the changes 7sl signal recognition particle cycle

The EMBO Journal vol.10 no.4 pp.767-777, 1991

Changes in 7SL RNA conformation during the signalrecognition particle cycle

Massimiliano Andreazzoli and Susan A.GerbilBrown University Division of Biology and Medicine, Providence,RI 02912, USA

1Corresponding author

Communicated by G.Blobel

The structure of7SL RNA has been probed by chemicalmodification followed by primer extension, using foursubstrates: (i) naked 7SL RNA; (ii) free signal recognitionparticle (SRP); (iii) polysome bound SRP; and (iv)membrane bound SRP. Decreasing sensitivity to chemicalmodification between these different substrates suggestsregions on 7SL RNA that: bind proteins associated withSRP might interact with ribosomes; and are protectedby binding to membranes. Other areas increase inchemical sensitivity, exemplified by a tertiary interactionpresent in naked 7SL RNA but not in free SRP. Suchchanges suggest that 7SL RNA changes its conformationduring the SRP cycle. These conformational changescould be a necessary component to move through the SRPcycle from one stage to the next.Key words: 7SL RNA conformation/SRP

IntroductionProtein synthesis and protein transport are importantprocesses within cells. A key player in these events ineukaryotes is signal recognition particle (SRP), whichfunctions as a cellular adaptor between the protein syntheticmachinery and the protein translocation apparatus of therough endoplasmic reticulum (ER) (for reviews see Walteret al., 1984; Walter and Lingappa, 1986; Hortsch andMeyer, 1986; Dobberstein, 1987; Walter, 1987). SRP iscomposed of 7SL RNA that is- 300 nt long (Walter andBlobel, 1982) and six polypeptides (Walter and Blobel,1980). These polypeptides comprise two monomers (pl9,p54) and two heterodimers (p9/14 and p68/72).What are the functions of 7SL RNA within the SRP? One

role is to act as a scaffold to which the SRP proteins bind.Each of the proteins interacts individually with 7SL RNAexcept for p54, whose binding is mediated by p19 (Walterand Blobel, 1983a). Homologous or heterologous SRP can

be reconstituted (Walter and Blobel, 1983a). The regionsof 7SL RNA bound by proteins were first deduced bymicrococcal nuclease cleavage that subdivides SRP into twodomains (Gundelfinger et al., 1983). The Alu domain (sonamed because it seems to have given rise during evolutionto the Alu interspersed repetitive sequences found in thehuman genome; Ullu and Tschudi, 1984) contains 100 ntof the 5' end of 7SL RNA base-paired with 50 nt of the 3'end of 7SL RNA; the S domain contains 150 nt of core

sequence lacking Alu repeat similarity (Ullu et al., 1982).p9/14 is bound to the Alu domain (Gundelfinger et al.,

Oxford University Press

1983), which seems necessary for elongation slow-down orarrest of protein synthesis (Siegel and Walter, 1985, 1986).The remaining four polypeptides are found in the S domainof SRP (Gundelfinger et al., 1983), and the exact bindingsites of p19 and p68/72 have been determined by a-sarcinfootprinting (Siegel and Walter, 1988a). p68/72 is neededfor protein translocation (Siegel and Walter, 1988b), and p54is important for signal recognition (Krieg et al., 1986; Kurz-chalia et al., 1986; Wiedmann et al., 1987; Siegel andWalter, 1988b).

In this paper we propose that in addition to a scaffold rolefor binding SRP proteins, 7SL RNA undergoes conforma-tional changes which might be a necessary component tomove through the SRP cycle. SRP cycles between severaldifferent states in the cell. In a postmitochondrial supematantunder physiological salt conditions (150 mM KOAc), 15%SRP is found as a free soluble form and the remaining SRPis divided about equally between ribosomes and microsomalmembranes (Walter and Blobel, 1983b). Soluble SRP looselyassociates with monosomes; polysomes synthesizingsecretory proteins cause increased binding affinity of SRPby three to four orders of magnitude (Walter et al., 1981).

In the first step of the SRP cycle, when canine SRP bindsto polysomes translating bovine pituitary preprolactin in awheat germ cell-free system, translation is arrested; thearrested polypeptide is 70 amino acids long of which onehalf is protected within the ribosome and the other half thathas emerged contains the N-terminal signal sequence (Walterand Blobel, 1981). Although synthesis of other secretoryproteins is also inhibited by canine SRP in wheat germcell-free systems (Walter and Blobel, 1981; Mueller et al.,1982; Ibrahimi, 1987; Lipp et al., 1987), the generalityof translational arrest has been questioned since it was notseen by canine SRP in rabbit reticulocyte or HeLa cellextracts (Meyer, 1985) nor by maize or wheat germ SRPin wheat germ extracts (Prehn et al., 1987; Campos et al.,1988). An explanation for some of these discrepancies isthat results lacking translational arrest were often confinedto steady state; in fact, a slow-down in kinetics of pre-prolactin synthesis by canine SRP in reticulocyte lysateshas recently been observed (Wolin and Walter, 1989). SRPcauses translational pausing at multiple sites in the nascentpolypeptide in both reticulocyte lysates (Wolin and Walter,1989) and wheat germ cell-free systems (Lipp et al., 1987).All of the SRP arrested nascent polypeptide resides in theP site of the ribosome, and it can be released by entry ofpuromycin into the vacant A site (Gilmore and Blobel, 1985).Though translational arrest is not essential for secretoryprotein transport to the ER, it seems to increase fidelity ofthe process (Siegel and Walter, 1985; Walter, 1987) as wellas keep the nascent polypeptide unfolded for easier entryinto the ER (Rapoport et al., 1987; Sanz and Meyer, 1988).Translational arrest may also regulate the synthesis of certainsecretory proteins such as insulin (Welsh et al., 1986).

