Periodic Conformational Changes in rRNA

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

Periodic Conformational Changes in rRNA: Monitoring the Dynamics of Translating Ribosomes Norbert Polacek,* Sebastian Patzke,† Knud H. Nierhaus,† and Andrea Barta*‡ * Institute of Biochemistry Vienna Biocenter University of Vienna Dr. Bohr-Gasse 9 A-1030 Vienna Austria † Max-Planck-Institut fu ¨ r Molekulare Genetik Ihnestrasse 73 D-14195 Berlin Germany

Summary In protein synthesis, a tRNA transits the ribosome via consecutive binding to the A (acceptor), P (peptidyl), and E (exit) site; these tRNA movements are catalyzed by elongation factor G (EF-G) and GTP. Site-specific Pb2ⴙ cleavage was applied to trace tertiary alterations in tRNA and all rRNAs on pre- and posttranslocational ribosomes. The cleavage pattern of deacylated tRNA and AcPhe-tRNA changed individually upon binding to the ribosome; however, these different conformations were unaffected by translocation. On the other hand, translocation affects 23S rRNA structure. Significantly, the Pb2ⴙ cleavage pattern near the peptidyl transferase center was different before and after translocation. This structural rearrangement emerged periodically during elongation, thus providing evidence for a dynamic and mobile role of 23S rRNA in translocation. Introduction The coupling of amino acids to form proteins is a crucial biological process performed by ribosomes in all living cells. The ribosomal elongation cycle is characterized by a series of activities in which the growing peptide chain is lengthened by one amino acid. Each new amino acid is delivered to the ribosome as an aminoacyltRNA•EF-Tu•GTP ternary complex, where it binds to the A site. Following peptide bond formation with the nascent peptide chain of P site–bound tRNA, the two tRNAs together with the mRNA must be translocated to the P and E sites, leaving the A site ready to accept the next ternary complex. This translocation is driven by the elongation factor G (EF-G) and GTP hydrolysis. The findings that, under certain in vitro conditions, translocation can also occur in the absence of EF-G (Pestka, 1969; Gavrilova and Spirin, 1971; Bergemann and Nierhaus, 1983) or mRNA (Belitsina et al., 1981) imply that this reaction is inherent to the ribosome itself and led to the view that the translation apparatus is a macromolecular machine. Therefore, moving ribosomal elements are to ‡ To whom correspondence should be addressed (e-mail: andrea@

bch.univie.ac.at).

be expected during the elongation cycle, and some reasonable candidates have been identified (for a review, see Wilson and Noller, 1998). Under in vivo conditions, the pretranslocation state is demarcated from the posttranslocation state by a huge activation energy barrier of about 120 kJ/mol (Schilling-Bartetzko et al., 1992) that “freezes” the tRNA–ribosome conformation in either state in the absence of elongation factors. Evidence for a structural switch that might be involved in “defreezing” the fixed complex derives from recent mutagenesis studies on 16S rRNA in the 912 region (Gabashvili et al., 1999a; Lodmell and Dahlberg, 1997). Other moving ribosomal elements that might be involved in translocation are the L7/L12 stalk on the 50S subunit (Agrawal et al., 1999), the head region of the 30S subunit (Agrawal et al., 1999), or even the whole 70S structure (O¨fverstedt et al., 1994). Most recently, the exciting insights into ribosome structure obtained by cryo-electron microscopy (Frank et al., 1995; Stark et al., 1995, 1997a; Malhotra et al., 1998) have been further extended by a wealth of interesting X-ray crystallography data of both subunits (Ban et al., 1999; Clemons et al., 1999; Tocilj et al., 1999) and the 70S ribosome (Cate et al., 1999). The next step in understanding translation will definitely come from elucidating ribosomal dynamics. Since conformational changes associated with translocation have not yet been shown in molecular terms, we set out to investigate this issue by applying a very sensitive structural probing technique, namely site-specific cleavage of RNA by Pb2⫹. For site-specific Pb2⫹ cleavage to occur, a number of structural prerequisites have to be met. A tightly bound Pb2⫹ hydroxyl ion has to be orientated in such a way that it can remove the proton from the 2⬘-OH of the ribose at the cleavage site. Nucleophilic attack of the resulting oxyanion at the adjacent phosphodiester bridge cleaves the RNA chain (Brown et al., 1985). This method of site-specific metal ion hydrolysis has already been successfully applied to probe for structural integrity and for tracing down metal ion binding pockets in various RNA molecules (Zito et al., 1993; Streicher et al., 1996; Winter et al., 1997). Due to its strict dependence on a specific tertiary fold, Pb2⫹ cleavage turned out to be a powerful tool in elucidating fine structural changes in RNA molecules (Gornicki et al., 1989; Behlen et al., 1990; Michalowski et al., 1996; Dorner and Barta, 1999). In these studies, it became evident that conformational alterations were reflected by a change in the cleavage rate or specificity. In previous work, we characterized the Pb2⫹ cleavage pattern in rRNAs in vacant ribosomes (Polacek and Barta, 1998) and have now expanded our structural investigations to ribosomes arrested in defined functional states of the elongation cycle. This enabled us to simultaneously probe the structures of rRNA and tRNA of ribosomal complexes in the same functional state. Our experiments show that tRNA structure changes upon binding to the ribosome, and EF-G-catalyzed transloca-

