Molecular Cell, Vol. 6, 159–171, July, 2000, Copyright 2000 by Cell Press

Periodic Conformational Changes in rRNA:Monitoring the Dynamics of Translating Ribosomes

be expected during the elongation cycle, and some rea-sonable candidates have been identified (for a review,see Wilson and Noller, 1998). Under in vivo conditions,

Norbert Polacek,* Sebastian Patzke,†Knud H. Nierhaus,† and Andrea Barta*‡

* Institute of BiochemistryVienna Biocenter the pretranslocation state is demarcated from the post-University of Vienna translocation state by a huge activation energy barrierDr. Bohr-Gasse 9 of about 120 kJ/mol (Schilling-Bartetzko et al., 1992)A-1030 Vienna that “freezes” the tRNA–ribosome conformation in eitherAustria state in the absence of elongation factors. Evidence for†Max-Planck-Institut fur Molekulare Genetik a structural switch that might be involved in “defreezing”Ihnestrasse 73 the fixed complex derives from recent mutagenesisD-14195 Berlin studies on 16S rRNA in the 912 region (Gabashvili etGermany al., 1999a; Lodmell and Dahlberg, 1997). Other moving

ribosomal elements that might be involved in translo-cation are the L7/L12 stalk on the 50S subunit (Agrawalet al., 1999), the head region of the 30S subunit (Agra-Summarywal et al., 1999), or even the whole 70S structure (Ofver-stedt et al., 1994). Most recently, the exciting insightsIn protein synthesis, a tRNA transits the ribosome viainto ribosome structure obtained by cryo-electron mi-consecutive binding to the A (acceptor), P (peptidyl),croscopy (Frank et al., 1995; Stark et al., 1995, 1997a;and E (exit) site; these tRNA movements are catalyzedMalhotra et al., 1998) have been further extended by aby elongation factor G (EF-G) and GTP. Site-specificwealth of interesting X-ray crystallography data of bothPb21 cleavage was applied to trace tertiary alterationssubunits (Ban et al., 1999; Clemons et al., 1999; Tociljin tRNA and all rRNAs on pre- and posttranslocationalet al., 1999) and the 70S ribosome (Cate et al., 1999).ribosomes. The cleavage pattern of deacylated tRNAThe next step in understanding translation will definitelyand AcPhe-tRNA changed individually upon binding tocome from elucidating ribosomal dynamics. Since con-the ribosome; however, these different conformationsformational changes associated with translocation havewere unaffected by translocation. On the other hand,

translocation affects 23S rRNA structure. Significantly, not yet been shown in molecular terms, we set out tothe Pb21 cleavage pattern near the peptidyl transferase investigate this issue by applying a very sensitive struc-center was different before and after translocation. tural probing technique, namely site-specific cleavageThis structural rearrangement emerged periodically of RNA by Pb21.during elongation, thus providing evidence for a dy- For site-specific Pb21 cleavage to occur, a number ofnamic and mobile role of 23S rRNA in translocation. structural prerequisites have to be met. A tightly bound

Pb21 hydroxyl ion has to be orientated in such a waythat it can remove the proton from the 29-OH of theIntroductionribose at the cleavage site. Nucleophilic attack of theresulting oxyanion at the adjacent phosphodiesterThe coupling of amino acids to form proteins is a crucialbridge cleaves the RNA chain (Brown et al., 1985). Thisbiological process performed by ribosomes in all livingmethod of site-specific metal ion hydrolysis has alreadycells. The ribosomal elongation cycle is characterizedbeen successfully applied to probe for structural integ-by a series of activities in which the growing peptiderity and for tracing down metal ion binding pockets inchain is lengthened by one amino acid. Each new aminovarious RNA molecules (Zito et al., 1993; Streicher et al.,acid is delivered to the ribosome as an aminoacyl-1996; Winter et al., 1997). Due to its strict dependence ontRNA•EF-Tu•GTP ternary complex, where it binds toa specific tertiary fold, Pb21 cleavage turned out to bethe A site. Following peptide bond formation with thea powerful tool in elucidating fine structural changes innascent peptide chain of P site–bound tRNA, the twoRNA molecules (Gornicki et al., 1989; Behlen et al., 1990;tRNAs together with the mRNA must be translocated toMichalowski et al., 1996; Dorner and Barta, 1999). Inthe P and E sites, leaving the A site ready to accept thethese studies, it became evident that conformationalnext ternary complex. This translocation is driven byalterations were reflected by a change in the cleavagethe elongation factor G (EF-G) and GTP hydrolysis. Therate or specificity.findings that, under certain in vitro conditions, transloca-

In previous work, we characterized the Pb21 cleavagetion can also occur in the absence of EF-G (Pestka, 1969;pattern in rRNAs in vacant ribosomes (Polacek andGavrilova and Spirin, 1971; Bergemann and Nierhaus,Barta, 1998) and have now expanded our structural in-1983) or mRNA (Belitsina et al., 1981) imply that this

reaction is inherent to the ribosome itself and led to the vestigations to ribosomes arrested in defined functionalview that the translation apparatus is a macromolecular states of the elongation cycle. This enabled us to simul-machine. Therefore, moving ribosomal elements are to taneously probe the structures of rRNA and tRNA of

ribosomal complexes in the same functional state. Ourexperiments show that tRNA structure changes upon‡ To whom correspondence should be addressed (e-mail: andrea@

bch.univie.ac.at). binding to the ribosome, and EF-G-catalyzed transloca-

Molecular Cell160

Figure 1. Functional Ribosomal Complexes

Pretranslocational complexes (PRE) (A andC) carry deacylated tRNA at the P site andAcPhe-tRNA at the A site. Posttranslocationalcomplexes (POST) (B and D) are character-ized by deacylated tRNA bound to the E siteand AcPhe-tRNA bound to the P site. Pi com-plexes (E) (i for initiating) carry one singletRNA at the P site. See the insert for the differ-ent radioactive labels applied.

tion does not further influence tRNA conformation. On with 32P was bound to the A site of poly(U)-programmedthe other hand, ribosome structure is affected by trans- ribosomes where the P site was blocked by prior bindinglocation, indicating that parts of 23S rRNA move. This of deacylated tRNAPhe (Figure 1A). Nonbound tRNAs weremovement was shown to be reversible, implying recur- removed by gel filtration. During EF-G-catalyzed translo-rent structural shifting of 23S rRNA features during the cation the two tRNAs move to the P and E sites, respec-elongation cycle. tively (Figure 1B). According to the puromycin reaction,

the homogeneity of the PRE and POST complexes wasbetter than 85% (see Experimental Procedures).Results

To gain insight into the conformation of deacylatedtRNAPhe at the P site in PRE and at the E site in POSTExperimental Designcomplexes, deacylated [32P]-59-end-labeled tRNAPhe wasWe report herein the investigation of structural changesbound to the P site followed by binding of Ac[14C]Phe-of ribosomes and their tRNA ligands in defined functionaltRNAPhe to the A site (Figure 1C). Since under the appliedstates before and after EF-G-catalyzed translocation. Toconditions (see Experimental Procedures) the P site isthis end, pre- and posttranslocational complexes werenot completely blocked with deacylated [32P]-tRNAPhe,constructed at near in vivo ionic concentration (polyamineAcPhe-tRNAPhe can also bind to the P site, which resultssystem in the presence of 6 mM Mg21), and the confor-in a mixture of Pi and PRE complexes. However, onlymation of tRNA as well as of rRNA was probed by site-the fate of the deacylated [32P]-tRNAPhe in the PRE andspecific Pb21 cleavage.POST complex is of interest in this experiment and isThe elongating ribosome exists in at least two func-analyzed in the Pb21 cleavage experiments. Successfultionally different states. The pretranslocational statetranslocation was monitored by a significant increase(PRE state) carries a deacylated tRNA at the P site andin the formation of Ac[14C]Phe-puromycin after EF-G in-a peptidyl tRNA at the A site in (see Experimental Proce-cubation (see Experimental Procedures).dures and Figure 1). This state resembles the situation