In the second step of the SRP cycle, elongation arrest is

767

M.Andreazzoli and S.A.Gerbi

relieved when SRP interacts with the SRP receptor (Gilmoreet al., 1982a,b), also called docking protein (Meyer et al.,1982), which is an integral protein of the rough ER. TheSRP receptor can bind the signal sequence, but has no affinityfor the ribosome (Gilmore and Blobel, 1983). However,arrested nascent polypeptide interacts with the rough ER onlyin the context of the ribosome (Gilmore and Blobel, 1985).The ribosome remains associated with the rough ER forcotranslational translocation of the nascent polypeptide intothe lumen of the rough ER (Gilmore and Blobel, 1983).Finally, once SRP has handed over the arrested ribosomecomplex to the SRP receptor, the SRP can recycle.To address the role of 7SL RNA in the SRP cycle, we

have studied the conformation of 7SL RNA during differentparts of this cycle. The sequence of 7SL RNA is known forseveral organisms, and phylogenetic comparisons haverevealed compensatory base changes in support of a proposedsecondary structure model (Zwieb, 1985, 1989; Larsen andZwieb, 1991). There have been only limited experimentaldata to support the theoretical secondary structure model,utilizing nuclease digestion (Gundelfinger et al., 1983, 1984;Haas et al., 1988) or a-sarcin cleavage (Siegel and Walter,1988a). Electron spectroscopic imaging shows 7SL RNAto be an elongated rod that spans the 6 nm wide and 24 nmlong SRP (Andrews et al., 1985, 1987); cross-linking sug-gests that stem IV of 7SL RNA folds back upon stem II tofit this three-dimensional structure (Zwieb and Schiiler,1989).We report here experimental evidence, based on chemical

modification, for the structure of 7SL RNA from differentsubstrate forms. Our data on naked 7SL RNA indicate thatsome of the theoretically proposed stems have opened, andreveal a hitherto undiscovered long-range tertiary interaction.Compensatory base changes support this tertiary interaction,which may play a role in SRP biogenesis. Previously, gelelectrophoresis of wild-type and mutant naked 7SL RNAshowed it could exist in various conformations (Zwieb andUllu, 1986). In order to examine whether conformationalchanges occur during the SRP cycle, we performed chemicalmodifications on 7SL RNA derived from soluble SRP,polysome bound SRP and membrane bound SRP. Our datashowing 7SL RNA conformational changes in these differentstates suggest that 7SL RNA could play a role in progressionthrough the SRP cycle. Moreover, sites within 7SL RNAthat are protected by SRP proteins, polysomes or roughendoplasmic reticulum membranes are indicated by compar-ison between the different states.

ResultsWe have assayed the chemical accessibility of 7SL RNA indifferent forms of the SRP cycle. For this purpose, SRPwas isolated from dog pancreas in three forms: soluble,membrane bound and ribosome bound (Walter and Blobel,1983b). In addition, naked 7SL RNA was obtained bydeproteinization of soluble SRP. Each of these forms wasthen reacted with chemicals that modify single strandednucleotides. The specificity of the chemicals used is asfollows: DMS methylates adenine at NI and more slowlycytosine at N3; CMCT modifies uracil at N3 and moreslowly guanine at Nl, and kethoxal modifies guanine at Nland N2. After modification the RNA was purified, denaturedand primer extended with reverse transcriptase; chain

elongation by this enzyme pauses or stops one nucleotidebefore the modified base (Hagenbiichle et al., 1978; Youvanand Hearst, 1979). The modified base can be unambiguouslyidentified by comparison to sequencing reactions in adjacentlanes. A control ('O') of untreated RNA was electrophoresedin parallel with treated samples to identify stops in reversetranscription due to inherent secondary structure, nicks, orpost-transcriptional modification of the RNA. A secondcontrol ('stop') to monitor chemical modification caused byresidual chemical in the subsequent RNA purification stepsconsisted of samples given chemical after instead of beforethe stop buffer. Examples of modification by the threechemicals are shown in Figures 1 and 3. Bands that areunique to the chemically modified sample lane or distinctlystronger than background bands in the control lane arescored. Sample lanes of different substrates can only becompared to one another if the amounts of sample loadedare the same. Table I summarizes all the modification datafor the different substrates.

Chemical modification of naked 7SL RNAAs expected, naked 7SL RNA shows the highest degree ofchemical modification relative to the other substratesanalyzed (Figure 1). As shown in Figure 2, 27% of the naked7SL RNA nucleotides are modified, mostly coinciding withpositions predicted to be single stranded according to themost recent, phylogenetically derived secondary structuremodel (Larsen and Zwieb, 1991). However, in a few casesareas predicted to be double stranded in the model arechemically modified (brackets in Figure 2). These regionsusually fall at the end of helical stems or next to bulges,both of which are areas that favor 'breathing'. In othercases, some nucleotides predicted to be single strandedwere not modified by the chemicals. These bases may beinvolved in long-range interactions; we will focus onone such interaction later. Two bases are hypersensitiveto chemical modifications: A75 and G150. A75 is hyper-sensitive in all SRP substrates examined, supporting the ideaproposed previously based on the accessibility of this baseto micrococcal nuclease and a-sarcin that A75 is extremelyexposed in the intact SRP (Gundelfinger et al., 1983; Siegeland Walter, 1988a). In contrast, G150 is only moderatelyaccessible once proteins are bound in solulble SRP, probablybecause it is protected by p19 as will be discussed below.

Comparison between naked 7SL RNA and soluble SRPBases that are exposed in naked 7SL RNA but protected inthe soluble SRP (Figure 1A,B,D, and filled circles in Figure4A) are candidates for protein binding sites. An alternativepossibility is that conformational changes in 7SL RNAwere induced by association with proteins thereby causingincreased protection at certain sites in 7SL RNA, even ifthese sites do not bind the proteins directly themselves. p19and p68/72 are the only proteins whose footprints on the7SL RNA have been published so far (Siegel and Walter,1988a). p19 protects nucleotides 147-153 and 191 -200;p68/72 protects scattered nucleotides from 101-106,129-138, 162-167, 172-183, 202-209, 233-239 and251 -253 (Siegel and Walter, 1988a). These footprints wereobtained using ce-sarcin which cuts both base-paired andsingle stranded nucleotides; the latter which becomeprotected by p19 and p68/72 generally coincide with residueswe find protected from chemical modification in soluble SRP

768

7SL RNA conformation

Table I.