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Figure 1. Functional Ribosomal Complexes Pretranslocational complexes (PRE) (A and C) carry deacylated tRNA at the P site and AcPhe-tRNA at the A site. Posttranslocational complexes (POST) (B and D) are characterized by deacylated tRNA bound to the E site and AcPhe-tRNA bound to the P site. Pi complexes (E) (i for initiating) carry one single tRNA at the P site. See the insert for the different radioactive labels applied.

tion does not further influence tRNA conformation. On the other hand, ribosome structure is affected by translocation, indicating that parts of 23S rRNA move. This movement was shown to be reversible, implying recurrent structural shifting of 23S rRNA features during the elongation cycle. Results Experimental Design We report herein the investigation of structural changes of ribosomes and their tRNA ligands in defined functional states before and after EF-G-catalyzed translocation. To this end, pre- and posttranslocational complexes were constructed at near in vivo ionic concentration (polyamine system in the presence of 6 mM Mg2⫹), and the conformation of tRNA as well as of rRNA was probed by sitespecific Pb2⫹ cleavage. The elongating ribosome exists in at least two functionally different states. The pretranslocational state (PRE state) carries a deacylated tRNA at the P site and a peptidyl tRNA at the A site in (see Experimental Procedures and Figure 1). This state resembles the situation immediately after peptide bond formation. After EF-G catalysis, the ribosome enters the posttranslocational state (POST state) whereby the deacylated tRNA is thought to be transferred to the E site and the peptidyl tRNA resides in the P site. Only during initiation of protein synthesis does the ribosome contain a single tRNA at the P site, and hence this complex is referred to as Pi complex (i for initiating) (Figure 1). Probing tRNA Structure In order to probe the structure of AcPhe-tRNAPhe, [14C]labeled N-AcPhe-tRNAPhe that was further 5⬘ end labeled

with 32P was bound to the A site of poly(U)-programmed ribosomes where the P site was blocked by prior binding of deacylated tRNAPhe (Figure 1A). Nonbound tRNAs were removed by gel filtration. During EF-G-catalyzed translocation the two tRNAs move to the P and E sites, respectively (Figure 1B). According to the puromycin reaction, the homogeneity of the PRE and POST complexes was better than 85% (see Experimental Procedures). To gain insight into the conformation of deacylated tRNAPhe at the P site in PRE and at the E site in POST complexes, deacylated [32P]-5⬘-end-labeled tRNAPhe was bound to the P site followed by binding of Ac[14C]PhetRNAPhe to the A site (Figure 1C). Since under the applied conditions (see Experimental Procedures) the P site is not completely blocked with deacylated [32P]-tRNAPhe, AcPhe-tRNAPhe can also bind to the P site, which results in a mixture of Pi and PRE complexes. However, only the fate of the deacylated [32P]-tRNAPhe in the PRE and POST complex is of interest in this experiment and is analyzed in the Pb2⫹ cleavage experiments. Successful translocation was monitored by a significant increase in the formation of Ac[14C]Phe-puromycin after EF-G incubation (see Experimental Procedures). After establishing Pi, PRE, and POST complexes, Pb(OAc)2 was added to a final concentration of 2 mM, and the cleaved tRNA was run on a 13% polyacrylamide gel. The cleavage pattern of ribosome-bound tRNA was compared to that obtained in solution. AcPhe-tRNAPhe and deacylated tRNAPhe showed indistinguishable Pb2⫹ cleavage patterns in solution with the strongest cuts located 5⬘ to positions D16 and G18 in the D loop. Intermediate cleavages were observed at U8, A9, G10, C17, A21, G46, X47, and C49, and weak cuts were mapped to A14, G15, and D20 (Figure 2). In general, all cleavages were inhibited when the tRNA was bound to the ribo-

