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Gel electrophoretic technique for separating crosslinked RNAs
./. Mol. Hiol. (1982)
159. 151-166
Gel Electrophoretic Technique for Separating Crosslinked RNAs Application to Improved Electron Microscopic Analysis of Psoralen Crosslinked 16 S Ribosomal RNA
We have developed a gel electrophoresis technique for separating crosslinked RSX molecules into a series of discrete fractions. The gel used is polyacrplamide made ill formamide and low salt designed to denature the RNA during electrophoresis. The mobility depends upon the positiotr of crosslinking within each moleeult~. as demonstrated by electron microscopy of RX.4 cluted from the gel. In genrral. molecules with large loops elect~rophorese more slowly than molecules with small loops or uncrosslinked molecules. We have used this technique to re-examine the psoralen crosslinking pattern of Eschwichin coli 16 S rihosomal RNA in inactivat4 30 S rihosomal suhunits. To determine the correct orientation of each type of rrosslink. we have covalently attached I)NA restriction fragments to the RNA so that the polarity of the RNA in the mieroseope would he known. Our previous major conclusions are c~onfirmed : the predominant long-distance crosslink detwted hy gel electrophoresis involves a residue rlose t,o the 3’ end and a residue approximately 600 nucleotides away : the formamidejpolyacr~lamide gel is able to separate two closely spaced 1 100.nueleotide interactions hegmning close to the 3’ end. which were reported as OIW interaction hefore: and an interaction joining t,h(* ends is detected as hefore. However. one lowfrequency crosslinked interactiorr. between positions 9.50 and 1400. and possibly another lowfrequenc~ interaction. between posit,ions 550 and 870. are determined to I)e in the opposite polarit,y t,o that deseribetl previously.
1. Introduction Our
int,erest,
has been
that) would
reveal
ribonucleie
acids.
of RSA-RNA
the development
details
of the secondary
Potentially.
psoralen
and tert’iary photochemical
erosslinking structure
into
a method
of single-st~randed
crosslinking
can
provide
strand folds back to interact with itself. This would be a very useful t,ool in understanding t’he struct’ure and dynamics of a molecule such as a ribosomal RNA. The greatest obstacle in realizing this end is in not having a truly expedient and rapid method for evaluating the types and freyucncies of the crosslinks made in t)he RSA. It has been our desire t’o find a gel
direct
evidence
of the positions
where
the single
Ial
I 32
I’
I,. L\‘OI,l,RS%IE:N
i\SI)
(’
I< (‘.AN’I’OI~
electrophoresis tnet,hod that could separate cwsslittked t~~olrc~nlw. This swtttecl possible, because t)he structural differences lwtwecw diff&ent types of crosslinked tnolecules should he tnost, apparent whett the molewles are denatured. and there a.re several tnet,hods for performing electrophoresis under denaturing cottditiotts. In t,his paper we show that it is possible to separate a mixture ofc~rosxlittkt4 16 S rKNA into a series of dist,inct species by e~ec~rophorrxis through a fortttatttitl~~ denat,uring medium. Llntreat,ed 16 S rRI\‘A electrophoreses as a single. f&t mobilit? species under these conditions. The new slow. tnohilit~y bands found whctt crosslinked samples are electrophoresed contain molecules that have long-dist.attw crosslinks at, different positions. 5Iolecules t’hat have large loops (or double loofw). in general. tnove tnore slowly than tnolec~nles wit,h smaller loops. Thereforc~. 1)~. inspecting the pattern on t)he gel, we are able to attticaipate the ttuntlw and size ot’ the different features that will be found in the mixture. Before electron tttic~rosc~~pic analysis. t.he crosslinked RSA sample is preparatively sorted into a set of fractions. This reduces t,he heterogeneity of the types of tttolwules seen in the ttticwwopt~ al ottc time and thereby greatly simplifies the analysis. Our earlier cxperitnents (Wollenzien rt (II.. 197!Im,b: Thatt~tttatta rt I//.. 1!479) wtlr(b hegun on Ewh~Prichin w/i 16 S rlCiYA brcausc a partial fwinrary srqurtrcv~ (Ehrestna~tm rl ~1.. 197.5) had been completed at tha,t titttc. Since then. t)he cotrtpletc~ primary sequence has been determined hy two groups (Krosius (‘I nl.. 1978: C‘arlwtl rt /xl., 1978) and this has unleashed a tt~t~tnendous advance in underst~andittg the organization of t’his molecule (see Soiler & b’oese. 1SSl). III particular. it has allowed several detailed secondary structure predictions of t,hr tnoleculr (~Votw rf al.. 1980: (ilotz & Kritnacotnhe. 1980: Stiegler rt ~1.. 1981 ). These prrdictiotts all agree with one attot,her to a large extent, Ijut are not in agreement with the psoralett crosslinking data we have presented previously. Eretl given the tttrwrtaittty of’ thca measurements we made in the rlectron microscope. there is essentially no matchul) of the long-distance cottt,a.cts we detected with those predicted 1)~sthe models. However. several matchups c~ould be tnadr if thr polarity assigntttents \\‘t’ hat1 made were reversed. Our feature I would then tttat~clt. ivithitt error. the 27 37 ’ 54i 5.~6 interaction, a base-pairing interaction twt,weetr rwidttes 27 through 37 \vit 11 residues 517 through 556 (for further description of’this ttomenc&turt~. SW Noller 8~ FVoese, 1981 ): our fbat,ure II would match the int~eractiott 17--20’915 318: anti ollt’ feature ,X1 would tnatch the ittt,eractiott 563-569 ’ 879M385. All of’ these propowd base-paired interactions contain oppositcl adjawttt tlridine residws. tvttich arc attractive sites for psoralett f)h(~to(~hetrric,aI crosslinkittg (Tttotrrpsott c,t t/l.. I!)81 ). Thus. n-c were in t,tttb dilrtntt~a of correlating our rcsrtlt~.* with tlrfw other tttotlt*ls only if our polarity assignments were ittwrtwt To try to r&olve this discrepancy. wt’ have de\-eloped a tnorc satisfactor.v method of determining the polarity of c~rosslittked features in the electron fragment from c~lotietl microscope (Wollrtiziet~ & (‘antor, 198%). A small restriction ribosomal D?iA is covalently attached to the crosslinked 16 S rRS=\ before preparing t,he sample for elect~rott microscopy. The fragment 1seen as an extra stna~ll strand at, the position where it hybridizes. allows the polarity of the 16 S rKXA to be known. The sample that we have st,udied for this report is I8 S rllN.4 that had Iwen
RNA
CROSSLINK
ELE(‘TKOPHOKF,SIS
BNI)
MI(‘ROS(‘OP‘1
I .53
crosslinked in the inactivated 30 S ribosomal subunit. This sample was chosen because we expected from our previous work (Thammana et al.. 1979) that it would contain a much simpler pattern of products than crosslinked free 16 S rRN.4. and thus would be easier to analyze.
2. Materials and Methods (a) F’rrpnratio,l
of thr
KAY.4
30 S rihosomal subunits isolated from E. coli MRE 800 cells. according to the met,hod of Amils rt nl. (1978), were a gift from Dr Y. Gloria Chu. They were psoralen crosslinked in inartivation buffer (0.5 miv-magnesium acetate. 100 mm-ammonium acetate. 10 rnMet al.. 1979). except that [3H]AMTt (HRI Tris.HU. pH 72) as described (Thammana Associates; spec. act. 95 x lo5 rts/min per pg) was used at a final concentration of 40 ~g/ml. and the samples were irradiated in 1.5-m] Eppendorf tubes at 6°C for 10 min in the device described by Isaacs et nl. (1977). The subunits were then digested for 30 min with 0..5 mg of
prf-incubated
proteinase K/ml
(EM Biochemicals)
in thr presence of lyb (W/V) sodium
sulfate (Hilz et al.. 1975) hefore extraction with phenol and precipitation n-it,h The incorporation of [ 3H]AMT into the RNA was determined after an additional prtbcipitation with ethanol. The I6 S rRNA was checked for intactness and contamination by electrophoresis on 20/6 to 15% (W/V) gradient polyacrylamide gels (Amils et al.. 1979). dodecyl et’hanol.