Nucleotide no. Chemical 7SL Sol. SRP Pol. SRP Mem. SRP

G13G14G16U25A26G27G33U35A36U38C39G40G41G44G45A49A53A56A59A67G73A75G76U77U78C84C92U97G98C99C1OOGIOIA102U103G107Ul 10GI 13A115A118Al 19G120U121U122C123G125A127U128A130A131U132A133U134G138A139G146G147G148A149G150A157A163G164C169U171A172

KKKCMCTDMSKKCMCTDMSCMCTDMSKKKKDMSDMSDMSDMSDMSKDMSKCMCTCMCTDMSDMSCMCTKDMSDMSKDMSCMCTKCMCTKDMSDMSDMSKCMCTCMCTDMSKDMSCMCTDMSDMSCMCTDMSCMCTKDMSKKKDMSKDMSDMSKDMSCMCTDMS

+ -+ -++ -+ +(+) -+ -

(+) ++ -+ +++ +(+) -(+) -(+) -(+) -+ -

+ -+ ++ -(+) (+)- +

+++ ++++ -+ +++ ++ +- (+)

(+) -++ +- (+)- +

- (+)- +

- (+)+ -- +

(+) -++ +++ ++ -

(+) -(+) -(+) -- (+)(+) -(+ )++ -

(+) (+)++ -

+ (+)+ -

- (+)+ (+)(+) -+ -++ -(+) (+)+++ (+)(+) -(+) -(+) -

+ -

(+) -

(+)+++

(+)+

(+)

+++

++++++

++

++

++

++

(+)++(+)

Table I. continued

Nucleotide no. Chemical 7SL Sol. SRP Pol. SRP Mem. SRP

G174G175A176G178A183C185C186G187A192U195A199A200A201C202G203G204A205A208U211A213A214A215A216U226G227A228U229A231G232U233A234G235U236G239A240U241C242C244

(+)

(+)

(+)

(+)

(+)

++(+)++

++++

+++

++

+

+++

(+)+ +++ ++

- +

(+)(+)(+)+ +

(+)

- (+)

(+)- (+)

(+) ++ +(+) (+)

KKDMSKDMSDMSDMSKDMSCMCTDMSDMSDMSDMSKKDMSDMSCMCTDMSDMSDMSDMSCMCTKDMSCMCTDMSKCMCTDMSKCMCTKDMSCMCTDMSDMS

(+)(+)

(+)

+++

(+)++(+)(+)+

(+)(+)++

(+)(+)(+)++

(+)

(++

(+)

(+)(+)

(+)

++

++(+)

(++

(+)(+)

+

(+)++(+)

(+)

++

(+)(+)++(+)+

(+)

++++(+)

(+)

(+)(+)

++++++

(+)

(+)

(+)

++

(+)

(+)

++

(+)+

List of bases which are accessible to the chemical indicated (DMS,CMCT, K = Kethoxal) in 7SL RNA from naked 7SL RNA (7SL),soluble SRP (Sol. SRP), polysome bound SRP (Pol. SRP) andmembrane bound SRP (Mem. SRP). The level of sensitivity tomodification was scored as: none -; weak (+); moderate +; strong+ +; hypersensitive + + +. In the few cases where a base wasmodified by more than one chemical, only the results from thechemical with the greatest specificity are listed. Bases upon whichreverse transcriptase pauses or stops to give background bands incontrol lanes of more than one chemical reaction, and in all foursubstrates are C46, U47, C84, U90, U108, C126, A130, U171, U181(nine bases = 3% 7SL RNA residues). Additional bases which hadendogenous stops in controls of just one chemical reaction for : 2substrates were: C22, C71, A75, A118, A133, U142, G155, A163,A231 (nine bases = 3% 7SL RNA residues).

as compared to naked 7SL RNA (Figure 4A; Table I). Inaddition, we see increased protection by proteins for118-123 and 227-229 on the opposing side of the stemwhich was not previously found in the a-sarcin footprints;the smaller chemicals we used here may penetrate morereadily to areas not accessible to the larger a-sarcin enzyme.p9/14 is bound to the Alu domain (Gundelfinger et al.,

1983), but its precise footprint has not yet been published.Our data show increased protection in the Alu domain ofsoluble SRP relative to naked 7SL RNA of residues 13-16

769


B SOL. 7SL-SRP RNA

CMCT o2 0 Z3 3ACGU

.. 1.1 lo1

22

* i3 2 --- --- *

'I-3 4

MLr0;~L

A G

'S; RNA D SOL. 7SL--

SRP RNA'F-I

.., tN,- -1-41N 0± 0° KETHOXALX

5 I-

'i- ACGU c"

* X ''w "........

- - G 187

..)

Fig. 1. Autoradiographs showing primer extension products of Kethoxal, CMCT and DMS treated 7SL RNA from naked 7SL RNA and solubleSRP. Lanes containing dideoxy sequencing reaction products, which were generated using a complementary 5' end labeled DNA primer, reversetranscriptase and 7SL RNA as a template, are indicated as A, C, G, U and correspond to reactions that received ddT, ddG, ddC and ddA. Controllanes marked 'O' and 'STOP' indicate untreated samples and samples to which the stop buffer was added prior to incubation with the chemical,respectively. Bands that are unique to the chemically modified sample lane or distinctly stronger than background bands in the control lanes arelabeled with a filled circle. The modified nucleotides are identified on one side of each autoradiograph. (A) and (D) Kethoxal modification. Samplesin lanes marked 1/5 were treated with 1/5 volume of kethoxal stock solution. (B) CMCT modification. Samples in lanes marked 2/3 received 2/3volume of CMCT stock solution. (C) DMS modification. Samples in lanes marked 1 received 1 ml of undiluted DMS stock solution, while samplesmarked 1/2 were treated with 1 ,ul of a 1:2 dilution of DMS stock solution. Indicated at the top of each lane is the potassium concentration to whichthe sample was adjusted prior to the chemical modification; the modification pattern was generally the same regardless of potassium concentration.GIOI was only weakly modified with kethoxal, as reported in Table I.