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Figure 2. Pb2⫹ Cleavage Patterns of Unbound and Ribosome-Bound tRNAPhe (A) Sites of Pb2⫹-induced cleavages are indicated by arrows in the secondary structure of tRNAPhe. Strong, medium, and weak cuts are defined as having more than 10-fold, between 3- and 10-fold, and less than 3-fold increased signal strength of the cleavage band compared to the corresponding band in uncleaved tRNA. Autoradiograms of both 5⬘-end-labeled AcPhe-tRNAPhe (B) and deacylated tRNAPhe (C) are shown, which were cleaved with 2 mM Pb(OAc)2 for 15 min at 25⬚C in solution (lane 2) or bound to ribosomes in the PRE state (lane 3) or POST state (lane 4). Lane 1, no Pb2⫹ added; lane 5, alkaline hydrolysis ladder (H); and lane 6, limited hydrolysis by RNase T1. The asterisk indicates a band at ␺55 that turned out to be independent of Pb2⫹ addition and is most probably a degradation product.

some, but the degree of inhibition varied for each cleavage site and was different for AcPhe-tRNAPhe and deacylated tRNAPhe (Figure 3A). However, the cleavage inhibition patterns of AcPhe-tRNAPhe bound to the A site in the PRE and to the P site in the POST state turned out to be very similar (Figures 2B and 3A). In this case, the most efficient cleavage inhibition was obtained at D16 (9.2- to 9.7-fold). In contrast, ribosome-bound deacylated tRNAPhe showed a distinct inhibition pattern from AcPhe-tRNAPhe, with G18 being the most efficiently inhibited cleavage site (9.0- to 9.5-fold). Again, the cleavage

pattern of deacylated tRNAPhe hardly changed upon translocation from the P to the E site (Figures 2C and 3A). The Pb2⫹ cleavage inhibition pattern of AcPhe-tRNAPhe in the Pi state (Figure 1E) was similar to that obtained with AcPhe-tRNAPhe at the APRE or PPOST site (data not shown). Likewise, deacylated tRNAPhe in the Pi complex revealed a pattern that was similar to that seen with deacylated tRNA at the P site of PRE complexes or E site of POST complexes (data not shown). These findings imply that the Pb2⫹ cleavage pattern and hence the conformation of ribosome-bound tRNA mainly depend

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Figure 3. Alteration of tRNA Cleavage Efficiencies upon Ribosome Binding (A) Inhibition of tRNA Pb2⫹ cleavages upon ribosome binding. The numbers give the fold inhibition of either AcPhe-tRNAPhe (left) bound to the APRE or PPOST site, or deacylated tRNAPhe (right) bound at the PPRE or EPOST site, both compared to unbound tRNAs. Significant differences between ribosome-bound AcPhetRNAPhe and deacylated tRNAPhe are bold. Values presented here are averages from 2–3 independent experiments. Numbers in ⌬AP and ⌬PE were calculated separately for every binding experiment and then averaged. (B) Degree of Pb2⫹ cleavage inhibition upon ribosome binding for each site is indicated in the 3D structure model. The inhibition pattern of AcPhe-tRNAPhe bound to the APRE or PPOST site (left) and of deacylated tRNAPhe bound to the PPRE or E site (right) are shown. See insert for the color code.

on the charging state of the tRNA molecule. These results indicate that the aminoacyl group triggers the ribosome-bound tRNA to adopt a specific tertiary fold, which is independent of the binding site and is not influenced by translocation. Probing rRNA Structure In addition to the structural investigations on tRNA during translocation, we further addressed the question as to whether the ribosome itself undergoes conformational changes upon translocation. Therefore, Pi, PRE, and POST complexes were constructed followed by the addition of 10 mM Pb2⫹ in order to cleave the rRNAs. Eighty to one hundred percent of ribosomes bind AcPhe-tRNAPhe in the Pi complex, as measured by membrane filtration. PRE complexes were constructed with Ac[14C]Phe-tRNAPhe at the A site and unlabeled deacylated tRNAPhe at the P site. In this case, 60%–88% of ribosomes bind AcPhe-tRNAPhe, and around 90% of bound tRNAs could be translocated. Subsequent to Pb2⫹ cleavage, the rRNAs of ribosomes in the different functional states were extracted and used as template for reverse transcription. No cleavages could be mapped on 5S rRNA. The Pb2⫹ cleavage pattern of 16S rRNA showed no differences in the Pi, PRE, or POST complex compared to that obtained in vacant ribosomes. The efficiency and location of 11 cleavage sites in 16S rRNA (G144, G211, G240, U245, C522, U531, A532, G1182, U1183, A1257, and A1285) were determined to be unaffected (Figure 4 and Table 1). These findings imply that 16S rRNA structure