(11) Formamid~/polyacrylanlidr
gel r/fxtrophowsis
A volume (300 ml) of formamide (Matheson. Coleman and Bell) was deionized by stirring for 3 h with 12 g of AU5OLX8 mixed bed resin (Bio Rad). A 35’?, (n/v) aqlamide solution was then prepared by dissolving 13.5 g of acrylamide and 2.25 g of bisacrylamide (Eastman. once recrystallized from chloroform and acetone, respectively) in the deionized formamidc. bringing the final volume to 45 ml. (iels, 5.20,; to 5%% (w/v) in polyacrylamide, were cast with a gradient maker into four 8 cm x 8 cm x 0.3 cm cassettes held in a slab gel casting apparatus GS(‘-4 (Pharmacia). The dead volume in the casting apparatus was filled with glass beads: 10 ml of 200/o (v/v) ethanol in the apparatus became an overlay when the gradient was made. The 5.23, solution contained t,he following components: 9.75 ml of the 359” acrylamide. 55 ml of deionized formamide, 65 ~1 of 2 M-sodium phosphate (pH 65). 145 ~1 of I~,i~~~‘.S’-tetraethglmethylenediamine. 280 ~1 of 32y0 (w/v) ammonium persulfate. The 5%oi, solution contained thr following components: 1083 ml of the 350,; acrylamide solution. 52 ml of deionized formamide. 1.95 g of sucrose, 65 ~1 of 2 M-sodium phosphate (pH 65). 145 ~1 of S.,V.S’..\“tetraethylmethylenediamine, 280 ~1 of 32”,& ammonium persulfate. The sucrose acts to stabilize the gradient during polymerization and was dissolved in the formamide before t.he other components were added. The 5.2”;0 solution was placed in the mixing side of tjhr, gradient maker and the gradient was allowed to flow into the bottom of the apparatus until th(s top of the overlay reached the edge of the cassettes. After polymerization. the overla? solution was rflmoved, the gels were wrapped carefully and were stored in the cold. Reforc, use. a cassette was placed in the electrophoresis apparatus and a well-forming gel \vas added. It contained: 0.41 ml of 30% acrglamide (300,, acrylamide/l50,b bisacrylamidr (\v/v in M-at,er)). 5 ~1 of 2 iv-sodium phosphate (pH 6%). 2.0 ml of water. 2.5 ml of hot, 1”,, (y/v) agarose. 8 ~1 of iV.LV,L%“.S’-t~etraethylmeth~lenediamine~ and 40 ~1 of 7.5(;,, (WV) ammonium persulfate. Before loading the samples. the gels were equilibrated in the cold n-it)h electrophoresis buffer (cold 2 miv-sodium phosphate. pH 6.5). Electrophoresis was for 12 h at 23 V/cm. After electrophoresis, gels were washed for 1 h in 5 mm-sodium phosphate before being stained with ethidium bromide. Analytical gels were phot,ographed on a
t .~hhrt~viation
used : AMT. aminomrth~ltrimeth~lpsorale~~
151
I’. I,. U’OI,I,ESZIES
ASI)
(‘. Ii
(‘ASTOR
trdnsillutnirtator box. Prrparative gels \vrrv visualizt~tl 1vit.h ii :
The method for covalcntly attaching a l)NA rrst~rivtion fragttttbttt t,tr the RNA molt~~ul~~ts described in detail elsc\vhere (Wollrnzirtt K- (‘antor. lW2). For thtwh c~xpt~rimt~nts. 0.5 ~1 c>t 016 pg of RNA/ml in formamidr~ \vas hyhridizrd to att equal volumes of thtl ltsoralrtt monoadduct DNA restriction fragment (at a,pprox. 02 pg/pl in WK (0.1.5 It-sodium c*hltrrid(~ I5 mw-sodium citratcl. IO mwdisodium EIJTA)). All of t,h(s hybridizations wc’rv lwrformtSd \vith the fragment ~otnplt~m(~ntar~ to nuclrotides 962 to I322. \vhic*tt is pcwrrated 1)~ ‘/‘UC/I endonuclraw digestion of plasmid pC”S3 (a gift from I)r (‘athtnriw Squires) csxwpt ott(‘. v, 1lic.h was performed with thv fragment (~otnl~lrtnt~ntnr~ to ttuc*lwtidcs 8!) to 123. ~hictt ih generated 1)~ Mboll cttdottucleaw dig&ion of plasmid pCS1 I ( ISrosius (t c/l.. 197X: \Vollenzirtt & Cant,or. 1982). After c~rosslinking atrd prwipitation u ith c~thxnol. ttw crosslinked hybrids \vvrv resuspc~nded in I ~1 of \\.atcsr Itrforv Ittvparatiotr for c‘l
Ikwaturation of the R3.A samples and t,hc spreadittg c~cttulitions arc’ a modification cjt’ t)ut previous t,rchniqw (LVollrnzirn r/ (I/.. 197%). RNA (1 ~1 of 0.16 &PI itt formamide) or the rwuspended DNA-RSA hybrid \vas mixed \vith 19.5 PI of formamide (vacuum distilled) attcl was heated t,o ,‘,i’(’ for 5 mitt lwfore l.I.5~1 of titrmaltlt~h~dt~ (heatc4 in a walrd vial in :I boiling waterbath for 30 min) and I.0 ~1 of 60 m~-?uTa~HP0, was addrd. The mixture \+.a~: hrated for an additional IO mirt and was then yuenc~hrd ott iw. Formamidr~ uwd irt thtl hyperphast. contains ltc~t~zyldialkplammonium chloride (HAC‘. a gift from I)r Thro Kollrr) t)o 135~1 of formamidr was added 1 ~1 of l~rt~z~ldialk~latntttottittt~~ cahloridr \vhich ha:: a concentratiott of 9 ;I 2h0 utrit,s (approx. 4.5 mg/ml). For t,htB RX.4 AIonto. thtx hvlwrphaw contained 3.5~1 of formamidr (\l-ith lwtwyldialkylammot~iutn chloridc~). 5 ~1 of iG!l ttnffbr (0.1 wtrirthanolatnitw (titrat,csd with HC’I to pH X+). 04)I sS~,E:I)T.-\). .i ~1 of the drnaturation mix. attd 5 ~1 of I mg cytochrorrtc~ c/ml (Sankyo. (‘Sl3r c.lcaved). For the 1)S.j RNA hybrid, the hyperphase cwntahted 15 1~1of formamidr (tvith l~rt~z~ltiialk~larrttrtot~ittttt chloride). 2..7pl of EM 1tufft.r. 5~1 of t,hr drnat~uratiott tnik. and 2.;)r ~1 of c*ytochrotttt~ < .)r p I of the hypwpha,sr \vas applird on a glass slitIt%to a dr~iottiwtl solution. In both vasw. ,..t water hypophase. After 1 mitt. thv tno~lolayrr was picked up on parlotlion-c.ovrrPtl grids awl ~vas M-ashrd for I mitt in .XP,, rthanol to rctnovc polyacrylatct drttris lwforr lwing rinsed in 90(‘, ethanol. stained with uranyl arrtattl. and hriefl? d&ainrd. The grids \~c~rc’-shacio\~c~cl \vith IV/I’d before viwittg.
The grids wcrv photographed in a Zeiss EnlO S-2 (~lrctrott ttticrosc.opts attcl thta Itroitv3rd nrgativrs \vertl t,rawd with a Nutnoniw measuring devic.e. All of the rnolwulcs \vc’ havfa traced have lwen reduwd to a standard pattwn. Sinw I crosslittk itt a tnoleculr forms I opvtt loop with as many as 2 t,ails. 3 measurcmcnts are ncedrd t,cr sprcif:\- t,hr positiotl 16 t.lrv c*rosslink : the distatrcv from the 5’ end (or the ~losrr end) to tht, I)asc~of the loop. t J-w rirc*umfrrencr of tttcl loop. and t,hc distattcv from the I)aw of the looI) to the other cstttl. ?;otc% that in this pattvrn rithvr (or both) of t,hr t,ails can Iw wro Thr data wt‘rt‘ wllwteti itttd
RN.4
C’ROSSLINK
EI,ECTROPHOKESIS
ANI)
#TCROSCOP\
I .i5
processed by a VACSjl l/780 computer (Digital Equipment Corporation). For each molec~ulr. the crosslink is then described by a pair of co-ordinates: the X co-ordinate is the position of the beginning of the loop measured in nucleotides, based on the total length of the molecule: the Y co-ordinate is the position of the end of the loop, also measured in nucleotides. The dat’a were first displayed as a correlation plot of Y versus 9. Tdentifying clusters of data on this plot is equivalent to finding a group of molecules that have similar loops. Tht data were then further processed by dividing the XY space by a rectangular grid. The darkness of each rectangle indicates the number of data points contained in that area. Sinctr. in principlr, X can have a value as large as 1541 and Y can have a value as large as 1542 and Y is always greater than X, the plot of data in which the polarit? of the molecules is used will occupy the half of a square (1542 nucleotides on an edge) that 1s above the diagonal line 1.=x. of the molecuk~s. We are able t,o perform this analysis even if we do not know the polarity In this case. t,he measurements of X and Y are made to the closer end of the molecule: thts middle of the loop made by the crosslink has a value less than half the length of the molcrulr. [II t)erms of the co-ordinates X and Y. this is equivalent t,o (X+ I’)/2 < 1542/2. This means. of course. that we do not know whet,her a particular loop is closer to the 5’ or 3’ end of thr molrc*ule. Operationally. the plot of Y ~rsus X becomes folded across the line Y = 1542 - S and occupies l/4 of the square that is 1542 on one edge. In practice. molecules that have not been hybridized are first measured and an unoriented (t,hr polarity is not known) plot of Y vwsus X is constructed. Molecules that have been hybridized are t,hen measured. These are first plotted as if we do not know tht, polarity. These plots should contain similar groups of data if the hybridized molecules are representative, of the unhybridized molecules (see Results). The plot of Y 1lersu.s X for the hybridized molerules can then be unfolded to determine the polarity of the characteristic groups of data. A more complete description of this technique will be given elsewhere (1’. L. \Vollenzien. R. Murphy & C. R. Cantor, unpublished results).