NAKED 7SL RNA

00

* 00

30 '::) 50 6l0- Iii 290

20- - 280

: 10

0

0.0 0

70 a00 80 90I

270 260

o W-ISO

140- 0

- -160

'10 0, 00

100 0.I 110 120 130-;- 17,0.L.oRo .o

0* °* **- 0

50 * 240 230 220C

0180-0

0o-l 1 IV0

210- -,g

[ 00.

200

Fig. 2. Chemical modification of naked 7SL RNA. The secondary structure drawn here for canine 7SL RNA is that proposed by Larsen and Zwieb(1991); base pairs for which phylogenetic proof of compensatory base changes exist are joined by a dash (G-C, A-U) or a dot (G -U). Stems are

numbered with Roman numerals as done previously by Poritz et al. (1988). * weak, 0 moderate, 0 strong, * hypersensitive modification isindicated adjacent to the modified base; Table I lists the chemical used for modification at each position shown. Brackets denote areas whereproposed base-paired stems seem to have opened up and are available for modification. The tertiary interaction between the '100' and '200' regionsis marked.

770

K..-.f.~-i %*. ,;3 .

1.* ¢ x ; .,

R p-. )

_. 0 0AzCLC3. un

!.:. --- . :::.

J 3.. . "w:

Alak

Table II. A potential tertiary interaction in the 7SL RNA

MEMBRIBOUIhSR

26 9 GA'02 GGG|l,GGCU, G CCC

GA

26 9CCCGA0 UC GGGU197GGCU94 GG CCCG

26 [J0CCGA103j UGGGLIGG.CU,l ACCC

26 1 99CCG1AUCGGGU19SG G C U,lGG CCC G

24 197UCGAmIuC GGGUL194GGCiU191GGUCCG

25 F97UGU1UOc|196G U G G193l24 U 63UCGA66 UCUC

A [,,GGUU GGAG

KETHOXAL 0 i

G 98--

( 101 - .

G 107--

C 113- _

SOLUBLE ; LMBRANESRP OUND

SRP

CMCT 0. . .i3 3, 3AC I

: -JL.Y SOME SOLUBLEBOUND SRP

SRPcL

1g O 1O0

1 10

.. _

POLY SOMEBOUNDSRP2 CL

3 F-G U (A

|9UUCC92 G UU

L17GAGIG,1 U GA

°UU G G103 U|179GGCC176 GG

Plants

Zeamays 21 {02GCCG1 GCCUU GGU197 CGGA

Wheat 26 |9GCUG102 GCCUG,j,4UGGUS,ICGGAU

Cineraria rAhybrida 30 I2ACCG's CCUGGC

GGAUUG

Tomato 34 G I5GUUG89 IG CUUAU 9CGGU,9, CAGAU

Bscteris

Halobacteriumhalobium 27 °6IIC G U Gl

,9,GCAC 19JBacukssubtilis 15 UU '122UGGG'2ICCUGAG I23GCCU,Oj GGACEscherichiacoil 14 UI'2U GG14.5 S RNA 63G C C s1

Compensatory base changes supporting the proposed tertiary interactionbetween bases near positions 100 and 200. The sequences listed fordifferent species are from Zweib (1988, 1989). Alignment wasdeduced by secondary structure parameters: (i) the 3' end of the boxed'200' region is the first unpaired base (usually a G) in the loop ofstem IV that proceeds via three paired bases to the 5' end of the box;(ii) the nucleotide distance beyond the 3' end of the boxed '100'region to the 3' end of the stem II is indicated.

and residues spanning 26-45 (Figure 4A; Table I),suggesting that these could be binding regions for p9/14.Other nucleotides are protected in the deproteinized RNA

but exposed in soluble SRP (open circles in Figure 4A). Themain example is shown in Figure IC where the protectionof C99, C100, GIOl and A102 in naked 7SL RNA and therelative exposure in soluble SRP are evident. We proposethat the above mentioned sequence (Figure 2 boxed sequence

U 7 .*

f-%. bl.MEW-4ABOUND

DMS r

A 1B3 __

.:v 1 8 S -_102 - -

A 192

4E.- SOLUBLE POLYSOMESRP BOUND

SRPX C

A :'-; G U CA c10

:..l*L--.

A 199---A 200

A 208

A 213 _

A 214A 215A 216

*:.. W .4

*::me.:- ,ft

Fig. 3. Autoradiographs showing primer extension products ofKethoxal, CMCT and DMS treated 7SL RNA from soluble, polysomebound and membrane bound SRP. Refer to Figure 1 for furtherdetails. (A) Kethoxal modification. (B) CMCT modification. The lanesof polysome bound SRP were exposed for longer than the other lanesshown. (C) DMS modification.