After establishing Pi, PRE, and POST complexes,immediately after peptide bond formation. After EF-GPb(OAc)2 was added to a final concentration of 2 mM,catalysis, the ribosome enters the posttranslocationaland the cleaved tRNA was run on a 13% polyacrylamidestate (POST state) whereby the deacylated tRNA isgel. The cleavage pattern of ribosome-bound tRNA wasthought to be transferred to the E site and the peptidylcompared to that obtained in solution. AcPhe-tRNAPhetRNA resides in the P site. Only during initiation of pro-and deacylated tRNAPhe showed indistinguishable Pb21tein synthesis does the ribosome contain a single tRNAcleavage patterns in solution with the strongest cutsat the P site, and hence this complex is referred to aslocated 59 to positions D16 and G18 in the D loop. Inter-Pi complex (i for initiating) (Figure 1).mediate cleavages were observed at U8, A9, G10, C17,A21, G46, X47, and C49, and weak cuts were mappedProbing tRNA Structureto A14, G15, and D20 (Figure 2). In general, all cleavagesIn order to probe the structure of AcPhe-tRNAPhe, [14C]-

labeled N-AcPhe-tRNAPhe that was further 59 end labeled were inhibited when the tRNA was bound to the ribo-

Structural Dynamics of Translating Ribosomes161

Figure 2. Pb21 Cleavage Patterns of Un-bound and Ribosome-Bound tRNAPhe

(A) Sites of Pb21-induced cleavages are indi-cated by arrows in the secondary structureof tRNAPhe. Strong, medium, and weak cutsare defined as having more than 10-fold, be-tween 3- and 10-fold, and less than 3-foldincreased signal strength of the cleavageband compared to the corresponding bandin uncleaved tRNA. Autoradiograms of both59-end-labeled AcPhe-tRNAPhe (B) and deac-ylated tRNAPhe (C) are shown, which werecleaved with 2 mM Pb(OAc)2 for 15 min at258C in solution (lane 2) or bound to ribo-somes in the PRE state (lane 3) or POST state(lane 4). Lane 1, no Pb21 added; lane 5, alka-line hydrolysis ladder (H); and lane 6, limitedhydrolysis by RNase T1. The asterisk indi-cates a band at c55 that turned out to beindependent of Pb21 addition and is mostprobably a degradation product.

some, but the degree of inhibition varied for each cleav- pattern of deacylated tRNAPhe hardly changed upon trans-location from the P to the E site (Figures 2C and 3A).age site and was different for AcPhe-tRNAPhe and deacyl-

ated tRNAPhe (Figure 3A). However, the cleavage inhibi- The Pb21 cleavage inhibition pattern of AcPhe-tRNAPhe

in the Pi state (Figure 1E) was similar to that obtainedtion patterns of AcPhe-tRNAPhe bound to the A site inthe PRE and to the P site in the POST state turned out with AcPhe-tRNAPhe at the APRE or PPOST site (data not

shown). Likewise, deacylated tRNAPhe in the Pi complexto be very similar (Figures 2B and 3A). In this case, themost efficient cleavage inhibition was obtained at D16 revealed a pattern that was similar to that seen with

deacylated tRNA at the P site of PRE complexes or E(9.2- to 9.7-fold). In contrast, ribosome-bound deacyl-ated tRNAPhe showed a distinct inhibition pattern from site of POST complexes (data not shown). These find-

ings imply that the Pb21 cleavage pattern and hence theAcPhe-tRNAPhe, with G18 being the most efficiently inhib-ited cleavage site (9.0- to 9.5-fold). Again, the cleavage conformation of ribosome-bound tRNA mainly depend

Molecular Cell162

Figure 3. Alteration of tRNA Cleavage Effi-ciencies upon Ribosome Binding

(A) Inhibition of tRNA Pb21 cleavages uponribosome binding. The numbers give the foldinhibition of either AcPhe-tRNAPhe (left) boundto the APRE or PPOST site, or deacylated tRNAPhe

(right) bound at the PPRE or EPOST site, bothcompared to unbound tRNAs. Significant dif-ferences between ribosome-bound AcPhe-tRNAPhe and deacylated tRNAPhe are bold. Val-ues presented here are averages from 2–3independent experiments. Numbers in DAPand DPE were calculated separately for everybinding experiment and then averaged.(B) Degree of Pb21 cleavage inhibition uponribosome binding for each site is indicated inthe 3D structure model. The inhibition patternof AcPhe-tRNAPhe bound to the APRE or PPOST

site (left) and of deacylated tRNAPhe bound tothe PPRE or E site (right) are shown. See insertfor the color code.

on the charging state of the tRNA molecule. These re- does not change significantly during EF-G-catalyzedtranslocation, at least in those regions where Pb21 cleav-sults indicate that the aminoacyl group triggers the ribo-

some-bound tRNA to adopt a specific tertiary fold, which age could be detected. In addition, the 13 sites of strandscission mapped to the 59 half (domains I–III) of 23Sis independent of the binding site and is not influenced

by translocation. rRNA do not change during translocation (G141, A332,G388, A505, G785, A792, C889, C890, A1133, U1523,G1524, G1555, and C1646). Furthermore, eight cleavageProbing rRNA Structuresites located in domain V (G2307, C2440, U2441, C2573,In addition to the structural investigations on tRNA dur-U2585, C2610, and C2611) and domain VI (U2833) ofing translocation, we further addressed the question as23S rRNA showed unaltered cleavage efficiency in anyto whether the ribosome itself undergoes conforma-of the tested complexes. However, a weak Pb21 cleav-tional changes upon translocation. Therefore, Pi, PRE,age inhibition at A1966 in domain IV of 23S rRNA in theand POST complexes were constructed followed by thePi complex, which was already observed in a previousaddition of 10 mM Pb21 in order to cleave the rRNAs.study (Polacek and Barta, 1998), was reproduced usingEighty to one hundred percent of ribosomes binda different buffer system. Cleavage efficiency at A1966AcPhe-tRNAPhe in the Pi complex, as measured by mem-decreases on average about 28% (Table 1). Remarkably,brane filtration. PRE complexes were constructed withthis effect was not seen in the POST complex, althoughAc[14C]Phe-tRNAPhe at the A site and unlabeled deacyl-AcPhe-tRNAPhe was also present in the P site in thisated tRNAPhe at the P site. In this case, 60%–88% ofstate. It therefore carries with it the suggestion that theribosomes bind AcPhe-tRNAPhe, and around 90% ofP site in the Pi complex somehow differs from the P sitebound tRNAs could be translocated. Subsequent toin the POST complex. Interestingly, much more pro-Pb21 cleavage, the rRNAs of ribosomes in the differentnounced effects on the Pb21 cleavage pattern could befunctional states were extracted and used as templateidentified in the POST state ribosome at positions C2347for reverse transcription.(domain V), C2626, G2694, and U2695 (domain VI) ofNo cleavages could be mapped on 5S rRNA. The Pb21