3. Results (a) Formamidelpobyacrylamide
gel elertrophoresis
30 S ribosomal subunits in inactivation buffer were incubated with 40 pg AMT/ml and were crosslinked with a saturating amount’ of 365 nm light. The RNA recovered from the subunits incorporated 10.1 AMT molecules per 16 S rR,NA molecule. This level of AMT incorporation is very similar to the amount’ of hydroxymethyltrimethylpsoralen incorporation under the same conditions (Thammana et al., 1979). In the RNA, both before and after psoralen crosslinking, there are small amounts of 23 S rRNA, 5 S rRNA and RNA intermediate in size between 16 S and 23 S, all of which must derive from contaminating 50 S ribosomal subunits (results not shown). Figure 1 shows the results of formamide/polyacrylamide gel electrophoresis of this crosslinked RNA sample and a control, untreated 16 S rRNA sample. The cont’rol 16 S rRNA electrophoreses as a fast mobility band (there is also a small amount of contaminating 23 S rRNA). whereas the crosslinked 16 S rRNA separates into a series of bands. The most intense band has the same mobi1it.y as untreat,ed 16 S rRNA. A sample (100 pg) of this RNA was electrophoresed on an identical gel (using a single wide well) for the purposes of obtaining RNA from each of the bands. After staining. the gel was visualized with a 375 nm mineral light to avoid photoreversal
I .ifi
of the psoralen crosslinks (Rabin & (‘rothers. 1979 : and our results. not shown). The gel was separated into seven fractions by t,he cuts indicat’ed in Figure 1. and the RKA was electroeluted from t,he gel slices. After extraction with phenol and precipitation with ethanol. the fractions were resuspended in wat’er and W~I‘P checked for their concentration and molecular weight by elwtrophorcsis OH polyacrylamide gels (results not shown), and were a~lso rF,-rlectrophoresrd 011 formamide/polyacr~lamide gels to contirm the identity of each fraction (Fig. 2). The characteristics of the fractions are given in Table 1. (h) ~detrt~ficatiot~ ctf thr crosslittks
hy r/wtrotr
ttricrosrop!y
All of these fractions have been examined in the electron microscope to determine if the RN.4 has unusual structures. &a&ion 1 contains a wry heterogeneous mixture of different types of molwules. On the polyacrylamide gels t,his fract’ion electrophoreses with bhe mobility of 23 S rRSA. In the microscope.
RSA
(‘ROSSLINK
EI,ECTROl’HC)RESIS
AND
MI(‘ROS(‘OPV
157
FIG:. 2. Re-electrophoresis of the fractionated crosslinked 16 S rRNA. Approximately 02 pg portions of the resuspended RNA samples (except for fraction 1) were re-elrctrophoresed on 2 formamidr,ipolva~r~lamide gels to check their composition. Lanes numbered 1 t,o 7 correspond to thv fractions I to 5. The starting crosslinked 16 S rRNA was electrophomsrd in the lanes lettered S for c~omparisotl.
RS-3 recovered from preparative Fraction no.
Yield (f%)
formamidelpolyacrylamide
Molecular weight identity (S)
t Small amounts of photoreversal
in these fractions.
Surnbrr bands
of
gel
It58
I'. I, b'OI,L~N%IEN
ASI) ('. It. ('AN'l'OI<
rnany of’the molecules contain more than two ends. Thesr are probably crosslinked 23 S rRNA molecules, which are frequently broken. We have made no further attempt t,o characterize this fraction. Molecules in fractions 6 and 7 both appear to be linear in the electron microscope. Local structures not resolved in the electron microscope would make the molecules appear short,er than untreated 16 S rRNA : however. we have not attempted to detect any systematic shortening of the contour length of these molecules, since at this level of psoralen incorporation we would not expect, more than S’$;, shortening based on previous result,s for 16 S rRNA crosslinked in reconstitution buffer (Wollenzien it al.. 1979aJ). This difference is about one standard deviat,ion in the dist)ribut~ion of contour lengths of the molecules, so the difference would be very difficult, to det#ect with any degree of certainty. Fractions 2, 3, 4 and 5 all contain molecules wibh recognizable crosslinked features. To determine t,he number of different features, each fraction was prepared for electron microscopy first without hybridization to short. complementary restriction fragments and then wit’h hybridization and crosslinking t,o these restriction fragments. For t,he unhybridized RNA, every field in which at least three crosslinked measurable molecules were found was photographed. even if t,hertb were already good examples of that type of molecule. In t,his way, t)he molecules measured reflect the frequency distribution of the different’ types on the grid. Thtx molecules were analyzed by encoding each crosslink into a pair of c-o-ordinates : t)hr X co-ordinate is the position of the beginning of the crosslinked loop, t)he 1’ coordinate is the end of the loop. The distribution of different types of crosslinked molecules can be seen on a plot of Y versus X (see Materials and Methods). ;\ clustering of data in a part’icular area indicates that there are many molecules with similar loops. Tt is inferred that a specific crosslink (or a few very closely spactaci crosslinks) is responsible for the appearance of the similar loops, and thus t h< average co-ordinates of t,he cluster of data will give the position of t)he c,rosslink. To check that, the molecules that have h,vbridized with a DNA4 fragment art‘ representative molecules, the analysis was first prrformed on them as if wf’ did not know the polarit,?. In this case. the correlation plot should Iook very similar to t hc> plot done for the unhybridized molecules. The molec*ules wer(’ then a~nalyzeti using the known polarity of each. The correlation plot then unfolds to show th(l c.c)r’recat orient’ation of eacshcharacterist,ic crosslink. Figure 3 shows t,hc>correlatjion plots for fractions 1 t,hrough 5. The three :LIVHSIII the correlation plot, of unh,vbridized molecules from fraction 2 are carntered at 0” 1542, 6” 1088 and a loose collection of data around 283’ 1025. The interaction 0” 1542 cannot have a polarity assigned t,o it. Molecules that caontjain t,htb 1 I(H)nucleotide loop hybridize to the restriction fragment,. and the loop involves t ht> 3’ end in t,he oriented molecules. This allows t,he avrrage crosslink position to be assigned as bet,ween residues 454 and 1536. Molec~ules that would fall into t hcl third area do not hybridize and are probably not I6 S rRN.4. ln t,hr cborrrlation plot of’ t)hr hybridized molecules there are several rxamples of rnolec-ulrs with ftiaturtss at (orient,ed) 493”975 and 61%” 1325. \\‘e will not consider these molecules as containing real crosslinks. since the loops were not seen in the original or subsequent) screening of t,he unhybridized molec~ules. A summary of t’hese features is
RNA ('ROSSLISK
E1,ECTKOl'HOI~ESIS
ASI)
S11('I~OS('Ol'\
l.i!)
given in Table 2. The frequency of each t)ype of crosslink is estimated on t’hr frequency in the correlation plot and the amount of R’KA in the fraction. In addition, fraction 2 contains two classes of molecules that have crosslinks: the interaction O”1540 together with the prominent feature of fractions 5 and 4, and the interaction 450”1540 (or the interaction 510-1540 from fraction 3) toget)her with the prominent feature of fractions 5 and 4. The one prominent area in the correlation plot, of unhybridized molecules from fraction 3 is centered at 6^ 1033. These molecules hybridize the restriction fragment and the interaction involves the 3’ end of the molecule, which makes it OCOUI between residues 509” 1542. This is a different interaction from that det’ected in fraction 2. since these fractions do not show cross-contamination on t,he formamide/polyacrylamide gel. The three areas in the correlation plot’ of unhybridized molecules from fraction 4 are centered at 2”609. 146”588 and 550”873. All three classes of molecules hybridize to the restriction fragments; the first, two features are closer to the 3’ end. which puts them at 933”‘1542 and 954^ 1396. There is a single example that places the interaction 550^873 somewhat closer to the 5’ end, so it tentatively remains at this co-ordinate. In addition. there are several examples of hybridized molecules containing an interaction that, is approximately 6” 1181, and two hybridized molecules that, contain an interaction at, approximately 0^675. The interaction 6”llSl is not seen in the unhybridized molecules of fraction 4, and the interaction 0^675 is not seen in the hybridized molecules of fraction 5. Fraction 5 contains molecules with features centered at 2”606 and there are also some molecules centered at 97”600. The correlation plot of the hybridized molecules places both of these features closer to the 3’ end. so the average positions are 936^ 1542 and 945” 1445. Since the molecules in fraction 5 are a subset’ of those found in fraction 4, it is likely that the interaction 945^ 1445 corresponds t’o thr int,eraction 954” 1396.
4. Discussion The improvement in t’hese experiments comes from the ability to elertrophoretically separate different crosslinked species and determine the orient,ation of each feature by covalent attachment of a DNA restriction fragment’ to a known position on the RNA. We now have a method independent of electron microscopic inspection to determine the number of new types of molecules present, in a mixture after crosslinking (see Fig. 4). We can greatly enrich for a particular type of molecule before determining its identity. This allows us to examine low-frequency species without the fear that their presence in the electron microscope is artifact’ual. Tn addit’ion. mobility on the formamide/polyacrylamide gel is quite sensitive to differences in t’he sizes or positions of features, even when t,he differences are difficult to detect in the electron microscope. However, since the change in electrophoretic mobility depends on the size of the loop in the molecule. t.his gel system probably would obscure the fact that there were short-range interactions that were crosslinked by psoralen. in addition to the long-range interactions, which are crosslinked and seen. For discussing the crosslinks detected in these samples. we wish to adopt a
t‘ll. :i
(a)
x
3, hybridlzed used
Fract\on Polarity
X
2, hybridized Fraction
.___ If
0
:1
%
I
j c, :: ‘”
4
‘0 0
8 LD A
A
notation derived from that of Keller & Woese (1981). A prefix EPs will be used before the approximate co-ordinates of the crosslink: E signifies that the ident,itication has been made in the electron microscope nith what,ever errors are inherent in this method : Ps indicates that the crosslinks are generated by psoralrn. The co-ordinates are measured only to the nearest) ten bases. because we have no reason t,o expect the accuracy to be better t’han this. We have been able to ident,ify six psoralencrosslinked interaCtions in t’he 16 S rRKiA from inactivated ribosomal subunits. In addition, WE see several types of doubly crosslinked molecules. which are expected. There are several other types of features that were encountered after the hybridization and crosslinking to restriction fragments but these other features were not seen in the initial screening of the fractions. so we do not consider them t’o be significant. Of the six interact,ions. four correspond to features we have already reported (Thammana crosslinked interaction El’s 930” 1540 Pt (rl., 1979). The most prominent corresponds to the feature previously estimated to be 570 nucleotjides in size. which was determined to involve the 3’ end of the molecule by our previous hpbridizatjion t,est, (F\~ollenzien rt nl., 1979h). The interactions EPs 450A 1540 and EPs 510A I.540 t,ogether cwrrespond to the feature previously estimated to be 1040 nacleot~ides in size and involving the same end (the 3’ end) as the most prominent feature. The int,eraction EPs 0” 1540 corresponds to the end t,o end crosslink seen previously. The int,eraction EPs 950^1400 csorresponds fairly well in size to a feat,urt measured in the 16 S rRNA crosslinked in solution, feature VII (U’ollenzien rt ~1.. 1979a). and also seen in the crosslinked 16 S rRNA from inactivated subunits. However. feature VII had been assigned by the previous hybridizabion t,est to be csloser t,o the 5 end of the molecule (U’ollenzien rt N/., 1979b). Likewise. the interact,ion EPs 550”870 corresponds fairly well in size to a feature measured in the 16 S rRXA crosslinked in solution, feature XI, and also seen in the crosslinked 16 S r-RN.4 from inactivated subunits. However. by looking at molecules t,hat had two
Flc:. 4. Separation of diffvrrnt types of crosslinked RSA molrculcs on thr formamjde/polva.c.r?-lamidl, gel. l\lolrwAes with the 6 types of rrosslinks we have identified we shown. The identity of thr crosslinks that the molecules contain are. from I)ot,tom to top: W’s 930A1540, Et’s 950A1400. EPs 560^870. KPs :it0^1A40.E:l’~450^1540(ri~ht.on topnw). W%OAl;i40( second from right. on top row). Also shonn irr thr top POWare 1 types of molecules with double features.