'100', stem U1), is involved in a long-range pairing interactionwith the sequence 3'-GGCU-5' found between positions 198and 195 (Figure 2 boxed sequence '200', stem IV). Thisstatement is based on the following observations. (i) In naked7SL RNA, sequence '200' must not be base-paired to itsneighboring, complementary sequence (202 -204), since thelatter is chemically modified (Figure ID; Table I).Moreover, neither the '100' nor the '200 sequences are

accessible to chemicals in naked 7SL RNA (except for U195that is moderately modified by CMCT), suggesting they

771

Organism nt to end of stem 11 *100j rejg*n'200' region

A


Animals

Human - A

Human - B

Rat

Dog

Xenopus

Drosophila

Schizosaccharomycespombe

Yarrowialipolytica 12

Trypanosoma

R

I


A.COMPARISON BETWEEN NAKED 7SL RNA *-150

AND SOLUBLE SRP .140-

- -160

0-40 120 1300 17030--. 50 60 70 %. 80 90 0 110

20__290 280 270 260 250 K 0 220290~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~20 20 20 *240 230 220

20. - 280 180

-.10 0

7SL RNA 210- - ,190F

Proteins 0 0

SOLUBLE SRP

MEMBRANE POLYSOMEBOUND BOUNDSRP SRP 200

B.COMPARISON BETWEEN SOLUBLE SRP -1

AND POLYSOME BOUND SRP ll

140-

0 II0- -40 100

30--: so 120o 130-01101703 510 7/.OT °§0go 900 °0 .X0o 'I °'0 .

o , , , , ,,., I 0

- jid d 290 270 260 250 240 p230 22020- - 280 -1I0

. -10

7SL-RNA

|_ProtI* ns210- - 190

SOLUBLE SRP

MEMBRANE POLYSOMEBOUND BOUNDSRP SRP 2

200

Fig. 4. Differences in chemical sensitivity (minor, medium, major) of a given nucleotide between the compared substrates are indicated on thesecondary structure of canine 7SL RNA (drawn as in Figure 2). A minor difference is one degree of difference between the two substrates (e.g.weak modification in one and moderate modification in the second). A medium difference is two degrees of difference between the two substrates(e.g. no modification in one and moderate modification in the second). A major difference is three degrees of difference between the two substrates(e.g. strong modification in one and no modification in the second). (A) Nucleotide sensitive in naked 7SL RNA and protected in soluble SRP:minor *, medium \, major 0 Nucleotide sensitive in soluble SRP and protected in naked 7SL RNA: minor 0, medium P, major 0. (B) Nucleotidesensitive in soluble SRP and protected in polysome bound SRP: minor *, medium ', major 0. Nucleotide sensitive in polysome bound SRP andprotected in soluble SRP: minor 0, mediumrn/, major 0.

could be base-paired to each other. (ii) The complementarityof region '100' with region '200' can be found for all known7SL RNAs, and often the complementarity can be extendedbeyond the four base-pair core of this interaction. Note thatthe extended interaction 99-108/199-188 is flanked onboth sides by chemically sensitive bases, but itself isprotected except for A192 which is bulged in the tertiaryinteraction (Figure 2; Table II). The only other base thatis chemically modified in this region is U195; phylogeneticcomparisons suggest that this position is less crucial thanthe rest of the four base core for the tertiary interaction(Table II).The interaction of the '100' region with the '200' region

can be found only in naked 7SL RNA but not in intact SRPin any of its functional states, suggesting it might play a rolein correct assembly of the RNP.

Comparison between soluble SRP and polysomebound SRPThe method of Gunning et al. (1981) was followed to obtaina pellet we refer to as the 'polysome' fraction of SRP; thispellet contained primarily polysomes and some ribosomes.We used this fraction to assay the effect that binding toribosomes has on SRP conformation. We had expectedpolysome bound SRP to be less sensitive to chemicalmodification than soluble SRP due to protection by boundribosomes. However, the converse was true, the chemicalaccessibility of polysome bound SRP was greater than solubleSRP (Figure 3; Table I). Some nucleotides that wereprotected in soluble SRP become accessible to chemicals inpolysome bound SRP, while most of the nucleotides thatwere already exposed become even more exposed (Figure4B, open circles especially nos 99, 103, 171, 185, 231 and

772


A.-_150

COMPARISON BETWEEN POLYSOME BOUND SRP -AND MEMBRANE BOUND SRP I

140- 00

- -160

01I0 100 120130-1 170

0 . .

290 0 270 2 25000 2*0~~~~~~~~~~'.~~~~~~~~~0 0

--10

7SL RNA210- _190

Proteins 0

SOLUBLE55P~~~~~~~~~~~~SOLUBLE SRP

MEM BRANE POLYSOMEBOUND 4 BOUNDSRP SRP

IV200

B.COMPARISON BETWEEN MEMBRANE BOUND SRP

AND SOLUBLE SRP140- 0

- 60

040 100 120/ 130-- 170~~~~~~I I-

030- - 50 60 70 000 6 °s 90110 o -

I. 0/ 0 O I. On'000 *o. 0.~~~~0.0

i I IIIII*I.III/1/ III B Il I I .I II II .I I III II I III ,.I I.I X, Oi*1 1 00

290 270 260 250 2202 220-. 26 220 :..- 0

_10 O

- .007SL-RNA 210-- 1190

Proteins

SOLUBLE SRP

MEMBRANE POLYSOMEBOUND * BOUNDSRP SRP

IV260

Fig. 5. Differences in chemical sensitivity of a given nucleotide between the compared substrates are indicated. Details as in Figure 3. (A) Nucleotidesensitive in polysome bound SRP and protected in membrane bound SRP: minor *, medium X, major 0. Nucleotide sensitive in membrane boundSRP and protected in polysome bound SRP: minor 0, medium A, major 0. (B) Nucleotide sensitive in membrane bound SRP and protected insoluble SRP: minor 0, medium A, major 0. Nucleotide sensitive in soluble SRP and protected in membrane bound SRP: minor *, medium ',major 0.

232). These nucleotides undergo conformational changeswhen SRP interacts with polysomes, such that polysomebound SRP obtains a more open conformation than was truefor soluble SRP. Of all the bases that were chemicallymodified in polysome bound SRP, two-thirds of themshowed increased chemical sensitivity relative to solubleSRP.The remaining one-third bases that were chemically

modified in polysome bound SRP showed decreasedchemical sensitivity relative to soluble SRP (Figure 4B, filledcircles). These are candidates for possible ribosome bindingsites, and are mainly clustered near the end of stem II(98-107, 233-239) and in the middle of stems III(138-139) and IV (182-192, 213-214). Alternativelypolysome binding might alter overall SRP conformation (e.g.conformational changes in SRP proteins), so that the 7SLRNA sites just listed become more protected rather thanthemselves being ribosome binding sites. Still anotherpossibility is that these sites on 7SL RNA become protectedby the nascent peptide with its signal sequence.