23S rRNA. Cleavage efficiency at C2347 was enhancedcleavage pattern of 16S rRNA showed no differences inabout 3-fold compared to that seen in all other statesthe Pi, PRE, or POST complex compared to that ob-(Figure 5 and Table 1). The three new POST-specifictained in vacant ribosomes. The efficiency and locationcleavages in domain VI are located near the highly con-of 11 cleavage sites in 16S rRNA (G144, G211, G240,served a-sarcin loop (nucleotides 2646–2674), which isU245, C522, U531, A532, G1182, U1183, A1257, andknown to be part of the EF-G binding site. To test if theA1285) were determined to be unaffected (Figure 4 and

Table 1). These findings imply that 16S rRNA structure observed effects on Pb21 hydrolysis are solely the result

Structural Dynamics of Translating Ribosomes163

Figure 4. Mapping of Pb21 Cleavage Sites in16S rRNA by Primer Extension

Vacant ribosomes (lane 2) as well as Pi com-plexes (lane 3), PRE complexes (lane 4), andPOST complexes (lane 5) were cleaved with10 mM Pb21 for 5 min at 258C. Lane 1 showsthe reverse transcription of uncleaved rRNA.AC (lanes 6 and 7) denote dideoxy sequenc-ing lanes.

of factor binding or due to a translocation event, EF-G complex contained tRNAfMet at the E site and Ac[14C]Phe-tRNAPhe at the P site while the Val codon was invadingwas added to empty ribosomes as well as to Pi com-

plexes carrying AcPhe-tRNAPhe at the P site. These two the A site. Binding tests revealed that 75%–82% of ribo-somes bind AcPhe-tRNAPhe, and the specificity of thecomplexes cannot enter the POST state. No effects on

the cleavage pattern could be observed in either of the complexes was 91% according to the puromycin reac-tion (Figure 7D). Subsequently, EF-G was removed bytwo cases (data not shown). To exclude the possibility

that the enhanced cleavage pattern was solely due to gel filtration, which did not affect the charging state ofthe ribosome (data not shown). To reenter the PRE statean occupied E site, both the P and E site were filled

with deacylated tRNA by incubating poly(U)-programmed (PRE II), the A site was filled with ternary complex con-taining [3H]Val-tRNAVal (Figure 7A). Membrane filtrationribosomes with 2–4 times molar excess of tRNA. No

effect on the cleavage rates at C2347, C2626, G2694, indicated that up to 50% of the ribosomes bind Val-tRNAVal (Figure 7D). The 13% decrease of Ac[14C]Pheand U2695 was seen (data not shown). These observa-

tions strongly hint at translocation as the cause for the counts most likely accounted for a loss of dipeptidyl-tRNA from the A site immediately after peptide bondaltered Pb21 cleavage pattern. Another control experi-

ment was performed aiming at elucidating the authentic- formation (Rheinberger and Nierhaus, 1990). Anothertranslocation finally drives the PRE II complexes intoity of the PRE and POST complexes in the presence of

2 mM Pb21 for 15 min (tRNA cleavage conditions) or the POST II state, where the dipeptidyl-tRNA becomespuromycin reactive again. Homogeneity of the latter10 mM Pb21 for 5 min (rRNA cleavage conditions). No

significant tRNA release was observed in any case (data complexes was from 70% to 87% (Figure 7D).All of the constructed complexes (PRE, POST, PREnot shown), suggesting that Pb21 addition does not

change the functional state of the ribosome. II, POST II), which mimic more than one complete elon-gation cycle, were probed with Pb21. The weak POST-In light of these observations, it is likely that parts of

the 50S subunit, including regions of domain V and VI specific cleavages at C2626, G2694, and U2695 in do-main VI of 23S rRNA could not be detected in ribosomalof 23S rRNA (Figure 6), undergo structural changes upon

EF-G-promoted translocation. complexes with heteropolymeric mRNA, for reasonscurrently not understood. However, the cleavage rateIf this proposed structural rearrangement in 23S rRNA

indeed reflects different functional states, it should fol- at C2347 in domain V of 23S rRNA increased significantlyin the POST complex. Moreover, as soon as Val-tRNAVallow the functional oscillation of ribosomes in the elonga-

tion cycle. To address this important question, ribo- was bound to the A site, the ribosome flipped back intothe PRE state (PRE II) with a concomitant loss of thesomal complexes with a heteropolymeric mRNA that

contains the three unique codons Met, Phe, and Val enhanced signal at C2347, resulting in a cleavage effi-ciency comparable to the starting complex. Signifi-were constructed. This mRNA enabled us to construct

two defined consecutive PRE and POST complexes in cantly, driving this PRE II complex into the POST II staterestored the POST-specific cleavage enhancement al-one experiment (Figure 7A). PRE state ribosomes carried

deacylated tRNAfMet at the P site and Ac[14C]Phe-tRNAPhe most to the same extent (Figures 7B and 7C).These findings show that the POST-specific Pb21 cleav-at the A site. Subsequent to translocation, the POST

Molecular Cell164

Table 1. Pb21 Cleavage of rRNA

Position Vacant Pi PRE POST

23S rRNAI G141 1 0.93 6 0.02 1.04 6 0.01 0.94 6 0.1

A332 1 1.10 6 0.04 1.08 6 0.01 0.90 6 0.02G388 1 1.00 6 0.01 1.02 6 0.11 0.99 6 0A505 1 1.14 1.15 6 0.11 1.00 6 0.11

II G785 1 0.94 6 0.09 0.92 6 0.08 0.93 6 0.02A792 1 0.97 6 0.02 1.04 6 0.05 1.08 6 0.05C889/C890 1 0.96 6 0.05 ND 0.96 6 0.06A1133 1 1.02 6 0.09 1.04 6 0.15 1.06 6 0.05

III U1523 1 1.13 6 0.04 1.16 6 0.07 1.15 6 0.03G1524 1 1.16 6 0.06 1.07 6 0.09 0.91 6 0.12G1555 1 1.07 6 0.22 1.06 6 0.1 1.11 6 0.06C1646 1 1.03 6 0.13 1.02 6 0.05 0.94 6 0.12

IV A1966 1 0.72 6 0.04 1.03 6 0.08 0.99 6 0.05V G2307 1 0.93 6 0.1 1.07 6 0.18 1.04 6 0.19

C2347 1 1.13 6 0.09 1.10 6 0.1 3.20 6 0.9C2440 1 0.90 6 0.05 1.14 6 0.09 1.10 6 0.09U2441 1 1.03 6 0.06 0.88 6 0.09 0.92 6 0.27C2573 1 0.99 6 0.03 0.94 6 0.2 0.80 6 0.01U2585 1 0.92 6 0.05 1.06 6 0.01 0.90 6 0.07C2610/C2611 1 1.06 6 0.14 1.09 6 0.06 1.10 6 0.07

VI C2626 0 0 0 1G2694/U2695 0 0 0 1U2833 1 1.01 6 0.07 1.17 6 0.08 1.15 6 0.01

16S rRNA59 dom G144 1 0.89 6 0.09 0.96 6 0.04 0.98 6 0.01

G211 1 0.94 6 0.07 0.96 6 0.05 1.05 6 0.16G240 1 1.03 6 0.07 0.97 6 0.07 1.10 6 0.1U245 1 0.90 6 0.07 1.00 6 0.1 1.10 6 0.13C522 1 0.85 6 0.07 0.91 6 0.07 0.87 6 0.09U531/A532 1 0.85 6 0.13 0.93 6 0.02 0.93 6 0.13