164
I’. I,. \VOI,LEN%IEN
ANI)
(‘. t<. (‘.SS’I’OR
crosslinks. feature XI had been assigned to be cslosrr to thr 3’ end of th(, molwulr. Because the map of 1I interactions found in 16 S rlCNA crosslinked in solution (protein-free) was built up by observing molecules with two features and was self consistent, correcting the polarity of feature VII and XI would mean that eit,her the most frequent features crosslinked in t,he protein-free I6 S rRKA4 are different from those crosslinked in the subunit, or the original map has inconsistencies. \i\‘c expect to learn more about t’his when we w-examine 16 S rRNA crosslinked in solution. We have not found in gel-fractionated molwules two low-frqutw~~y fi,at urw WV previously reported seeing : an interaction involving one end of t,he molwulr and some residue about 240 nucleot~ides away (f+ature I\‘), previously reported in ()..5”,, of the molecules crosslinked under these conditions, and an interaction srparat~rtl by 210 nucleotides (feat)ure 1’1). which was enclosed 1)~ feature 1’11. pwviousl> reported in 2.20z, of the molecules. The failure to see t,hese features could rrwdt from differences in the crosslinked samI+. since the frequency of these features LVRS quite dependent on the crosslinking cwlditions (Thammana P/ ~1.. 1979). The pat)tern of psoralrn crosslinks indicates t)ha,t large regions of the 16 S rKS,\ are not accessible to crosslinking. These regions irwlude thr 5’.t,erminal domain defined by the interact)ion 27 -37 A547LX6 (for a description of this and subsequent domains, see Noller 8 Myoest~.1981). cxoept for positions at approximat’ely 4.50 and 510, the entire central domain, defined by the intrracbion 567-570A8XO--886. and the 3’ major domain. definrd by thr interaction 926-933^ 1384-l 391. Two of thrx features we have reported here probably c*orrtaspond to crosslinks made at t,hr baw pairing interactions t,hat define these domains. If allowa,nce is madr for the accuracy of the measurements made in the electron microscope. t,he feature El’s 950” 1400 fits reasonably wrll t)hc interaction 925-933” 1384-1391. and the ti~aturt~ EPs 550*X70 fits the interaction 567-670”88OC%6. The 3’ minor domain, &I thrb other hand. is highly accessible to crosslinking. Thr four most frequent crosslinks it1 the inactivated subunit) involve t,his region. In none of the molecules that contain these interactions has the rnolecdr Iwrtl seen to form a theta figure. =\ theta figure c~)uld be indicatJiv(> of a trapprti topological knot (Cant,or rf cl/.. 1980). In pa,rt’icular. the end to end carosslink must enclose nearly the entirr moleculr. so we can cwnclude that thew are tlo topologically linked interac%ions anylvhere in the molecule. The four most frequent int~cractions cwsslinked in the ina.c*tivch30 S subunit arc’ not found in the consensus tnodtll of t,he 16 S rRSA (Soiler & \Voew. 1981 ). 1tI addition, t,here are long-rangr secwndary strwtuw ff~aturrs in t’hr rnodrl that \voultl bc expected to be crosslinked but are not. For rsarnpl~~. the interac+ion 27 37 ‘517 556, which is irilmediately adjacent t,o the intrraction X4- 570’ X80-886. has not been detected among the crosslinkcd molwules. It is rasy to reason that, RNA RNA duplexes that arc in close association with ribosomal proteins c~mlti be mwlh poorer sites for psoralen intercalation and crosslinking than duplexes that are not. A clear example of this type of psoralen behavior occurs in chromatin. in which the DNA bet,ween the nucleosomw is a much better substrat,e for wosslinkinp than the DNA associat’ed with the nwleosome wre (wiesrhahn rf trl.. 1977). There are two explanations of wh), the prominent f’soralerl-c,rosslinkrct
KN’X
(‘HOSSI,INK
~I,~(‘TIZO1’HOltESIS
ASI)
RlI(‘I~OS(‘OI’1
I ti.i
interactions are not found in the secondary structure model. The model may he grossly incomplete or incorrect, but this seems unlikely considering the quality and quantity of the data that has been used to generat’e it, and considering how well the different types of data are in agreement. Alternat’ively. the crosslinks may occur in addit’ional short helices or tertiary structure not’ included in the present model. The terminal 50 nucleotides contain many positions that are both highly conserved (see Schnare B Gray. 1981) and chemically modifiable. By comparison to transfer RSA sequences and structure, these characteristics are a signature for the involvement of these residues in tert’iary structure (Noller C! Woese. 1X-41). It is known from the size of the 30 S subunit and the projected size of the srtwndary structure model that t’he model will have to he condensed by, at least 1an additional factor of two in both width and height. The four prominent pxoralen cwsslinks report’ed in these experiments indicate that the three-dimensional strrwture cont,ains interactions that put the 3’ minor domain int,o close eontact with other regions of the molecule. These interactions may be important, for the function of the subunit. The topography of the messenger binding sit,e (Shine H: Dalgamo. 1971: St&z & ,Jakes. 3975) anti the peptidvl tRX.4 bintling site (Taylor Pt ~1.. 1981 ). both of which are located in the 3’ mirier domain. could he controlled from the body of’ t,hr subunit’ by these RN.4 cont’a,cts. \Vt* are grat~~ful to I)r Rolwrt Llurphy for assistanw itt analyzing thtb crwslinkittg data wtti to I)rs .lohtt Hearst, l’allaiah Thamtnatla attd b3izalwth Matthe\\ s for helpful critirisms of thr manuscript. This work \vas supported I)y a National lttstit,utrs of Htdth fellowship (AI06001 to l’.L.\V.) atrd a Sational Itrstjituttbs of Hralth rcwarch grattt, ((in1 19843 to (‘.K.(‘.l.
(‘antjor. (‘. R,.. Wollenzirn. P. I,. & Hearst. .J. E. (1080). AVcrc/. .-lcl:ds BPS. 8. IX&- 1872. (‘arlwn. I’.. Khrwmann. C.. Ehresmann, R. & Elwl. .1.-l’. (1978). FKKS Lrttr,rs. 94. 132~ 156. I)ierrrr. T. 0. (1973). =IM~. Riochr~~r. 55, 317-~320. Ehrwmanrt, C‘.. Stiegler. I’.. Mackit~. (:. A.. %itnmrrmantt. R. AA.. EM. J-1’. Hr Pr~lltwr. I’. (;lotz. (‘. 8: Brimacwmbe. R. (1980). Sd. Alcid.s Krs. 8, 2377 %4!1.‘3. 56. ICI-108. Hilt. H., If:iegers, V. B Atlamirtz. I’. (1975). Ir:rtr. .J. Rio~hrn/. Is;~i\~s. S. T.. Shtln. C‘. ,I.. Hearst. J. E. R: Rapoport, H. (I!tTi). Riochr,rttistr!y. 16. 1058 IO6k. Nollw. H. F. & L\‘oew. (‘. K. (1981). Scipuw. 212, 10:)-41 I. Ibl)itt. I). & (‘t-others. I). RI. (I!JT!)). SW/. .-lcids Kus. 7. 68!)~ 703. Schnare. 11. R’. & (iray. 11. \V. (I!)Xl). I’li:BS f,dtw.s. 128. 298 304. Shintb. .J. k I)algarno. I,. (1974). I’roc. Sut. .Awd. Sci.. I’.S.d. 71. 1312 1346. Stcit,z. .l. A. & -lakes, li. (1!)73). I’m-. Sut. 3rnd. Sci.. l’.S..-I, 72. X-558. Stivgltlr. I’.. C’arhon. P., %ukrr. M.. Eltel. .1.-l’. & Khtwmattrt. (‘. (l!ISl). Surl. .-lcids Kcs. 9, 215%Zli2. ‘I’:~ylor. 13. H.. Print. .J. 13.. Ofrtrgand. J. & %itttmc~rtnatttt. K. A. (1981). Biochr/,/istr,//. 20. ir,Xl 7.588.