Comparison between polysome bound SRP andmembrane bound SRPThe number of nucleotides susceptible to chemicalmodification is slightly higher in membrane bound SRP thanin polysome bound SRP. Half of the nucleotides modifiedin membrane bound SRP show an increase in sensitivitygoing from polysome bound to membrane bound SRP(Figure 5A, open circles). These bases appear to undergoconformational changes as a consequence of the interactionbetween SRP and its receptor on the membrane. Inparticular, GI 13 becomes increasingly exposed, going fromsoluble through polysome bound to membrane bound SRPwhere it is hypersensitive (Figure 3A). Other noteworthyexamples of increased sensitivity in membrane bound SRP,indicative of conformational changes, are nos 120, 164, 183,186, 241, 242 and especially 76, 98 and 101.The remaining half of the bases that are chemically

modified in membrane bound SRP show less sensitivityrelative to polysome bound SRP (Figure 5A, filled circles).These sites are clustered in stem II (73-110, 226-233) and

773


-iu doma.

09/14

Ip at--- O G

: GSUGfi* U,.

P.

p54

ELONGATION ARREST TRANSLOCATION PROMOTION SIGNAL RECOGNITION

Fig. 6. Schematic of 7SL RNA indicating regions deduced for functional importance. Superimposed under this figure (modified from Siegel andWalter, 1988a; 7SL RNA secondary structure drawn according to Larsen and Zwieb, 1991) are regions known by a-sarcin footprinting to bindp68/72 and p19 (Siegel and Walter, 1988a) and deduced from the present work to bind p9/14. Areas of increased protection from chemicalmodification by binding to polysomes are shaded; single bases that also become protected by polysomes, such as nos 33 and 73, are not highlighted.Areas of increased protection by binding to rough endoplasmic reticulum membranes are indicated by brackets.

the bottom of stem III (130-134), spanning regions knownto interact with p68/72 (Siegel and Walter, 1988a). Theincreased protection of these bases may be due either to(i) their direct protection by the SRP receptor or othercomponents on the rough ER membrane, or (ii) protectionby SRP proteins whose conformation could have been altereddue to membrane binding.

Comparison between membrane bound SRP andsoluble SRPThis comparison completes the SRP cycle and shows howthe chemical accessibility (and therefore the exposure of 7SLRNA in SRP) decreases when SRP is no longer interactingwith ribosomes or the SRP receptor (Figure 5B, opencircles). A small number of bases become more exposed insoluble SRP (Figure 5B, filled circles).

DiscussionA tertiary interaction in naked 7SL RNAThe secondary structure of naked 7SL RNA that can bederived from our experimental results of chemicalmodification is in good agreement with the theoretical modelderived by phylogenetic comparisons (Larsen and Zwieb,1991). However, a few areas predicted to be base-pairedhave 'breathed' open, as indicated by brackets in Figure2. One such area is near the tip of stem IV where bases202-204 are chemically modified; therefore, they mustnot be base-paired with the '200' region opposite them aspreviously supposed. The '200' region is generally protectedfrom modification, and we propose that it is involved in along-range tertiary interaction with the '100' region, whichis also protected from modification in naked 7SL RNA(boxed regions in Figure 2). Compensatory base changessupport the base-pairing between regions '100' and '200'(Table II). In fact, Table II shows that this interaction caneven be drawn for Escherichia coli 4.5S RNA which contains774

a region homologous to stem IV of eukaryotic 7SL RNAs(Poritz et al., 1988; Zwieb, 1988).The base-pairing we propose between the '100' and '200'

regions would only occur in naked 7SL RNA, as the '100'region becomes sensitive to chemical modification in solubleSRP, polysome bound SRP and membrane bound SRP(Table I). The tertiary interaction suggested by our data couldbe a prerequisite for correct biogenesis of SRP. The tertiaryinteraction seen in naked 7SL RNA remains present evenafter binding by p19 (data not shown), but perhaps bindingby other SRP proteins abolishes base-pairing between the'100' and '200' regions. For example, the subsequentbinding of p54 might modulate the association of p19 with7SL RNA. Similarly, the p54 homologue in E. coli (p48 inRomisch et al., 1989; equivalent to ffh protein in Bernsteinet al., 1989), known to bind to E.coli 4.5S RNA (Poritzet al., 1990; Ribes et al., 1990), might destroy thecomparable tertiary interaction that can be drawn for E. coli4.5S RNA (Table II). The addition of SRP proteins mightstabilize the close juxtaposition of stem II with stem IV (asindicated by cross-linking at other positions on stem II withstem IV; Zwieb and Schiiler, 1989) such that base-pairingbetween the '100' and '200' regions is now dispensable.7SL RNA sites protected by SRP proteinsComparison of chemical modification of naked 7SL RNAand soluble SRP indicates sites of increased protection dueto protein binding (Figure 4A). Figure 6 portrays all thededuced protein binding sites on 7SL RNA. These regionscoincide well with the a-sarcin footprints of p68/72 and p19(Siegel and Walter, 1988a). No footprint data have yet beenpublished for p9/14 known to associate with the Alu domainof 7SL RNA, but our results suggest p9/14 binds to stemI and the junction with stem II, both of which are in the Aludomain (Figure 4A).

7SL RNA sites protected by polysomesSRP association with polysomes may be mediated by severalinteractions. First, p54 located at one end of SRP binds the

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emerging signal sequence (Krieg et al., 1986; Kurzchaliaet al., 1986; Wiedmann et al., 1987; Siegel and Walter,1988b). Second, other parts of SRP such as the 7SL RNAcomponent may bind directly to the ribosome. Bases thatare more protected from chemical modification in polysomebound SRP than in soluble SRP (Figure 4B and shaded areasof Figure 6) are candidates for ribosome binding, thoughother explanations are also possible (see Results).