39 dom G1182/U1183 1 0.91 6 0.02 0.91 6 0.05 0.94 6 0.02A1257 1 0.90 6 0.07 0.91 6 0.03 0.89 6 0.05A1285 1 0.92 6 0.07 1.00 6 0.01 1.00 6 0.04

Band intensities were quantified and normalized to control bands. Lead cleavage efficiency of each site in vacant ribosomes was taken as1.00 and compared to the cleavage rate in Pi, PRE, and POST complexes. Italicized numbers indicate significant differences in the cut rate.Values shown here are averages from 2–4 independent experiments. Double cuts that could not be consistently resolved were quantified asone band. ND, not determined.

age signal in domain V of 23S rRNA appears periodically ribosomal elongation cycle, we applied Pb21 cleavage ofthe RNA backbone. The site specificity of Pb21 cleavageand can be followed throughout a whole elongation cy-

cle. This indicates that the translocation-dependent dependent on a correct tertiary fold allows it to serveas a method to monitor fine structural differences instructural rearrangements in 23S rRNA are reversible.RNA molecules (Gornicki et al., 1989; Behlen et al., 1990;Michalowski et al., 1996; Dorner and Barta, 1999).Discussion

Incubation of tRNAs with Pb21 revealed that unbounddeacylated tRNAPhe and AcPhe-tRNAPhe have indistin-Ribosomal protein synthesis requires a series of subse-

quent steps of initiation, tRNA binding, peptidyl transfer, guishable cleavage patterns. This indicates that thestructures of the tRNAs are similar in solution, at leastelongation factor binding, GTP hydrolysis, translocation,

and termination. Although much progress in elucidating in the elbow region, since all observed cuts clusteredthere in the tertiary structure (Figure 3B). The strongestthe structure of vacant ribosomes (Frank et al., 1995;

Stark et al., 1995) and subunits (Ban et al., 1999; Clem- cuts were mapped to positions D16 and G18 in the Dloop, which is in agreement with previously publishedons et al., 1999; Gabashvili et al., 1999b), as well those

complexed with functional ligands (tRNA [Agrawal et data (Marciniec et al., 1989). When bound to the ribo-some, the Pb21 cleavage patterns of deacylated tRNAPheal., 1996; Malhotra et al., 1998; Cate et al., 1999], IF-3

[McCutcheon et al., 1999], EF-G [Agrawal et al., 1998, and AcPhe-tRNAPhe were different. In general, cleavagerates at all positions were diminished, but to differing1999; Stark et al., 2000], and EF-Tu-GTP-aatRNA [Stark

et al., 1997b]) has recently been made by cryo-electron degrees. Similar effects were observed in a recent study,where tRNA Pb21 cleavage was inhibited due to bindingmicroscopic and X-ray crystallographic methods, the

dynamics of elongation and the structural components to the nuclear tRNA export receptor of Xenopus oocytes(Arts et al., 1998). The authors concluded that this mayof ribosomal peptidyl transferase are not yet character-

ized in molecular terms. reflect a structural change in either the D or the T loopof tRNA rather than a direct shielding against cleavage.In order to get insight into possible structural changes

in tRNA and rRNA in well-defined functional states of the In the case of ribosome-bound AcPhe-tRNAPhe, the most

Structural Dynamics of Translating Ribosomes165

Figure 5. Primer Extension Analysis ofPb21-Cleaved Ribosomal Complexes

Mapping of Pb21 cleavage sites in domain V(A) and domain VI (B) in 23S rRNA. Vacantribosomes (lane 2) as well as Pi complexes(lane 3), PRE complexes (lane 4), and POSTcomplexes (lane 5) were cleaved with 10 mMPb21 for 5 min at 258C. Lane 1 shows thereverse transcription of uncleaved rRNA. Aand C (lanes 6 and 7) denote dideoxy se-quencing lanes. Asterisks (*) indicate sitesof enhanced cleavage rate in the POSTcomplex.

pronounced cleavage inhibition was seen at D16 (Fig- hand, probes from the interior of the tRNA rather thanfrom the surface, therefore monitoring the tertiary foldures 2B and 3). Interestingly, the cleavage pattern does

not change significantly when AcPhe-tRNAPhe was of the molecule and not necessarily shedding light onits ribosomal environment. Since all characterized Pb21bound at the A site of pretranslocational ribosomes (PRE

state) or at the P site after EF-G-catalyzed transloca- sites are clustered around the elbow region of the tRNA,we cannot extrapolate our interpretation to other partstion (POST state). On the other hand, the strongest

Pb21 cleavage inhibition of ribosome-bound deacylated of the molecule, such as the acceptor stem and theanticodon stem loop.tRNAPhe was identified at G18 (Figures 2C and 3). It is

noteworthy that also in the case of deacylated tRNAPhe, As mentioned in the Introduction, the dynamic func-tional oscillation between the PRE and POST state hasthe cleavage pattern hardly changed upon translocation

from the P site (PRE state) to the E site (POST state). led to the hypothesis that the ribosome is a macromolec-ular machine (reviewed in Wilson and Noller, 1998). It isThe slightly different Pb21 cleavage pattern of AcPhe-

tRNAPhe and deacylated tRNAPhe bound to ribosomes in- implicit in this metaphor, however, that the ribosomeitself consists of dynamic and mobile features. This as-dicates that they adopt different conformations, which,

however, do not change during translocation. sumption, however, has not yet been demonstrated con-clusively, though it is integral to all current models ofRecent findings showed that the iodine cleavage pat-

terns of phosphorothioated tRNA at the A and P sites, the ribosomal elongation cycle. In the a2e model, amovable ribosomal domain is predicted that carries thealthough different from each other, also hardly changed

during translocation to the P and E sites (Dabrowski et two tRNAs from the PRE to the POST state (Dabrowskiet al., 1998). Also in the hybrid state model, in which theal., 1998). Based on these findings, a movable ribosomal

domain has been postulated to bind both tRNAs and movement of the tRNA on the small subunit is uncoupledfrom the tRNA movement on the large subunit, a re-carry them together from the A-P to the P-E sites, re-

spectively (termed a2e model). Our observations that arrangement of ribosomal features is to be expectedduring translocation (Moazed and Noller, 1989a; GreentRNA Pb21 cleavages are unaffected support this hy-

pothesis. The agreement seems to be particularly sig- et al., 1998). Cryo-electron microscopy investigationson PRE and POST state ribosomes have not yet reachednificant, since the applied experimental methods are

different. Iodine cleavage is suitable to probe for tRNA– the necessary resolution to unequivocally answer thisquestion on a molecular level (Stark et al., 1997a). How-ribosome contacts and gives evidence about the envi-

ronment of bound tRNAs. Pb21 cleavage, on the other ever, in recent publications, conformational changes

Molecular Cell166

Figure 6. Summary of Pb21 Cleavage Sites in the 39 Half of 23S rRNA of E. coli Obtained in Poly(U)-Programmed Ribosomal Complexes

Location of strand scissions in the secondary structure models (Gutell et al., 1994) are indicated by blue arrows. Differences in the cleavagerate at A1966 in the Pi complex and at C2347, C2626, U2694, and G2695 in the POST complex compared to vacant ribosomes are indicatedby red arrows (see also insert).