159. 151-166
Gel Electrophoretic Technique for Separating Crosslinked RNAs Application to Improved Electron Microscopic Analysis of Psoralen Crosslinked 16 S Ribosomal RNA
We have developed a gel electrophoresis technique for separating crosslinked RSX molecules into a series of discrete fractions. The gel used is polyacrplamide made ill formamide and low salt designed to denature the RNA during electrophoresis. The mobility depends upon the positiotr of crosslinking within each moleeult~. as demonstrated by electron microscopy of RX.4 cluted from the gel. In genrral. molecules with large loops elect~rophorese more slowly than molecules with small loops or uncrosslinked molecules. We have used this technique to re-examine the psoralen crosslinking pattern of Eschwichin coli 16 S rihosomal RNA in inactivat4 30 S rihosomal suhunits. To determine the correct orientation of each type of rrosslink. we have covalently attached I)NA restriction fragments to the RNA so that the polarity of the RNA in the mieroseope would he known. Our previous major conclusions are c~onfirmed : the predominant long-distance crosslink detwted hy gel electrophoresis involves a residue rlose t,o the 3’ end and a residue approximately 600 nucleotides away : the formamidejpolyacr~lamide gel is able to separate two closely spaced 1 100.nueleotide interactions hegmning close to the 3’ end. which were reported as OIW interaction hefore: and an interaction joining t,h(* ends is detected as hefore. However. one lowfrequency crosslinked interactiorr. between positions 9.50 and 1400. and possibly another lowfrequenc~ interaction. between posit,ions 550 and 870. are determined to I)e in the opposite polarit,y t,o that deseribetl previously.
1. Introduction Our
int,erest,
has been
that) would
reveal
ribonucleie
acids.
of RSA-RNA
the development
details
of the secondary
Potentially.
psoralen
and tert’iary photochemical
erosslinking structure
into
a method
of single-st~randed
crosslinking
can
provide
strand folds back to interact with itself. This would be a very useful t,ool in understanding t’he struct’ure and dynamics of a molecule such as a ribosomal RNA. The greatest obstacle in realizing this end is in not having a truly expedient and rapid method for evaluating the types and freyucncies of the crosslinks made in t)he RSA. It has been our desire t’o find a gel
direct
evidence
of the positions
where
the single
Ial
I 32
I’
I,. L\‘OI,l,RS%IE:N
i\SI)
(’
I< (‘.AN’I’OI~
electrophoresis tnet,hod that could separate cwsslittked t~~olrc~nlw. This swtttecl possible, because t)he structural differences lwtwecw diff&ent types of crosslinked tnolecules should he tnost, apparent whett the molewles are denatured. and there a.re several tnet,hods for performing electrophoresis under denaturing cottditiotts. In t,his paper we show that it is possible to separate a mixture ofc~rosxlittkt4 16 S rKNA into a series of dist,inct species by e~ec~rophorrxis through a fortttatttitl~~ denat,uring medium. Llntreat,ed 16 S rRI\‘A electrophoreses as a single. f&t mobilit? species under these conditions. The new slow. tnohilit~y bands found whctt crosslinked samples are electrophoresed contain molecules that have long-dist.attw crosslinks at, different positions. 5Iolecules t’hat have large loops (or double loofw). in general. tnove tnore slowly than tnolec~nles wit,h smaller loops. Thereforc~. 1)~. inspecting the pattern on t)he gel, we are able to attticaipate the ttuntlw and size ot’ the different features that will be found in the mixture. Before electron tttic~rosc~~pic analysis. t.he crosslinked RSA sample is preparatively sorted into a set of fractions. This reduces t,he heterogeneity of the types of tttolwules seen in the ttticwwopt~ al ottc time and thereby greatly simplifies the analysis. Our earlier cxperitnents (Wollenzien rt (II.. 197!Im,b: Thatt~tttatta rt I//.. 1!479) wtlr(b hegun on Ewh~Prichin w/i 16 S rlCiYA brcausc a partial fwinrary srqurtrcv~ (Ehrestna~tm rl ~1.. 197.5) had been completed at tha,t titttc. Since then. t)he cotrtpletc~ primary sequence has been determined hy two groups (Krosius (‘I nl.. 1978: C‘arlwtl rt /xl., 1978) and this has unleashed a tt~t~tnendous advance in underst~andittg the organization of t’his molecule (see Soiler & b’oese. 1SSl). III particular. it has allowed several detailed secondary structure predictions of t,hr tnoleculr (~Votw rf al.. 1980: (ilotz & Kritnacotnhe. 1980: Stiegler rt ~1.. 1981 ). These prrdictiotts all agree with one attot,her to a large extent, Ijut are not in agreement with the psoralett crosslinking data we have presented previously. Eretl given the tttrwrtaittty of’ thca measurements we made in the rlectron microscope. there is essentially no matchul) of the long-distance cottt,a.cts we detected with those predicted 1)~sthe models. However. several matchups c~ould be tnadr if thr polarity assigntttents \\‘t’ hat1 made were reversed. Our feature I would then tttat~clt. ivithitt error. the 27 37 ’ 54i 5.~6 interaction, a base-pairing interaction twt,weetr rwidttes 27 through 37 \vit 11 residues 517 through 556 (for further description of’this ttomenc&turt~. SW Noller 8~ FVoese, 1981 ): our fbat,ure II would match the int~eractiott 17--20’915 318: anti ollt’ feature ,X1 would tnatch the ittt,eractiott 563-569 ’ 879M385. All of’ these propowd base-paired interactions contain oppositcl adjawttt tlridine residws. tvttich arc attractive sites for psoralett f)h(~to(~hetrric,aI crosslinkittg (Tttotrrpsott c,t t/l.. I!)81 ). Thus. n-c were in t,tttb dilrtntt~a of correlating our rcsrtlt~.* with tlrfw other tttotlt*ls only if our polarity assignments were ittwrtwt To try to r&olve this discrepancy. wt’ have de\-eloped a tnorc satisfactor.v method of determining the polarity of c~rosslittked features in the electron fragment from c~lotietl microscope (Wollrtiziet~ & (‘antor, 198%). A small restriction ribosomal D?iA is covalently attached to the crosslinked 16 S rRS=\ before preparing t,he sample for elect~rott microscopy. The fragment 1seen as an extra stna~ll strand at, the position where it hybridizes. allows the polarity of the 16 S rKXA to be known. The sample that we have st,udied for this report is I8 S rllN.4 that had Iwen
RNA
CROSSLINK
ELE(‘TKOPHOKF,SIS
BNI)
MI(‘ROS(‘OP‘1
I .53
crosslinked in the inactivated 30 S ribosomal subunit. This sample was chosen because we expected from our previous work (Thammana et al.. 1979) that it would contain a much simpler pattern of products than crosslinked free 16 S rRN.4. and thus would be easier to analyze.
2. Materials and Methods (a) F’rrpnratio,l
of thr
KAY.4
30 S rihosomal subunits isolated from E. coli MRE 800 cells. according to the met,hod of Amils rt nl. (1978), were a gift from Dr Y. Gloria Chu. They were psoralen crosslinked in inartivation buffer (0.5 miv-magnesium acetate. 100 mm-ammonium acetate. 10 rnMet al.. 1979). except that [3H]AMTt (HRI Tris.HU. pH 72) as described (Thammana Associates; spec. act. 95 x lo5 rts/min per pg) was used at a final concentration of 40 ~g/ml. and the samples were irradiated in 1.5-m] Eppendorf tubes at 6°C for 10 min in the device described by Isaacs et nl. (1977). The subunits were then digested for 30 min with 0..5 mg of
prf-incubated
proteinase K/ml
(EM Biochemicals)
in thr presence of lyb (W/V) sodium
sulfate (Hilz et al.. 1975) hefore extraction with phenol and precipitation n-it,h The incorporation of [ 3H]AMT into the RNA was determined after an additional prtbcipitation with ethanol. The I6 S rRNA was checked for intactness and contamination by electrophoresis on 20/6 to 15% (W/V) gradient polyacrylamide gels (Amils et al.. 1979). dodecyl et’hanol.