It could be that 7SL RNA interacts directly with RNA ofthe ribosome to cause translational arrest. For example,an oligonucleotide for 97-110 of 7SL RNA can primesynthesis of 18S rRNA (Ullu and Weiner, 1985). Note thatthis 97-110 region includes nucleotides 98, 101 and 107which we find more protected from chemical modificationwhen SRP is bound to polysomes. In a second example,some species have sequence similarity between two regionsin 7SL RNA and 5S RNA, so 7SL RNA could compete for5S RNA binding sites on rRNA (Zwieb, 1985; Boehm,1987). However, the similarity between 7SL RNA and 5SRNA does not hold up in some plants (Haas et al., 1988).Nonetheless, it is intriguing to note that polysome protectionseen in our experiments for (i) nos 98, 101 and 107, and(ii) nos 233 and 239 are within or near the 5S-like regionsof 7SL RNA of nos 104-109 and 222 -263, respectively(Zwieb, 1985; Boehm, 1987). Curiously, both regions arein the S domain of 7SL RNA, rather than the Alu domainthat has been implicated in translational arrest (Siegel andWalter, 1985, 1986). Nucleotides 33 and 73 are the onlyones in the Alu domain where we find increased protectionfrom chemical modification after binding to polysomes. Thearea of the Alu domain containing nucleotide 33 mimics theshape of tRNA and could compete for tRNA binding siteson the ribosome (Zwieb, 1986). Site directed mutagenesisof each of the bases we find more protected in both the Aluand S domains in polysome bound SRP could reveal whichbases might in fact be recognition points on 7SL RNA forribosome binding.

7SL RNA sites protected by membranesThe brackets in Figure 6 indicate regions in 7SL RNA thatare more protected from chemical modification in membranebound SRP than in polysome bound SRP (see also FigureSA). These regions in 7SL RNA could either be (i) protecteddirectly by the SRP receptor or other components of therough ER (e.g. mp3O binds SRP in the absence of SRPreceptor, Tajima et al., 1986) or (ii) protected by SRPproteins whose conformation could have been altered dueto membrane binding. Notice that most of the bracketed areasof 7SL RNA (Figure 6) coincide with p68/72 footprintregions (Siegel and Walter, 1988a), thus supportingpossibility (ii).

Conformational changes in 7SL RNA during the SRPcycleThe areas discussed above become increasingly protectedfrom chemical modification during progression from onestage to the next of the SRP cycle. Other nucleotidesshow increased sensitivity moving through the SRP cycle,indicative of conformational changes in 7SL RNA. In fact,it has previously been demonstrated by gel electrophoresisthat 7SL RNA can exist in several conformations (Zwieb,1985; Zwieb and Ullu, 1986). One example of a con-formational change that we found is the tertiary interactionbetween the '100' and '200' regions seen for naked 7SL

RNA but not for soluble SRP, discussed above. In addition,several sites of increased sensitivity going from soluble SRPto polysome bound SRP (Figure 4B, open symbols), or frompolysome bound SRP to membrane bound SRP (Figure SA,open symbols), or from membrane bound SRP to solubleSRP (Figure SB, closed symbols) are scattered throughoutthe 7SL RNA molecule, though predominantly found in stemII and the no 170 junction between stems III and IV. Thesechanges in chemical sensitivity indicate conformationalchanges in 7SL RNA between each of these stages of theSRP cycle. One result of these changes is that soluble SRPis in a more closed, protected conformation than polysomebound SRP or membrane bound SRP. In other words, whenSRP is active during arrest of translation (polysome bound)or handing the signal complex over to the SRP receptor fortranslocation into the ER (membrane bound), the 7SL RNAmoiety is more exposed than when SRP is inactive (solubleSRP). Most of the sites indicative of conformational changeswere not protected by SRP proteins (i.e. not sensitive tochemicals in naked 7SL RNA and then protected in solubleSRP), making unlikely the alternative possibility that thenucleotides indicative of conformational changes onlybecome more sensitive because of displacement of SRPproteins.

Do RNA -RNA interactions drive some steps of theSRP cycle?RNA-RNA interactions are important for translation(reviewed by Dahlberg, 1989). For example, rRNA interactswith mRNA during initiation (Shine -Dalgarno interaction)and during elongation (seen by ribosomal frame shiftingexperiments; Weiss et al., 1987). In another example, tRNAinteracts with mRNA (anticodon -codon pairing) and withrRNA (seen by A, P and E site footprints; Moazed andNoller, 1986, 1989a). Intramolecular interactions withinrRNA may also be important; the conformation of rRNAchanges between inactive and active 30S ribosomal subunitsin prokaryotes (Moazed et al., 1986) and between subunitsand monosomes in eukaryotes (Stebbins-Boaz and Gerbi,1990). Moreover, analysis of tRNA hybrid sites suggeststhat tRNA translocation is accompanied by changes ininteractions between ribosomal subunits (Moazed and Noller,1989b).

Just as rRNA conformational changes may be impliedfor the ribosome cycle, so too our data presented heredemonstrate that 7SL RNA changes its conformationduring the SRP cycle. There could be an interplay betweenthe ribosome cycle and the SRP cycle via RNA-RNAinteractions that alter RNA conformation. We speculate thatchanges in rRNA conformation during the elongation phaseof the ribosome cycle could be responsible for tRNAtranslocation. Interaction of 7SL RNA with rRNA once SRPbinds polysomes could block further conformational changesin rRNA, resulting in arrest of translation. Zwieb (1989)has hypothesized specific ways by which 7SL RNA mayinteract with the ribosomes, resulting in translational arrest.This translational arrest would be relieved when the 7SLRNA-rRNA interaction is discontinued upon docking onthe rough ER. A major point to be made in our modelis that SRP binding to polysomes is mediated both byp54-signal peptide and 7SL RNA-rRNA interactions.Similarly, SRP release upon docking on the rough ERrequires that both sets of interactions be abolished.Our data presented here indicate that 7SL RNA changes

775


its conformation during the SRP cycle. It will be fascinatingin future experiments to discover if there is a causal linkbetween these conformational changes and progressionthrough the SRP cycle as hypothesized here, as well asto substantiate the proposed overlap via RNA-RNAinteractions between the SRP cycle and the ribosome cycle.