Structural Dynamics of Translating Ribosomes167

Figure 7. Probing rRNA Structure during Two Consecutive Translocation Steps

(A) Ribosomal complexes with a heteropolymeric mRNA (Met-Phe-Val) were constructed that mimic a complete round of elongation. Afterthe first translocation step, EF-G was removed by gel filtration.(B) Tracking the Pb21 cleavage efficiency at C2347 of 23S rRNA in PRE (lane 2), POST (lane 3), PRE II (lane 4), and POST II (lane 5) complexes.Lane 1 shows the reverse transcription of uncleaved rRNA. A and C (lanes 6 and 7) indicate sequencing lanes.(C) Pb21 cleavage efficiency at C2347 was quantified in the different ribosomal states whereby the relative cleavage rate in the starting PREcomplex was taken as 1.00. Values shown here are averages from four independent PRE II and POST II complexes. Cleavage rates in PREII/POST II were normalized to the same ribosomal occupancy with AcPhe-tRNA as seen in PRE/POST complexes (0.75).(D) Characterization of the probed ribosomal complexes. An aliquot of PRE or POST complexes contained 6 pmol of 70S ribosomes or 3pmol in the cases of PRE II or POST II complexes, respectively. The specific activities were 1073 dpm/pmol for Ac[14C]Phe-tRNAPhe and 2000dpm/pmol for [3H]Val-tRNAVal. The specificity of the investigated complexes was calculated from the puromycin reactivity of peptidyl-tRNAbefore and after EF-G-promoted translocation. Translocation efficiency was calculated as specified in the Experimental Procedures. nd, notdetermined.

mainly in the L7/L12 stalk and in the head of the small behavior in all of the investigated states (Figure 4 andTable 1). Since Pb21 cleavage was shown to be a verysubunit have been proposed upon EF-G-driven translo-

cation (Agrawal et al., 1999; Stark et al., 2000). sensitive tool to probe changes in tertiary structure, weconclude that the conformation of 16S rRNA near theWe therefore set out to address this issue by applying

Pb21 cleavage of rRNA in defined functional ribosomal sites of strand scission is very similar before and aftertranslocation. This might be at least the case for thosecomplexes (Figure 1). Pi, PRE, and POST complexes

have been constructed, and subsequent to Pb21 cleav- regions of 16S rRNA where Pb21 cleavage occurred,namely the 59 domain and 39 major domain. The factage, the patterns on the rRNAs were identified by primer

extension and compared to that of vacant ribosomes. that no cleavages were observed in the central domainand the 39 minor domain (including the decoding center)All 11 cleavages on 16S rRNA showed indistinguishable

Molecular Cell168

of 16S rRNA limits our interpretation of the possible Pb21 cleavage enhancement at C2347 in domain V ofstructural dynamics of the 30S subunit during translo- 23S rRNA was seen again (Figures 7B and 7C). As sooncation. as the A site was refilled with ternary complex containing

Since no Pb21 cleavages could be mapped to 5S Val-tRNAVal, the ribosome flipped back into the PRE staterRNA, we have no information about possible structural (PRE II complex in Figure 7A) with a simultaneous losschanges during translocation. However, in a recent of the cleavage enhancement at C2347. Since in thisprobing study, an unaltered contact pattern of the phos- state peptide bond formation has already occurred, onephate residues of 5S rRNA in PRE and POST states has can exclude peptidyl transfer as the cause for the ob-been reported (Shpanchenko et al., 1998). served structural changes in 23S rRNA. Forcing this PRE

23S rRNA was shown to be frequently targeted by II complex to undergo another round of translocationsite-specific metal ion hydrolysis (Polacek and Barta, (POST II state in Figure 7A) led to the reappearance1998), which is mirrored by the identification of Pb21 of the POST-specific Pb21 cleavage enhancement atcleavage sites dispersed over the whole molecule. This C2347 (Figures 7B and 7C). The observations presentedenabled us to investigate the effects of translocation on here show that parts of 23S rRNA can adopt two differentrRNA structure in all domains of 23S rRNA. Interestingly, conformations that are specific for the functional statethe Pb21 cleavage rate at position C2347 in domain V of the ribosome during the elongation cycle. It appearsincreases significantly, on average about 3-fold, in the that the functional oscillation between PRE and POSTPOST state (Figure 5A and Table 1). This site was also state ribosomes is mirrored by a recurrent structuraltargeted by Pb21 in human ribosomes, implying that this rearrangement of the large subunit rRNA.nucleotide resides in a structurally highly conserved part It is of note that the Pb21 cleavages in or near theof domain V of 23S rRNA (Polacek and Barta, 1998). central multibranched loop of domain V were shown toRecent cross-linking studies have placed C2347 of do- be very similar in all tested functional complexes (Tablemain V and adjacent regions in close proximity to 5S 1). This indicates that the structure of this domain, whichrRNA (Osswald and Brimacombe, 1999). An E site– is known to be an important part of the peptidyl trans-specific tRNA footprint on 23S rRNA (C2394) (Moazed ferase center, is not significantly affected in any of theand Noller, 1989b) also mapped close to the affected functional states. This is somehow unexpected, sincePb21 cleavage site of domain V in the POST state. How- tRNA footprinting (Moazed and Noller, 1989b) and pho-ever, control experiments revealed that filling the E site toaffinity labeling studies (Steiner et al., 1988) indicatedirectly with tRNAPhe or binding EF-G alone did not show interaction of some of the highly conserved residues ofthis enhanced cleavage pattern. Also, the Pi state with this loop with the aminoacyl end of tRNA.an AcPhe-tRNAPhe at the P site and a free A site did not Focusing our structural investigations with Pb21 si-show the enhanced cleavage at C2347, thus rendering multaneously on tRNA and rRNA in defined functionalit unlikely that the reduced cleavage rate seen in the states of the ribosomal elongation cycle led us to con-PRE state is due to shielding the Pb21 access by A clude that: (1) ribosome-bound tRNA adopts a differentsite–bound tRNA. Therefore, we conclude that the trans- conformation compared to tRNA free in solution; (2) thelocation step accounts for the observed alterations in tertiary fold of bound AcPhe-tRNAPhe and tRNAPhe is dis-the Pb21 cleavage rates and hence indicates structural tinguishable; (3) translocation does not change tRNAdifferences in 23S rRNA between PRE and POST com- structure; (4) most of the Pb21-sensitive regions of 16Splexes. Additionally, three new cleavages occurred in and 23S rRNA are unaffected by translocation; (5) somethe POST complex at C2626, G2694, and U2695 in do- parts of domain V and VI of 23S rRNA exist in differentmain VI (Figure 5B). These sites flank the evolutionarily structural conformations in the PRE and the POST state;highly constrained a-sarcin loop (C2646-G2674), which and (6) this flexible 23S rRNA element can periodicallyis a major component of the elongation factor binding shift between the two different states during elongation.site. The identified Pb21 cleavage enhancements in the

The latter finding provides evidence for a flexible andPOST state could be due to a conformational change

possibly mobile 23S rRNA element that is involved inthat either leads to a higher affinity metal ion binding

translocation and hints at a pulsing structural featurepocket for the attacking Pb21, or to a more suitablethat is beating in the catalytic heart of translating ribo-geometry and positioning of the Pb(OH)1 relative to thesomes. Our findings add motion and structural flexibility29-hydroxyl of the ribose and the targeted phosphodies-to the RNA components of the hitherto rather rigid pro-ter bond. Other sites of strand scission in domain V andtein-synthesizing “ribosome machine”.domain VI are unaffected by translocation, implying that

the proposed conformational rearrangement on 23SExperimental ProceduresrRNA in the POST state is localized.

