 253, 8759-8766. 6. 303tFi-3012.

Servire.

(:,1114826

and

TRITIVM

ENC’HANGE

OF

RIROSOM.41,

RN.4

(‘hang, F. N‘. (1973). J. Mol. Rio/. 78, 563% Chapman, N. M. Pr Noller, H. F. (1977). J. Mdl. Biol. 109, 131-149. Delihas. N.. Dunn, J. & Erdmann. V. A. (1975). FEBS L&w. 58, 76-80. Eikenberry. E. F.. Bickle, T. A., Traut. R. R. & Price, C. A. (1970). Eur. J. Biochem. 116. Erdmann, V. A. (1976). Pray. Svcl. ;Icid Rrs. 18, 45~90. Erdmann, V. .A.. Sprinzl, M. & Pongs, 0. (1973). Biochem. Biophys. Req. (‘ommun.

12. 113

54.

942

948.

Farher. Farber. Fruntrun. Forget. Fox. G. (:aml)le,

N. 31. & (lantor, C. R. (1980). Proc. Sut. .il~ad. %i.. I’.S.d. 77, Til35p5139. N. M. & (‘antor, C. R. (1981). J. Mol. Biol. 146, 223-239. J. & Monier, R. (1971). Biochrnzi~, 53, 657. B. C. $ Reynier, M. (1970). Biochem. Biophys. Res. Common. 39, 114. E. B Woese, C. R. (1975). ‘Vnturr (l~~/~dorc). 256, ,505~507. R. (“., Schoemaker. H. J. P., ,Iekowsky. E. & Schimmel. P. R. (1976). Biochm,istry, 15. 2791 m-2799. (iarrett. It. X. 8 Soiler, H. F. (1979). J. Mol. Biol. 132. 637-648. Gray, I’. S., Bellemare, C:., Monier. R.. (iarrett, R. A. 8: Stoffler. (:. (1973). J. Iwo/. Biol. 77. 133 1.52. (irummt. I’.. Grummt, I., Gross. H. J.. Sprinzl, M.. Richter. D. 8r Erdmann. V. A. (1974). FEBS Icttrrs. 42, 15-17. Herr. W. Ni Soiler. H. F. (1979). J. ~Vlol. Biol. 130, 421-432. Hori, H. & Osa\l;a, S. (1979). Proc. Sat. &ad. Sci.. 1r.S.A. 76, 381-385. Huang, K-H. & (‘antor, C. R. (1975). J. Mol. Biol. 97, 423-441. Kahashrva. C:. N.. Sandakhehiev. L. S. & Sevastyanov. A. P. (1971). FEBS kttrrs, 14, 161 I&t.

Mcl’hie. 1’. & (:ratzer, W. B. (1966). Biochemistry, 5, 131C1315. Sci., C’.S..-l 47. 1589~-1594. Xirrnberg. N. & Matthaei, W. (1961). P rot-. Sat. Acad. Xoller. H. F. bz Herr. W. (1974). J. ,llol. Biol. 90, 181-184. Ofengand. J. Xr Henes, C. J. (1969). J. Rio/. (‘hem. 244, 624M253. Richter. I).. Erdmann, V. A. 8 Sprinzl, M. (1973). Satxrr Srtc> Rio/. 246. 132-135. Santer. M. & Shane, S. (1977). J. Bacterial. 130, 9O(h910. Sarker. I’.. Yang. .J. T. 8r Doty. P. (1967). Riopolymers, 5, l-4. Schimmcl. 1’. R. (1977). Act. Chem. KPS. 10, 411-418. Schimmel. P. R. Hr Schoemaker, H. ,J. P. (1979). Methods Enzymol. 59, 332-350. Srhoemakrr, H. J. P. 8z Schimmel, P. R. (1976). J. Rio/. Chem. 251, 6823S6830. Schoemakrr. H. tJ. P.. Gamble, R. C’.. Budzik. G. P. & Kchimmel, I’. R. (1976). Bioch,emis/ry,

15. IMHl-2803. Sherman. M. I. B Simpson, 54. V. (1969). (‘old Spring Harbor Symp. Qunt. Biol. 34,22&221. Shimizo. S.. Hayashi, H. & Miura, K. (1970). J. Biol. Ch,em. 244, 6241-6253. Silberklang, M. (1976). Ph.D. thesis. Massachusetts Institute of Technology. Sprinzl, hr., Wagner. T.. Lorenz, 8. & Erdmann. 17. A. (1976). BiochrmGstry, 15, 3031-3039. Traub, D.. Mizushima, 8.. Lowry. C. V. 8r Nomura. ill. (1971). M&hods Enzymol. 20,391~407. Vatr Holde. K. E. & Hill, W. E. (1974). In Ribouomps (Nomura, M.. Tissi@res. A. & Lengyel. I’.. rds). pp. 75-79. Cold Spring Harbor Press. New York. Wishnia. A.. Boussert, A., Gaffe. M.. Desser. Ph. & (irunherg-Manago, $1. (1975). J. Mol. Hiol. 93 499-515. Wredr. P.. t;ongs. 0. & Erdmann, \‘. A. (1978). J. Mol. Biol. 120, 83-96. Zamir. ;\.. hliskin. R.. Vogel, Z. & El son. D. (1974). Methods Enzymol. 30. 406-426.