Materials and methods

Preparation of microsomal membranes, SRP and polysomesMicrosomal membranes were prepared as described by Walter and Blobel(1983c) and shown to be active by assaying translocation activity in wheatgerm extract (Promega) programmed with bovine preprolactin mRNAobtained by in vitro transcription of pSPBP4 (Siegel and Walter, 1988b;a generous gift from V.Siegel). SRP was solubilized from microsomalmembranes by salt extraction as described by Walter and Blobel (1983d).Both microsomal membranes and solubilized SRP were used for chemicalmodification experiments without further purification.

Polysomes were prepared essentially as described by Gunning et al. (1981).Briefly, canine pancreas was homogenized in the usual buffer but in thepresence of 100 mM potassium acetate, since at this concentration muchSRP is ribosome bound (Walter and Blobel, 1983b). The postmitochondrialsupernatant was layered on a discontinuous sucrose gradient consisting of2.0 M sucrose and 0.8 M sucrose containing the same ionic conditions asthe homogenization buffer. After centrifugation at 90 000 g for 18 h at 4°Cin a SW-28 rotor, the pellets were collected and resuspended in 50 mMtriethanolamine (TEA), pH 7.5, 100 mM potassium acetate, 250 mM sucroseand 1 mM DTT. Soluble SRP and membranes do not pellet under theseconditions (Gunning et al., 1981). This pellet, which we refer to as the'polysome' fraction, contains primarily polysomes (Falvey and Staehlin,1970); in our conditions it has -75% polysomes and 25 % monosomes asassayed by respinning the resuspended pellet in a 10-30% sucrose gradientand checking fractions for the presence of 7SL RNA by primer extension.Samples were stored at -20'C and generally used soon thereafter forchemical modification.

Preparation of naked 7SL RNATo prepare naked 7SL RNA, soluble SRP was extracted twice with phenol,followed by chloroform, 0.1% SDS. RNA was precipitated with 1/10 volumeof 2.5 M sodium acetate (pH 5.1) and 2.5 volumes of 95% ethanol at -20°Covernight, pelleted and resuspended in 50 mM TEA (pH 7.5), 50 mMpotassium acetate, 250 mM sucrose, 1 mM DTT. Samples were stored at-20'C and generally used soon thereafter for chemical modification andsequencing.

Reconstitution of p19 with 7SL RNANaked 7SL RNA prepared as described above was mixed with SRP proteinp19 (a generous gift from P.Walter) under reconstitution conditions (Siegeland Walter, 1985). The molar ratio of 7SL RNA to p19 was 1:17.5;therefore, p19 was in excess. We used primer extension to confirm thatp19 had indeed bound to 7SL RNA, and we obtained protection of 7SLRNA in regions predicted by the ca-sarcin footprint (Siegel and Walter,1988a).

Chemical modification of RNA substratesChemical modification of naked 7SL RNA, soluble SRP, membrane boundSRP and polysome bound SRP was carried out according to the proceduredescribed by Stebbins-Boaz and Gerbi (1990).

PrimersTwo 20-mer deoxyoligonucleotide primers, complementary to uniqueregions within the 7SL RNA, were synthesized using a Biosearch 8600DNA synthesizer. The sequences of the primers were: (7SL 272-253)5'-GGCTGGAGTGCAGTGGCTAT-3'; (7SL 187-168) 5'-CGGTTC-ACCCCTCCTTAGGC-3'. The primers were purified by gel electrophoresisthrough 20% polyacrylamide (19:1 bis-acrylamide), 7 M urea and 1 x TBE(0.8 M Tris-borate, 1 mM EDTA, pH 8.0). Primers were eluted fromgel fragments by incubating with 1 ml of 0.1 M ammonium bicarbonate.Urea was removed from the eluted primer by repeated precipitations with95% ethanol. Purified primers were resuspended in H20 at a finalconcentration of 20 pmol/Ail and stored at -20'C.

End-labeling, sequencing and primer extensionEnd-labeling of the primers and RNA sequencing were carried out asdescribed by Stebbins-Boaz and Gerbi (1990), while primer extensions of

chemically modified RNAs were performed according to the proceduredescribed by Lillie et al. (1986).

Gel electrophoresisPrimer extension products were run on 0.4 mm thick sequencing gels (8%polyacrylamide, 7 M urea, 1 x TBE) using a sharkstooth comb. Gels wererun at 20 mA, constant current, for 2.5-5 h and exposed to Kodak X-rayfilm at room temperature overnight (12-16 h). Adjustments in volume ofsamples loaded were made following a preliminary run in order to equalizethe relative signals between sample lanes; when background bands wereof equal intensity between lanes, samples were judged to be equivalent inloading. Modified bands can only be compared between different lanes whenidentical amounts of sample are loaded in each lane. Within a given lanethe strength of modification signal was judged to be weak, moderate, strongor hypersensitive by comparison between bands in that one lane.

AcknowledgementsWe are grateful to V.Ware, V.Siegel and B.Dobberstein for teachingus how to isolate SRP, to B.Stebbins-Boaz for instruction in chemicalmodification, to C.Zwieb for sharing unpublished data on 7SL RNAconformation, and to P.Walter for a gift of pI9. We thank the followingfor their helpful comments on this paper: M.Firpo, P.Grabowski, B.Hoffman, H.Hoffmann, P.Milos, R.Rivera-Leon, R.Savino and B.Stebbins-Boaz. The assistance of D.Angeloni with the figures was greatly appreciated.Our research is funded by NIH GM20261.

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Received on November 15, 1990; revised on Januarv 14, 1991

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the changes 7sl signal recognition particle cycle

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