Is this indeed a moving element of 23S rRNA that has Preparation of Reassociated Ribosomesthe potential of adopting different conformations before Ribosomal subunits from 400 g of frozen E. coli Can 20 cells wereand after translocation? And if so, does this 23S rRNA prepared as described (Bommer et al., 1997). 4460 A260 units of 30Selement flip back when the ribosome reenters the PRE and 7550 A260 units of 50S were reassociated in reassociation buffer

(20 mM HEPES/KOH [pH 7.6] [08C], 20 mM MgCl2, 30 mM KCl, andstate on its way through the elongation cycle? To test4 mM b-mercaptoethanol) for 60 min at 408C in a volume of 150 ml.this hypothesis, the rRNA fold was probed with Pb21

Portions of 4000 A260 units were subjected to a zonal centrifugationduring a complete elongation cycle (PRE→POST→PREat 20,000 rpm (Beckman Ti 15 rotor) for 16 hr at 48C on a 6%–38%

II→POST II). To this end, ribosomal complexes with a (w/v) sucrose gradient made up in reassociation buffer. Fractionsheteropolymeric mRNA that contains three unique co- of the 70S peak were pooled and ribosomes pelleted by ultracentrif-dons for Met, Phe, and Val were constructed. After con- ugation at 50,000 3 g for 24 hr at 48C. Ribosome pellets were

resuspended and pooled in reassociation buffer and allowed tostruction of the first POST complex, the POST-specific

Structural Dynamics of Translating Ribosomes169

reassociate again for 20 min at 408C to undo possible partial disso- 90% and was calculated as follows: % puromycin-reactive peptidyl-tRNA after EF-G addition/puromycin efficiency (z80%) 3 100.ciation that can occur during the zonal run. Ribosomes were then

dialyzed against buffer containing 20 mM HEPES/KOH (pH 7.6) (08C), To investigate rRNA structure during a complete elongation cycle,300 pmol of ribosomes was programmed with 2400 pmol of Met-6 mM MgCl2, 30 mM KCl, and 4 mM b-mercaptoethanol, divided

into small aliquots, quick frozen, and stored at –808C. Phe-Val-mRNA. This 49-nucleotide-long heteropolymeric mRNAcarries the three unique codons AUG-UUC-GUU in the middle andwas prepared as described for the MF-mRNA (Triana-Alonso et al.,

tRNAs 1995). In the first step, the P site was blocked by incubating withE. coli tRNAPhe, [32P]-59-end-labeled tRNAPhe, or tRNAVal was charged, 750 pmol of deacylated tRNAfMet in 312.6 ml of binding buffer for 15N-acetylated, and purified by reverse-phase HPLC as described min at 378C. To construct PRE complexes, the A site was filled by(Rheinberger et al., 1988). adding 540 pmol of Ac[14C]Phe-tRNAPhe (1073 dpm/pmol), and the

incubation continued for 30 min at 378C in a total volume of 625.2ml. POST complexes were established by incubation with EF-GtRNA/Ribosome Complexes for tRNA Cleavageas described above. Ribosomes (75%–82%) bound Ac[14C]Phe-Binding buffer conditions in all steps were constant 20 mM HEPES/tRNAPhe, and the homogeneity (ratio of puromycin reactivity beforeKOH (pH 7.6) (08C), 6 mM MgCl2, 150 mM NH4Cl, 2 mM spermidine,and after EF-G addition) of the complexes was 91%. After formation0.05 mM spermine, and 4 mM b-mercaptoethanol. PRE complexesof the POST state ribosomes, EF-G was removed by gel filtrationwere constructed by incubating 175 pmol of 70S ribosomes with(spun columns) before ternary complex binding. Ternary complex350 pmol of deacylated tRNAPhe and 350 mg of poly(U) in 87.5 ml of([3H]Val-tRNAVal•EF-Tu•GTP:70S 5 1.5:1; EF-Tu:aa-tRNA 5 1:8.1)binding buffer for 15 min at 378C. In the second step, the A site waswas preformed in binding buffer for 2 min at 378C before incubationfilled by adding 140 pmol of double-labeled Ac[14C]Phe-[32P]tRNAPhe

with an equal volume of the ribosomal complexes for 2–5 min at(14C:996.6 dpm/pmol; 32P: 9100 dpm/pmol) and incubated at 378C208C in order to refill the A site (PRE II complex). Subsequently, anfor 30 min in a total volume of 175 ml. Nonbound tRNA was removedaliquot of this PRE II complex was incubated with EF-G to formby gel filtration over a Sephacryl-S300 cDNA spun column (Phar-POST II complexes. Membrane filtration reveals that 41%–51% ofmacia). To establish the POST state, aliquots of the elute wereribosomes bound [3H]Val-tRNAVal (2000 dpm/pmol), and the homoge-incubated with EF-G (EF-G:70S 5 0.1, 115 mM GTP) for 10 min atneity of PRE II and POST II complexes was from 70%–87% ac-378C. The puromycin reaction was performed overnight at 08C incording to the puromycin reaction. Translocation efficiency wasthe presence of 0.8 mM puromycin. The reaction was stopped bybetter than 78% in the second step (calculated as above). The factaddition of 1 vol 0.3 M sodium acetate saturated with MgSO4 (pHthat more than 70% of POST complexes bound [3H]Val-tRNA that5.5), followed by extraction with ethyl acetate. Thirty percent ofsubsequently participates quantitatively in dipeptide bond formationribosomes bound Ac[14C]Phe-[32P]tRNAPhe as measured by mem-indicates that also the preceding translocation (PRE→POST) mustbrane filtration, and the site specificity of bound Ac[14C]Phe-have been better than 70%. Indeed, a translocation efficiency of[32P]tRNAPhe was better than 85% according to the puromycin re-85% was calculated (% puromycin-reactive tRNA after EF-G/puro-action.mycin efficiency (z30%–40%) 3 100), as the control experimentTo probe deacylated tRNAPhe structure at the P site in PRE andwith this mRNA resulted in a reduced puromycin efficiency of aboutat the E site in POST complexes, 135 pmol of [32P]-59-labeled tRNA30%. Note that puromycin values are kinetic values and that under(6800 dpm/pmol) was incubated in the first step with 270 pmol ofthe conditions employed, the plus EF-G values reflect an almostribosomes and 450 mg of poly(U) in a total volume of 112.5 ml. Inquantitative translocation (Beyer et al., 1994).the second step, 324 pmol of Ac[14C]Phe-tRNA (1008 dpm/pmol)

was added (total volume 225 ml). Ribosomes (25%–30%) bounddeacylated [32P]tRNAPhe, and 40%–70% carried Ac[14C]Phe-tRNAPhe. Pb21 Cleavage ProcedureSince under the applied conditions the P site is not completely Pb21 cleavage was initiated by adding freshly prepared Pb(OAc)2 toblocked with deacylated tRNA, AcPhe-tRNAPhe also binds to the P the desired ribosomal complex to a final concentration of 2 mMsite, which results in a mixed population of Pi and PRE complexes. (for tRNA cleavage) or 10 mM (for rRNA cleavage). Cleavage wasHowever, an increase in Ac[14C]-Phe-puromycin counts of 31%–60% performed at 258C for 15 min (tRNA) or 5 min (rRNA), respectively.after EF-G addition indicates quantitative translocation of PRE state The reaction was stopped by the addition of excess EDTA withribosomes. Under the near in vivo conditions applied, deacylated respect to divalent metal ions. Cleaved tRNAs were purified bytRNAPhe remains stongly bound to the E site after translocation as phenol extraction, precipitated with ethanol/0.3 M sodium acetateindicated by membrane filtration (32P counts: 25,082 dpm before, (pH 5.5), and resuspended in loading buffer, and 20,000–30,000 dpmand 28,042 dpm after translocation). was applied to a 13% denaturing polyacrylamide gel. Cleaved rRNAs

Pi complexes were constructed by incubating 20–40 pmol of dou- were purified as described (Winter et al., 1997), and 0.2–0.4 pmolble-labeled Ac[14C]Phe-[32P]tRNAPhe with 100 pmol of 70S ribosomes of rRNA was used as template for reverse transcription primed byand 250 mg of poly(U) for 15 min at 378C in 62.5 ml of binding buffer. DNA oligos (Winter et al., 1997), which was performed as describedInput AcPhe-tRNAPhe (90%–100%) was bound and 73%–80% of it (Polacek and Barta, 1998). Gels were scanned and quantified usingwas puromycin reactive, reflecting the efficiency of the puromycin a Molecular Dynamics PhosphorImager.reaction under the applied conditions (overnight incubation at 08C).