(11) Formamid~/polyacrylanlidr
gel r/fxtrophowsis
A volume (300 ml) of formamide (Matheson. Coleman and Bell) was deionized by stirring for 3 h with 12 g of AU5OLX8 mixed bed resin (Bio Rad). A 35’?, (n/v) aqlamide solution was then prepared by dissolving 13.5 g of acrylamide and 2.25 g of bisacrylamide (Eastman. once recrystallized from chloroform and acetone, respectively) in the deionized formamidc. bringing the final volume to 45 ml. (iels, 5.20,; to 5%% (w/v) in polyacrylamide, were cast with a gradient maker into four 8 cm x 8 cm x 0.3 cm cassettes held in a slab gel casting apparatus GS(‘-4 (Pharmacia). The dead volume in the casting apparatus was filled with glass beads: 10 ml of 200/o (v/v) ethanol in the apparatus became an overlay when the gradient was made. The 5.23, solution contained t,he following components: 9.75 ml of the 359” acrylamide. 55 ml of deionized formamide, 65 ~1 of 2 M-sodium phosphate (pH 65). 145 ~1 of I~,i~~~‘.S’-tetraethglmethylenediamine. 280 ~1 of 32y0 (w/v) ammonium persulfate. The 5%oi, solution contained thr following components: 1083 ml of the 350,; acrylamide solution. 52 ml of deionized formamide. 1.95 g of sucrose, 65 ~1 of 2 M-sodium phosphate (pH 65). 145 ~1 of S.,V.S’..\“tetraethylmethylenediamine, 280 ~1 of 32”,& ammonium persulfate. The sucrose acts to stabilize the gradient during polymerization and was dissolved in the formamide before t.he other components were added. The 5.2”;0 solution was placed in the mixing side of tjhr, gradient maker and the gradient was allowed to flow into the bottom of the apparatus until th(s top of the overlay reached the edge of the cassettes. After polymerization. the overla? solution was rflmoved, the gels were wrapped carefully and were stored in the cold. Reforc, use. a cassette was placed in the electrophoresis apparatus and a well-forming gel \vas added. It contained: 0.41 ml of 30% acrglamide (300,, acrylamide/l50,b bisacrylamidr (\v/v in M-at,er)). 5 ~1 of 2 iv-sodium phosphate (pH 6%). 2.0 ml of water. 2.5 ml of hot, 1”,, (y/v) agarose. 8 ~1 of iV.LV,L%“.S’-t~etraethylmeth~lenediamine~ and 40 ~1 of 7.5(;,, (WV) ammonium persulfate. Before loading the samples. the gels were equilibrated in the cold n-it)h electrophoresis buffer (cold 2 miv-sodium phosphate. pH 6.5). Electrophoresis was for 12 h at 23 V/cm. After electrophoresis, gels were washed for 1 h in 5 mm-sodium phosphate before being stained with ethidium bromide. Analytical gels were phot,ographed on a
t .~hhrt~viation
used : AMT. aminomrth~ltrimeth~lpsorale~~
151
I’. I,. U’OI,I,ESZIES
ASI)
(‘. Ii
(‘ASTOR
trdnsillutnirtator box. Prrparative gels \vrrv visualizt~tl 1vit.h ii :
The method for covalcntly attaching a l)NA rrst~rivtion fragttttbttt t,tr the RNA molt~~ul~~ts described in detail elsc\vhere (Wollrnzirtt K- (‘antor. lW2). For thtwh c~xpt~rimt~nts. 0.5 ~1 c>t 016 pg of RNA/ml in formamidr~ \vas hyhridizrd to att equal volumes of thtl ltsoralrtt monoadduct DNA restriction fragment (at a,pprox. 02 pg/pl in WK (0.1.5 It-sodium c*hltrrid(~ I5 mw-sodium citratcl. IO mwdisodium EIJTA)). All of t,h(s hybridizations wc’rv lwrformtSd \vith the fragment ~otnplt~m(~ntar~ to nuclrotides 962 to I322. \vhic*tt is pcwrrated 1)~ ‘/‘UC/I endonuclraw digestion of plasmid pC”S3 (a gift from I)r (‘athtnriw Squires) csxwpt ott(‘. v, 1lic.h was performed with thv fragment (~otnl~lrtnt~ntnr~ to ttuc*lwtidcs 8!) to 123. ~hictt ih generated 1)~ Mboll cttdottucleaw dig&ion of plasmid pCS1 I ( ISrosius (t c/l.. 197X: \Vollenzirtt & Cant,or. 1982). After c~rosslinking atrd prwipitation u ith c~thxnol. ttw crosslinked hybrids \vvrv resuspc~nded in I ~1 of \\.atcsr Itrforv Ittvparatiotr for c‘l
Ikwaturation of the R3.A samples and t,hc spreadittg c~cttulitions arc’ a modification cjt’ t)ut previous t,rchniqw (LVollrnzirn r/ (I/.. 197%). RNA (1 ~1 of 0.16 &PI itt formamide) or the rwuspended DNA-RSA hybrid \vas mixed \vith 19.5 PI of formamide (vacuum distilled) attcl was heated t,o ,‘,i’(’ for 5 mitt lwfore l.I.5~1 of titrmaltlt~h~dt~ (heatc4 in a walrd vial in :I boiling waterbath for 30 min) and I.0 ~1 of 60 m~-?uTa~HP0, was addrd. The mixture \+.a~: hrated for an additional IO mirt and was then yuenc~hrd ott iw. Formamidr~ uwd irt thtl hyperphast. contains ltc~t~zyldialkplammonium chloride (HAC‘. a gift from I)r Thro Kollrr) t)o 135~1 of formamidr was added 1 ~1 of l~rt~z~ldialk~latntttottittt~~ cahloridr \vhich ha:: a concentratiott of 9 ;I 2h0 utrit,s (approx. 4.5 mg/ml). For t,htB RX.4 AIonto. thtx hvlwrphaw contained 3.5~1 of formamidr (\l-ith lwtwyldialkylammot~iutn chloridc~). 5 ~1 of iG!l ttnffbr (0.1 wtrirthanolatnitw (titrat,csd with HC’I to pH X+). 04)I sS~,E:I)T.-\). .i ~1 of the drnaturation mix. attd 5 ~1 of I mg cytochrorrtc~ c/ml (Sankyo. (‘Sl3r c.lcaved). For the 1)S.j RNA hybrid, the hyperphase cwntahted 15 1~1of formamidr (tvith l~rt~z~ltiialk~larrttrtot~ittttt chloride). 2..7pl of EM 1tufft.r. 5~1 of t,hr drnat~uratiott tnik. and 2.;)r ~1 of c*ytochrotttt~ < .)r p I of the hypwpha,sr \vas applird on a glass slitIt%to a dr~iottiwtl solution. In both vasw. ,..t water hypophase. After 1 mitt. thv tno~lolayrr was picked up on parlotlion-c.ovrrPtl grids awl ~vas M-ashrd for I mitt in .XP,, rthanol to rctnovc polyacrylatct drttris lwforr lwing rinsed in 90(‘, ethanol. stained with uranyl arrtattl. and hriefl? d&ainrd. The grids \~c~rc’-shacio\~c~cl \vith IV/I’d before viwittg.
The grids wcrv photographed in a Zeiss EnlO S-2 (~lrctrott ttticrosc.opts attcl thta Itroitv3rd nrgativrs \vertl t,rawd with a Nutnoniw measuring devic.e. All of the rnolwulcs \vc’ havfa traced have lwen reduwd to a standard pattwn. Sinw I crosslittk itt a tnoleculr forms I opvtt loop with as many as 2 t,ails. 3 measurcmcnts are ncedrd t,cr sprcif:\- t,hr positiotl 16 t.lrv c*rosslink : the distatrcv from the 5’ end (or the ~losrr end) to tht, I)asc~of the loop. t J-w rirc*umfrrencr of tttcl loop. and t,hc distattcv from the I)aw of the looI) to the other cstttl. ?;otc% that in this pattvrn rithvr (or both) of t,hr t,ails can Iw wro Thr data wt‘rt‘ wllwteti itttd
RN.4
C’ROSSLINK
EI,ECTROPHOKESIS
ANI)
#TCROSCOP\
I .i5
processed by a VACSjl l/780 computer (Digital Equipment Corporation). For each molec~ulr. the crosslink is then described by a pair of co-ordinates: the X co-ordinate is the position of the beginning of the loop measured in nucleotides, based on the total length of the molecule: the Y co-ordinate is the position of the end of the loop, also measured in nucleotides. The dat’a were first displayed as a correlation plot of Y versus 9. Tdentifying clusters of data on this plot is equivalent to finding a group of molecules that have similar loops. Tht data were then further processed by dividing the XY space by a rectangular grid. The darkness of each rectangle indicates the number of data points contained in that area. Sinctr. in principlr, X can have a value as large as 1541 and Y can have a value as large as 1542 and Y is always greater than X, the plot of data in which the polarit? of the molecules is used will occupy the half of a square (1542 nucleotides on an edge) that 1s above the diagonal line 1.=x. of the molecuk~s. We are able t,o perform this analysis even if we do not know the polarity In this case. t,he measurements of X and Y are made to the closer end of the molecule: thts middle of the loop made by the crosslink has a value less than half the length of the molcrulr. [II t)erms of the co-ordinates X and Y. this is equivalent t,o (X+ I’)/2 < 1542/2. This means. of course. that we do not know whet,her a particular loop is closer to the 5’ or 3’ end of thr molrc*ule. Operationally. the plot of Y ~rsus X becomes folded across the line Y = 1542 - S and occupies l/4 of the square that is 1542 on one edge. In practice. molecules that have not been hybridized are first measured and an unoriented (t,hr polarity is not known) plot of Y vwsus X is constructed. Molecules that have been hybridized are t,hen measured. These are first plotted as if we do not know tht, polarity. These plots should contain similar groups of data if the hybridized molecules are representative, of the unhybridized molecules (see Results). The plot of Y 1lersu.s X for the hybridized molerules can then be unfolded to determine the polarity of the characteristic groups of data. A more complete description of this technique will be given elsewhere (1’. L. \Vollenzien. R. Murphy & C. R. Cantor, unpublished results).