does not change significantly during EF-G-catalyzed translocation, at least in those regions where Pb2⫹ cleavage could be detected. In addition, the 13 sites of strand scission mapped to the 5⬘ half (domains I–III) of 23S rRNA do not change during translocation (G141, A332, G388, A505, G785, A792, C889, C890, A1133, U1523, G1524, G1555, and C1646). Furthermore, eight cleavage sites located in domain V (G2307, C2440, U2441, C2573, U2585, C2610, and C2611) and domain VI (U2833) of 23S rRNA showed unaltered cleavage efficiency in any of the tested complexes. However, a weak Pb2⫹ cleavage inhibition at A1966 in domain IV of 23S rRNA in the Pi complex, which was already observed in a previous study (Polacek and Barta, 1998), was reproduced using a different buffer system. Cleavage efficiency at A1966 decreases on average about 28% (Table 1). Remarkably, this effect was not seen in the POST complex, although AcPhe-tRNAPhe was also present in the P site in this state. It therefore carries with it the suggestion that the P site in the Pi complex somehow differs from the P site in the POST complex. Interestingly, much more pronounced effects on the Pb2⫹ cleavage pattern could be identified in the POST state ribosome at positions C2347 (domain V), C2626, G2694, and U2695 (domain VI) of 23S rRNA. Cleavage efficiency at C2347 was enhanced about 3-fold compared to that seen in all other states (Figure 5 and Table 1). The three new POST-specific cleavages in domain VI are located near the highly conserved ␣-sarcin loop (nucleotides 2646–2674), which is known to be part of the EF-G binding site. To test if the observed effects on Pb2⫹ hydrolysis are solely the result

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Figure 4. Mapping of Pb2⫹ Cleavage Sites in 16S rRNA by Primer Extension Vacant ribosomes (lane 2) as well as Pi complexes (lane 3), PRE complexes (lane 4), and POST complexes (lane 5) were cleaved with 10 mM Pb2⫹ for 5 min at 25⬚C. Lane 1 shows the reverse transcription of uncleaved rRNA. AC (lanes 6 and 7) denote dideoxy sequencing lanes.

of factor binding or due to a translocation event, EF-G was added to empty ribosomes as well as to Pi complexes carrying AcPhe-tRNAPhe at the P site. These two complexes cannot enter the POST state. No effects on the cleavage pattern could be observed in either of the two cases (data not shown). To exclude the possibility that the enhanced cleavage pattern was solely due to an occupied E site, both the P and E site were filled with deacylated tRNA by incubating poly(U)-programmed ribosomes with 2–4 times molar excess of tRNA. No effect on the cleavage rates at C2347, C2626, G2694, and U2695 was seen (data not shown). These observations strongly hint at translocation as the cause for the altered Pb2⫹ cleavage pattern. Another control experiment was performed aiming at elucidating the authenticity of the PRE and POST complexes in the presence of 2 mM Pb2⫹ for 15 min (tRNA cleavage conditions) or 10 mM Pb2⫹ for 5 min (rRNA cleavage conditions). No significant tRNA release was observed in any case (data not shown), suggesting that Pb2⫹ addition does not change the functional state of the ribosome. In light of these observations, it is likely that parts of the 50S subunit, including regions of domain V and VI of 23S rRNA (Figure 6), undergo structural changes upon EF-G-promoted translocation. If this proposed structural rearrangement in 23S rRNA indeed reflects different functional states, it should follow the functional oscillation of ribosomes in the elongation cycle. To address this important question, ribosomal complexes with a heteropolymeric mRNA that contains the three unique codons Met, Phe, and Val were constructed. This mRNA enabled us to construct two defined consecutive PRE and POST complexes in one experiment (Figure 7A). PRE state ribosomes carried deacylated tRNAfMet at the P site and Ac[14C]Phe-tRNAPhe at the A site. Subsequent to translocation, the POST