Acknowledgments

tRNA/Ribosome Complexes for rRNA CleavageWe are grateful to Gregor Blaha for the help of preparing reassoci-Pi complexes were prepared by incubating 45 pmol of Ac[14C]Phe-ated ribosomes and to Ozlem Tastan for the Met-Phe-Val-mRNAtRNAPhe (1008 dpm/pmol) with 30 pmol of poly(U)-programmed (150clone and for help with tRNA charging. Our thanks are extended tomg) ribosomes for 15 min at 378C in a volume of 37.5 ml. BindingSilke Dorner, Uwe von Ahsen, Sean Connell, and Uli Stelzl for criticalcontrols revealed that 80%–100% of ribosomes bound AcPhe-review of the manuscript and to Viter Marquez and Uli Stelzl fortRNAPhe whereas 80% reacted with puromycin under the appliedstimulating discussions. This work was supported by grant NI 176/conditions indicating P site location.11-1 from the Deutsche Forschungsgemeinschaft to K. H. N. andThe PRE state was established by first blocking the P site of 60by grant P13651-GEN from the Austrian Science Foundation (FWF)pmol of 70S ribosomes with 72 pmol of deacylated tRNAPhe in 75 mlto A. B.of binding buffer in the presence of 300 mg of poly(U) for 15 min at

378C. Subsequently, 138 pmol of Ac[14C]Phe-tRNAPhe was added,Received December 27, 1999; revised May 16, 2000.and the incubation continued at 378C for 30 min in a reaction volume

of 150 ml. For translocation, an aliquot of the PRE complex wasReferencesincubated with EF-G (EF-G:70S 5 0.1, 115 mM GTP) for 10 min at

378C. Ribosomes (60%–88%) bound Ac[14C]Phe-tRNAPhe, and thehomogeneity of PRE and POST complexes was from 67%–75% as Agrawal, R.K., Penczek, P., Grassucci, R.A., Li, Y., Leith, A., Nier-

haus, K.H., and Frank, J. (1996). Direct visualization of A-, P-, andjudged by the puromycin reaction. Translocation efficiency was around

Molecular Cell170

E-site transfer RNAs in the Escherichia coli ribosome. Science 271, peptide-bond formation with an A-site substrate covalently linkedto 23S ribosomal RNA. Science 280, 286–289.1000–1002.

Agrawal, R.K., Penczek, P., Grassucci, R.A., and Frank, J. (1998). Gutell, R.R., Larsen, N., and Woese, C.R. (1994). Lessons from anVisualization of elongation factor G on the Escherichia coli 70S evolving rRNA: 16S and 23S rRNA structures from a comparativeribosome: the mechanism of translocation. Proc. Natl. Acad. Sci. perspective. Microbiol. Rev. 58, 10–26.USA 95, 6134–6138. Lodmell, J.S., and Dahlberg, A.E. (1997). A conformational switch inAgrawal, R.K., Heagle, A.B., Penczek, P., Grassucci, R.A., and Frank, Escherichia coli 16S ribosomal RNA during decoding of messengerJ. (1999). EF-G-dependent GTP hydrolysis induces translocation RNA. Science 277, 1262–1267.accompanied by large conformational changes in the 70S ribosome.

Malhotra, A., Penczek, P., Agrawal, R.K., Gabashvili, I.S., Grassucci,Nat. Struct. Biol. 6, 643–647.R.A., Junemann, R., Burkhardt, N., Nierhaus, K.H., and Frank, J.

Arts, G.J., Kuersten, S., Romby, P., Ehresmann, B., and Mattaj, I.W. (1998). Escherichia coli 70 S ribosome at 15 A resolution by cryo-(1998). The role of exportin-t in selective nuclear export of mature electron microscopy: localization of fMet-tRNAfMet and fitting oftRNAs. EMBO J. 17, 7430–7441. L1 protein. J. Mol. Biol. 280, 103–116.Ban, N., Nissen, P., Hansen, J., Capel, M., Moore, P.B., and Steitz, Marciniec, T., Ciesiolka, J., Wrzesinski, J., and Krzyzosiak, W.J.T.A. (1999). Placement of protein and RNA structures into a (1989). Identification of the magnesium, europium, and lead binding5 A-resolution map of the 50S ribosomal subunit. Nature 400, sites in E. coli and lupine tRNAPhe by specific metal ion-induced841–847. cleavages. FEBS Lett. 243, 293–298.Behlen, L.S., Sampson, J.R., DiRenzo, A.B., and Uhlenbeck, O.C. McCutcheon, J.P., Agrawal, R.K., Philips, S.M., Grassucci, R.A.,(1990). Lead-catalyzed cleavage of yeast tRNAPhe mutants. Bio- Gerchman, S.E., Clemons, W.M., Jr., Ramakrishnan, V., and Frank,chemistry 29, 2515–2523. J. (1999). Location of translational initiation factor IF3 on the smallBelitsina, N.V., Tnalina, G.Z., and Spirin, A.S. (1981). Template-free ribosomal subunit. Proc. Natl. Acad. Sci. USA 96, 4301–4306.ribosomal synthesis of polylysine from lysyl-tRNA. FEBS Lett. 131,

Michalowski, D., Wrzesinski, J., and Wlodzimierz, K. (1996). Cleav-289–292.

ages induced by different metal ions in yeast tRNAPhe U59C60Bergemann, K., and Nierhaus, K.H. (1983). Spontaneous, elongation mutants. Biochemistry 35, 10727–10734.factor G independent translocation of Escherichia coli ribosomes.

Moazed, D., and Noller, H.F. (1989a). Intermediate states in theJ. Biol. Chem. 258, 15105–15113.movement of transfer RNA in the ribosome. Nature 342, 142–148.

Beyer, D., Skripkin, E., Wadzack, J., and Nierhaus, K.H. (1994). HowMoazed, D., and Noller, H.F. (1989b). Interaction of tRNA with 23Sthe ribosome moves along the mRNA during protein synthesis. J.rRNA in the ribosomal A, P, and E sites. Cell 57, 585–597.Biol. Chem. 269, 30713–30717.Ofverstedt, L.G., Zhang, K., Tapio, S., Skoglund, U., and Isaksson,Bommer, U.A., Burkhardt, N., Junemann, R., Spahn, C.M.T., Triana-L.A. (1994). Starvation in vivo for aminoacyl-tRNA increases theAlonso, F.J., and Nierhaus, K.H. (1997). Ribosomes and polysomes.spatial separation between the two ribosomal subunits. Cell 79,In Subcellular Fractionation—A Practical Approach, J. Graham and629–638.D. Rickwood, eds. (Washington DC: IRL Press), pp. 271-301.