3. Results (a) Formamidelpobyacrylamide
gel elertrophoresis
30 S ribosomal subunits in inactivation buffer were incubated with 40 pg AMT/ml and were crosslinked with a saturating amount’ of 365 nm light. The RNA recovered from the subunits incorporated 10.1 AMT molecules per 16 S rR,NA molecule. This level of AMT incorporation is very similar to the amount’ of hydroxymethyltrimethylpsoralen incorporation under the same conditions (Thammana et al., 1979). In the RNA, both before and after psoralen crosslinking, there are small amounts of 23 S rRNA, 5 S rRNA and RNA intermediate in size between 16 S and 23 S, all of which must derive from contaminating 50 S ribosomal subunits (results not shown). Figure 1 shows the results of formamide/polyacrylamide gel electrophoresis of this crosslinked RNA sample and a control, untreated 16 S rRNA sample. The cont’rol 16 S rRNA electrophoreses as a fast mobility band (there is also a small amount of contaminating 23 S rRNA). whereas the crosslinked 16 S rRNA separates into a series of bands. The most intense band has the same mobi1it.y as untreat,ed 16 S rRNA. A sample (100 pg) of this RNA was electrophoresed on an identical gel (using a single wide well) for the purposes of obtaining RNA from each of the bands. After staining. the gel was visualized with a 375 nm mineral light to avoid photoreversal
I .ifi
of the psoralen crosslinks (Rabin & (‘rothers. 1979 : and our results. not shown). The gel was separated into seven fractions by t,he cuts indicat’ed in Figure 1. and the RKA was electroeluted from t,he gel slices. After extraction with phenol and precipitation with ethanol. the fractions were resuspended in wat’er and W~I‘P checked for their concentration and molecular weight by elwtrophorcsis OH polyacrylamide gels (results not shown), and were a~lso rF,-rlectrophoresrd 011 formamide/polyacr~lamide gels to contirm the identity of each fraction (Fig. 2). The characteristics of the fractions are given in Table 1. (h) ~detrt~ficatiot~ ctf thr crosslittks
hy r/wtrotr
ttricrosrop!y
All of these fractions have been examined in the electron microscope to determine if the RN.4 has unusual structures. &a&ion 1 contains a wry heterogeneous mixture of different types of molwules. On the polyacrylamide gels t,his fract’ion electrophoreses with bhe mobility of 23 S rRSA. In the microscope.
RSA
(‘ROSSLINK
EI,ECTROl’HC)RESIS
AND
MI(‘ROS(‘OPV
157
FIG:. 2. Re-electrophoresis of the fractionated crosslinked 16 S rRNA. Approximately 02 pg portions of the resuspended RNA samples (except for fraction 1) were re-elrctrophoresed on 2 formamidr,ipolva~r~lamide gels to check their composition. Lanes numbered 1 t,o 7 correspond to thv fractions I to 5. The starting crosslinked 16 S rRNA was electrophomsrd in the lanes lettered S for c~omparisotl.
RS-3 recovered from preparative Fraction no.
Yield (f%)
formamidelpolyacrylamide
Molecular weight identity (S)
t Small amounts of photoreversal
in these fractions.
Surnbrr bands
of
gel
It58
I'. I, b'OI,L~N%IEN
ASI) ('. It. ('AN'l'OI<
rnany of’the molecules contain more than two ends. Thesr are probably crosslinked 23 S rRNA molecules, which are frequently broken. We have made no further attempt t,o characterize this fraction. Molecules in fractions 6 and 7 both appear to be linear in the electron microscope. Local structures not resolved in the electron microscope would make the molecules appear short,er than untreated 16 S rRNA : however. we have not attempted to detect any systematic shortening of the contour length of these molecules, since at this level of psoralen incorporation we would not expect, more than S’$;, shortening based on previous result,s for 16 S rRNA crosslinked in reconstitution buffer (Wollenzien it al.. 1979aJ). This difference is about one standard deviat,ion in the dist)ribut~ion of contour lengths of the molecules, so the difference would be very difficult, to det#ect with any degree of certainty. Fractions 2, 3, 4 and 5 all contain molecules wibh recognizable crosslinked features. To determine t,he number of different features, each fraction was prepared for electron microscopy first without hybridization to short. complementary restriction fragments and then wit’h hybridization and crosslinking t,o these restriction fragments. For t,he unhybridized RNA, every field in which at least three crosslinked measurable molecules were found was photographed. even if t,hertb were already good examples of that type of molecule. In t,his way, t)he molecules measured reflect the frequency distribution of the different’ types on the grid. Thtx molecules were analyzed by encoding each crosslink into a pair of c-o-ordinates : t)hr X co-ordinate is the position of the beginning of the crosslinked loop, t)he 1’ coordinate is the end of the loop. The distribution of different types of crosslinked molecules can be seen on a plot of Y versus X (see Materials and Methods). ;\ clustering of data in a part’icular area indicates that there are many molecules with similar loops. Tt is inferred that a specific crosslink (or a few very closely spactaci crosslinks) is responsible for the appearance of the similar loops, and thus t h< average co-ordinates of t,he cluster of data will give the position of t)he c,rosslink. To check that, the molecules that have h,vbridized with a DNA4 fragment art‘ representative molecules, the analysis was first prrformed on them as if wf’ did not know the polarit,?. In this case. the correlation plot should Iook very similar to t hc> plot done for the unhybridized molecules. The molec*ules wer(’ then a~nalyzeti using the known polarity of each. The correlation plot then unfolds to show th(l c.c)r’recat orient’ation of eacshcharacterist,ic crosslink. Figure 3 shows t,hc>correlatjion plots for fractions 1 t,hrough 5. The three :LIVHSIII the correlation plot, of unh,vbridized molecules from fraction 2 are carntered at 0” 1542, 6” 1088 and a loose collection of data around 283’ 1025. The interaction 0” 1542 cannot have a polarity assigned t,o it. Molecules that caontjain t,htb 1 I(H)nucleotide loop hybridize to the restriction fragment,. and the loop involves t ht> 3’ end in t,he oriented molecules. This allows t,he avrrage crosslink position to be assigned as bet,ween residues 454 and 1536. Molec~ules that would fall into t hcl third area do not hybridize and are probably not I6 S rRN.4. ln t,hr cborrrlation plot of’ t)hr hybridized molecules there are several rxamples of rnolec-ulrs with ftiaturtss at (orient,ed) 493”975 and 61%” 1325. \\‘e will not consider these molecules as containing real crosslinks. since the loops were not seen in the original or subsequent) screening of t,he unhybridized molec~ules. A summary of t’hese features is
RNA ('ROSSLISK
E1,ECTKOl'HOI~ESIS
ASI)
S11('I~OS('Ol'\
l.i!)
given in Table 2. The frequency of each t)ype of crosslink is estimated on t’hr frequency in the correlation plot and the amount of R’KA in the fraction. In addition, fraction 2 contains two classes of molecules that have crosslinks: the interaction O”1540 together with the prominent feature of fractions 5 and 4, and the interaction 450”1540 (or the interaction 510-1540 from fraction 3) toget)her with the prominent feature of fractions 5 and 4. The one prominent area in the correlation plot, of unhybridized molecules from fraction 3 is centered at 6^ 1033. These molecules hybridize the restriction fragment and the interaction involves the 3’ end of the molecule, which makes it OCOUI between residues 509” 1542. This is a different interaction from that det’ected in fraction 2. since these fractions do not show cross-contamination on t,he formamide/polyacrylamide gel. The three areas in the correlation plot’ of unhybridized molecules from fraction 4 are centered at 2”609. 146”588 and 550”873. All three classes of molecules hybridize to the restriction fragments; the first, two features are closer to the 3’ end. which puts them at 933”‘1542 and 954^ 1396. There is a single example that places the interaction 550^873 somewhat closer to the 5’ end, so it tentatively remains at this co-ordinate. In addition. there are several examples of hybridized molecules containing an interaction that, is approximately 6” 1181, and two hybridized molecules that, contain an interaction at, approximately 0^675. The interaction 6”llSl is not seen in the unhybridized molecules of fraction 4, and the interaction 0^675 is not seen in the hybridized molecules of fraction 5. Fraction 5 contains molecules with features centered at 2”606 and there are also some molecules centered at 97”600. The correlation plot of the hybridized molecules places both of these features closer to the 3’ end. so the average positions are 936^ 1542 and 945” 1445. Since the molecules in fraction 5 are a subset’ of those found in fraction 4, it is likely that the interaction 945^ 1445 corresponds t’o thr int,eraction 954” 1396.
4. Discussion The improvement in t’hese experiments comes from the ability to elertrophoretically separate different crosslinked species and determine the orient,ation of each feature by covalent attachment of a DNA restriction fragment’ to a known position on the RNA. We now have a method independent of electron microscopic inspection to determine the number of new types of molecules present, in a mixture after crosslinking (see Fig. 4). We can greatly enrich for a particular type of molecule before determining its identity. This allows us to examine low-frequency species without the fear that their presence in the electron microscope is artifact’ual. Tn addit’ion. mobility on the formamide/polyacrylamide gel is quite sensitive to differences in t’he sizes or positions of features, even when t,he differences are difficult to detect in the electron microscope. However, since the change in electrophoretic mobility depends on the size of the loop in the molecule. t.his gel system probably would obscure the fact that there were short-range interactions that were crosslinked by psoralen. in addition to the long-range interactions, which are crosslinked and seen. For discussing the crosslinks detected in these samples. we wish to adopt a
t‘ll. :i
(a)
x
3, hybridlzed used
Fract\on Polarity
X
2, hybridized Fraction
.___ If
0
:1
%
I
j c, :: ‘”
4
‘0 0
8 LD A
A
notation derived from that of Keller & Woese (1981). A prefix EPs will be used before the approximate co-ordinates of the crosslink: E signifies that the ident,itication has been made in the electron microscope nith what,ever errors are inherent in this method : Ps indicates that the crosslinks are generated by psoralrn. The co-ordinates are measured only to the nearest) ten bases. because we have no reason t,o expect the accuracy to be better t’han this. We have been able to ident,ify six psoralencrosslinked interaCtions in t’he 16 S rRKiA from inactivated ribosomal subunits. In addition, WE see several types of doubly crosslinked molecules. which are expected. There are several other types of features that were encountered after the hybridization and crosslinking to restriction fragments but these other features were not seen in the initial screening of the fractions. so we do not consider them t’o be significant. Of the six interact,ions. four correspond to features we have already reported (Thammana crosslinked interaction El’s 930” 1540 Pt (rl., 1979). The most prominent corresponds to the feature previously estimated to be 570 nucleotjides in size. which was determined to involve the 3’ end of the molecule by our previous hpbridizatjion t,est, (F\~ollenzien rt nl., 1979h). The interactions EPs 450A 1540 and EPs 510A I.540 t,ogether cwrrespond to the feature previously estimated to be 1040 nacleot~ides in size and involving the same end (the 3’ end) as the most prominent feature. The int,eraction EPs 0” 1540 corresponds to the end t,o end crosslink seen previously. The int,eraction EPs 950^1400 csorresponds fairly well in size to a feat,urt measured in the 16 S rRNA crosslinked in solution, feature VII (U’ollenzien rt ~1.. 1979a). and also seen in the crosslinked 16 S rRNA from inactivated subunits. However. feature VII had been assigned by the previous hybridizabion t,est to be csloser t,o the 5 end of the molecule (U’ollenzien rt N/., 1979b). Likewise. the interact,ion EPs 550”870 corresponds fairly well in size to a feature measured in the 16 S rRXA crosslinked in solution, feature XI, and also seen in the crosslinked 16 S r-RN.4 from inactivated subunits. However. by looking at molecules t,hat had two
Flc:. 4. Separation of diffvrrnt types of crosslinked RSA molrculcs on thr formamjde/polva.c.r?-lamidl, gel. l\lolrwAes with the 6 types of rrosslinks we have identified we shown. The identity of thr crosslinks that the molecules contain are. from I)ot,tom to top: W’s 930A1540, Et’s 950A1400. EPs 560^870. KPs :it0^1A40.E:l’~450^1540(ri~ht.on topnw). W%OAl;i40( second from right. on top row). Also shonn irr thr top POWare 1 types of molecules with double features.