Osswald, M., and Brimacombe, R. (1999). The environment of 5SBrown, R.S., Dewan, J.C., and Klug, A. (1985). Crystallographic andrRNA in the ribosome: cross-links to 23S rRNA from sites withinbiochemical investigation of the lead(II)-catalyzed hydrolysis ofhelices II and III of the 5S molecule. Nucleic Acids Res. 27, 2283–yeast phenylalanine tRNA. Biochemistry 24, 4785–4801.2290.Cate, J.H., Yusupov, M.M., Yusupova, G.Z., Earnest, T.N., and Noller,Pestka, S. (1969). Studies on the formation of transfer ribonucleicH.F. (1999). X-ray crystal structures of 70S ribosome functional com-acid-ribosome complexes. VI. Oligopeptide synthesis and translo-plexes. Science 285, 2095–2104.cation on ribosomes in the presence and absence of soluble transferClemons, W.M., Jr., May, J.L., Wimberly, B.T., McCutcheon, J.P.,factors. J. Biol. Chem. 244, 1533–1539.Capel, M.S., and Ramakrishnan, V. (1999). Structure of a bacterial

30S ribosomal subunit at 5.5 A resolution. Nature 400, 833–840. Polacek, N., and Barta, A. (1998). Metal ion probing of rRNAs: evi-dence for evolutionarily conserved divalent cation binding pockets.Dabrowski, M., Spahn, C.M., Schafer, M.A., Patzke, S., and Nierhaus,RNA 4, 1282–1294.K.H. (1998). Protection patterns of tRNAs do not change during

ribosomal translocation. J. Biol. Chem. 273, 32793–32800. Rheinberger, H.J., Geigenmuller, U., Wedde, M., and Nierhaus, K.H.(1988). Parameters for the preparation of Escherichia coli ribosomesDorner, S., and Barta, A. (1999). Probing ribosome structure byand ribosomal subunits active in tRNA binding. Methods Enzymol.europium-induced RNA cleavage. Biol. Chem. 380, 243–251.164, 658–670.Frank, J., Zhu, J., Penczek, P., Li, Y., Srivastava, S., Verschoor, A.,Rheinberger, H.J., and Nierhaus, K.H. (1990). Partial release ofRadermacher, M., Grassucci, R., Lata, R.K., and Agrawal, R.K.AcPhe-Phe-tRNA from ribosomes during poly(U)-dependent poly-(1995). A model of protein synthesis based on cryo-electron micros-(Phe) synthesis and the effects of chloramphenicol. Eur. J. Biochem.copy of the E. coli ribosome. Nature 376. 441-444.193, 643–650.Gabashvili, I.S., Agrawal, R.K., Grassucci, R., Squires, C.L., Dahl-

berg, A.E., and Frank, J. (1999a). Major rearrangements in the 70S Schilling-Bartetzko, S., Bartetzko, A., and Nierhaus, K.H. (1992). Ki-ribosomal 3D structure caused by a conformational switch in 16S netic and thermodynamic parameters for tRNA binding to the ribo-ribosomal RNA. EMBO J. 18, 6501–6507. some and for the translocation reaction. J. Biol. Chem. 267, 4703–

4712.Gabashvili, I.S., Agrawal, R.K., Grassucci, R., and Frank, J. (1999b).Structure and structural variations of the Escherichia coli 30 S ribo- Shpanchenko, O.V., Dontsova, O.A., Bogdanov, A.A., and Nierhaus,somal subunit as revealed by three-dimensional cryo-electron mi- K.H. (1998). Structure of 5S rRNA within the Escherichia coli ribo-croscopy. J. Mol. Biol. 286, 1285–1291. some: iodine-induced cleavage patterns of phosphorothioate deriv-

atives. RNA 4, 1154–1164.Gavrilova, L.P., and Spirin, A.S. (1971). Stimulation of “non-enzymic”translocation in ribosomes by p-Chloromercuribenzolate. FEBS Stark, H., Mueller, F., Orlova, E.V., Schatz, M., Dube, P., Erdemir,Lett. 17, 324–326. T., Zemlin, F., Brimacombe, R., and van Heel, M. (1995). The 70S

Escherichia coli ribosome at 23 A resolution: fitting the ribosomalGornicki, P., Baudin, F., Romby, P., Wiewiorowski, M., Kryzosiak,RNA. Structure 3, 815–821.W., Ebel, J.P., Ehresmann, C., and Ehresmann, B. (1989). Use of

lead(II) to probe the structure of large RNA’s. Conformation of the Stark, H., Orlova, E.V., Rinke-Appel, J., Junke, N., Mueller, F., Rod-39 terminal domain of E. coli 16S rRNA and its involvement in building nina, M., Wintermeyer, W., Brimacombe, R., and van Heel, M.the tRNA binding sites. J. Biomol. Struct. Dyn. 6, 971–984. (1997a). Arrangement of tRNAs in pre- and posttranslocational ribo-

somes revealed by electron cryomicroscopy. Cell 88, 19–28.Green, R., Switzer, C., and Noller, H.F. (1998). Ribosome-catalyzed

Structural Dynamics of Translating Ribosomes171

Stark, H., Rodnina, M.V., Rinke-Appel, J., Brimacombe, R., Win-termeyer, W., and van Heel, M. (1997b). Visualization of elongationfactor Tu on the Escherichia coli ribosome. Nature 389, 403–406.

Stark, H., Rodnina, M.V., Wieden, H.-J., van Heel, M., and Win-termeyer, W. (2000). Large-scale movement of elongation factorG and extensive conformational change of the ribosome duringtranslocation. Cell 100, 301–309.

Steiner, G., Kuechler, E., and Barta, A. (1988). Photo-affinity labellingat the peptidyl transferase centre reveals two different positions forthe A- and P-sites in domain V of 23S rRNA. EMBO J. 7, 3949–3955.

Streicher, B., Westhof, E., and Schroeder, R. (1996). The environ-ment of two metal ions surrounding the splice site of a group I intron.EMBO J. 15, 2556–2564.

Tocilj, A., Schlunzen, F., Janell, D., Gluhmann, M., Hansen, H.A.,Harms, J., Bashan, A., Bartels, H., Agmon, I., Franceschi, F., andYonath, A. (1999). The small ribosomal subunit from thermus ther-mophilus at 4.5 A resolution: pattern fittings and the identificationof a functional site. Proc. Natl. Acad. Sci. USA 96, 14252–14257.

Triana-Alonso, F.J., Dabrowski, M., Wadzack, J., and Nierhaus, K.H.(1995). Self-coded 39-extension of run-off transcripts produces ab-errant products during in vitro transcription with T7 RNA polymerase.J. Biol. Chem. 270, 6298–6307.

Wilson, K.S., and Noller, H.F. (1998). Molecular movement insidethe translational engine. Cell 92, 337–349.

Winter, D., Polacek, N., Halama, I., Streicher, B., and Barta, A. (1997).Lead-catalysed specific cleavage of ribosomal RNAs. Nucleic AcidsRes. 25, 1817–1824.

Zito, K., Huttenhofer, A., and Pace, N.R. (1993). Lead-catalyzedcleavage of ribonuclease P RNA as a probe for integrity of tertiarystructure. Nucleic Acids Res. 21, 5916–5920.