164
I’. I,. \VOI,LEN%IEN
ANI)
(‘. t<. (‘.SS’I’OR
crosslinks. feature XI had been assigned to be cslosrr to thr 3’ end of th(, molwulr. Because the map of 1I interactions found in 16 S rlCNA crosslinked in solution (protein-free) was built up by observing molecules with two features and was self consistent, correcting the polarity of feature VII and XI would mean that eit,her the most frequent features crosslinked in t,he protein-free I6 S rRKA4 are different from those crosslinked in the subunit, or the original map has inconsistencies. \i\‘c expect to learn more about t’his when we w-examine 16 S rRNA crosslinked in solution. We have not found in gel-fractionated molwules two low-frqutw~~y fi,at urw WV previously reported seeing : an interaction involving one end of t,he molwulr and some residue about 240 nucleot~ides away (f+ature I\‘), previously reported in ()..5”,, of the molecules crosslinked under these conditions, and an interaction srparat~rtl by 210 nucleotides (feat)ure 1’1). which was enclosed 1)~ feature 1’11. pwviousl> reported in 2.20z, of the molecules. The failure to see t,hese features could rrwdt from differences in the crosslinked samI+. since the frequency of these features LVRS quite dependent on the crosslinking cwlditions (Thammana P/ ~1.. 1979). The pat)tern of psoralrn crosslinks indicates t)ha,t large regions of the 16 S rKS,\ are not accessible to crosslinking. These regions irwlude thr 5’.t,erminal domain defined by the interact)ion 27 -37 A547LX6 (for a description of this and subsequent domains, see Noller 8 Myoest~.1981). cxoept for positions at approximat’ely 4.50 and 510, the entire central domain, defined by the intrracbion 567-570A8XO--886. and the 3’ major domain. definrd by thr interaction 926-933^ 1384-l 391. Two of thrx features we have reported here probably c*orrtaspond to crosslinks made at t,hr baw pairing interactions t,hat define these domains. If allowa,nce is madr for the accuracy of the measurements made in the electron microscope. t,he feature El’s 950” 1400 fits reasonably wrll t)hc interaction 925-933” 1384-1391. and the ti~aturt~ EPs 550*X70 fits the interaction 567-670”88OC%6. The 3’ minor domain, &I thrb other hand. is highly accessible to crosslinking. Thr four most frequent crosslinks it1 the inactivated subunit) involve t,his region. In none of the molecules that contain these interactions has the rnolecdr Iwrtl seen to form a theta figure. =\ theta figure c~)uld be indicatJiv(> of a trapprti topological knot (Cant,or rf cl/.. 1980). In pa,rt’icular. the end to end carosslink must enclose nearly the entirr moleculr. so we can cwnclude that thew are tlo topologically linked interac%ions anylvhere in the molecule. The four most frequent int~cractions cwsslinked in the ina.c*tivch30 S subunit arc’ not found in the consensus tnodtll of t,he 16 S rRSA (Soiler & \Voew. 1981 ). 1tI addition, t,here are long-rangr secwndary strwtuw ff~aturrs in t’hr rnodrl that \voultl bc expected to be crosslinked but are not. For rsarnpl~~. the interac+ion 27 37 ‘517 556, which is irilmediately adjacent t,o the intrraction X4- 570’ X80-886. has not been detected among the crosslinkcd molwules. It is rasy to reason that, RNA RNA duplexes that arc in close association with ribosomal proteins c~mlti be mwlh poorer sites for psoralen intercalation and crosslinking than duplexes that are not. A clear example of this type of psoralen behavior occurs in chromatin. in which the DNA bet,ween the nucleosomw is a much better substrat,e for wosslinkinp than the DNA associat’ed with the nwleosome wre (wiesrhahn rf trl.. 1977). There are two explanations of wh), the prominent f’soralerl-c,rosslinkrct
KN’X
(‘HOSSI,INK
~I,~(‘TIZO1’HOltESIS
ASI)
RlI(‘I~OS(‘OI’1
I ti.i
interactions are not found in the secondary structure model. The model may he grossly incomplete or incorrect, but this seems unlikely considering the quality and quantity of the data that has been used to generat’e it, and considering how well the different types of data are in agreement. Alternat’ively. the crosslinks may occur in addit’ional short helices or tertiary structure not’ included in the present model. The terminal 50 nucleotides contain many positions that are both highly conserved (see Schnare B Gray. 1981) and chemically modifiable. By comparison to transfer RSA sequences and structure, these characteristics are a signature for the involvement of these residues in tert’iary structure (Noller C! Woese. 1X-41). It is known from the size of the 30 S subunit and the projected size of the srtwndary structure model that t’he model will have to he condensed by, at least 1an additional factor of two in both width and height. The four prominent pxoralen cwsslinks report’ed in these experiments indicate that the three-dimensional strrwture cont,ains interactions that put the 3’ minor domain int,o close eontact with other regions of the molecule. These interactions may be important, for the function of the subunit. The topography of the messenger binding sit,e (Shine H: Dalgamo. 1971: St&z & ,Jakes. 3975) anti the peptidvl tRX.4 bintling site (Taylor Pt ~1.. 1981 ). both of which are located in the 3’ mirier domain. could he controlled from the body of’ t,hr subunit’ by these RN.4 cont’a,cts. \Vt* are grat~~ful to I)r Rolwrt Llurphy for assistanw itt analyzing thtb crwslinkittg data wtti to I)rs .lohtt Hearst, l’allaiah Thamtnatla attd b3izalwth Matthe\\ s for helpful critirisms of thr manuscript. This work \vas supported I)y a National lttstit,utrs of Htdth fellowship (AI06001 to l’.L.\V.) atrd a Sational Itrstjituttbs of Hralth rcwarch grattt, ((in1 19843 to (‘.K.(‘.l.
(‘antjor. (‘. R,.. Wollenzirn. P. I,. & Hearst. .J. E. (1080). AVcrc/. .-lcl:ds BPS. 8. IX&- 1872. (‘arlwn. I’.. Khrwmann. C.. Ehresmann, R. & Elwl. .1.-l’. (1978). FKKS Lrttr,rs. 94. 132~ 156. I)ierrrr. T. 0. (1973). =IM~. Riochr~~r. 55, 317-~320. Ehrwmanrt, C‘.. Stiegler. I’.. Mackit~. (:. A.. %itnmrrmantt. R. AA.. EM. J-1’. Hr Pr~lltwr. I’. (;lotz. (‘. 8: Brimacwmbe. R. (1980). Sd. Alcid.s Krs. 8, 2377 %4!1.‘3. 56. ICI-108. Hilt. H., If:iegers, V. B Atlamirtz. I’. (1975). Ir:rtr. .J. Rio~hrn/. Is;~i\~s. S. T.. Shtln. C‘. ,I.. Hearst. J. E. R: Rapoport, H. (I!tTi). Riochr,rttistr!y. 16. 1058 IO6k. Nollw. H. F. & L\‘oew. (‘. K. (1981). Scipuw. 212, 10:)-41 I. Ibl)itt. I). & (‘t-others. I). RI. (I!JT!)). SW/. .-lcids Kus. 7. 68!)~ 703. Schnare. 11. R’. & (iray. 11. \V. (I!)Xl). I’li:BS f,dtw.s. 128. 298 304. Shintb. .J. k I)algarno. I,. (1974). I’roc. Sut. .Awd. Sci.. I’.S.d. 71. 1312 1346. Stcit,z. .l. A. & -lakes, li. (1!)73). I’m-. Sut. 3rnd. Sci.. l’.S..-I, 72. X-558. Stivgltlr. I’.. C’arhon. P., %ukrr. M.. Eltel. .1.-l’. & Khtwmattrt. (‘. (l!ISl). Surl. .-lcids Kcs. 9, 215%Zli2. ‘I’:~ylor. 13. H.. Print. .J. 13.. Ofrtrgand. J. & %itttmc~rtnatttt. K. A. (1981). Biochr/,/istr,//. 20. ir,Xl 7.588.