complex contained tRNAfMet at the E site and Ac[14C]PhetRNAPhe at the P site while the Val codon was invading the A site. Binding tests revealed that 75%–82% of ribosomes bind AcPhe-tRNAPhe, and the specificity of the complexes was 91% according to the puromycin reaction (Figure 7D). Subsequently, EF-G was removed by gel filtration, which did not affect the charging state of the ribosome (data not shown). To reenter the PRE state (PRE II), the A site was filled with ternary complex containing [3H]Val-tRNAVal (Figure 7A). Membrane filtration indicated that up to 50% of the ribosomes bind ValtRNAVal (Figure 7D). The 13% decrease of Ac[14C]Phe counts most likely accounted for a loss of dipeptidyltRNA from the A site immediately after peptide bond formation (Rheinberger and Nierhaus, 1990). Another translocation finally drives the PRE II complexes into the POST II state, where the dipeptidyl-tRNA becomes puromycin reactive again. Homogeneity of the latter complexes was from 70% to 87% (Figure 7D). All of the constructed complexes (PRE, POST, PRE II, POST II), which mimic more than one complete elongation cycle, were probed with Pb2⫹. The weak POSTspecific cleavages at C2626, G2694, and U2695 in domain VI of 23S rRNA could not be detected in ribosomal complexes with heteropolymeric mRNA, for reasons currently not understood. However, the cleavage rate at C2347 in domain V of 23S rRNA increased significantly in the POST complex. Moreover, as soon as Val-tRNAVal was bound to the A site, the ribosome flipped back into the PRE state (PRE II) with a concomitant loss of the enhanced signal at C2347, resulting in a cleavage efficiency comparable to the starting complex. Significantly, driving this PRE II complex into the POST II state restored the POST-specific cleavage enhancement almost to the same extent (Figures 7B and 7C). These findings show that the POST-specific Pb2⫹ cleav-

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Table 1. Pb2⫹ Cleavage of rRNA

23S rRNA I

II

III

IV V

VI

16S rRNA 5⬘ dom

3⬘ dom

Position

Vacant

Pi

PRE

G141 A332 G388 A505 G785 A792 C889/C890 A1133 U1523 G1524 G1555 C1646 A1966 G2307 C2347 C2440 U2441 C2573 U2585 C2610/C2611 C2626 G2694/U2695 U2833

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1

0.93 1.10 1.00 1.14 0.94 0.97 0.96 1.02 1.13 1.16 1.07 1.03 0.72 0.93 1.13 0.90 1.03 0.99 0.92 1.06 0 0 1.01

⫾ 0.02 ⫾ 0.04 ⫾ 0.01

G144 G211 G240 U245 C522 U531/A532 G1182/U1183 A1257 A1285

1 1 1 1 1 1 1 1 1

0.89 0.94 1.03 0.90 0.85 0.85 0.91 0.90 0.92

POST ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.01 0.01 0.11 0.11 0.08 0.05

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.15 0.07 0.09 0.1 0.05 0.08 0.18 0.1 0.09 0.09 0.2 0.01 0.06

⫾ 0.07

1.04 1.08 1.02 1.15 0.92 1.04 ND 1.04 1.16 1.07 1.06 1.02 1.03 1.07 1.10 1.14 0.88 0.94 1.06 1.09 0 0 1.17

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.96 0.96 0.97 1.00 0.91 0.93 0.91 0.91 1.00

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.09 0.02 0.05 0.09 0.04 0.06 0.22 0.13 0.04 0.1 0.09 0.05 0.06 0.03 0.05 0.14

0.09 0.07 0.07 0.07 0.07 0.13 0.02 0.07 0.07

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

⫾ 0.08

0.94 0.90 0.99 1.00 0.93 1.08 0.96 1.06 1.15 0.91 1.11 0.94 0.99 1.04 3.20 1.10 0.92 0.80 0.90 1.10 1 1 1.15

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.98 1.05 1.10 1.10 0.87 0.93 0.94 0.89 1.00

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.04 0.05 0.07 0.1 0.07 0.02 0.05 0.03 0.01

0.1 0.02 0 0.11 0.02 0.05 0.06 0.05 0.03 0.12 0.06 0.12 0.05 0.19 0.9 0.09 0.27 0.01 0.07 0.07

⫾ 0.01 0.01 0.16 0.1 0.13 0.09 0.13 0.02 0.05 0.04

Band intensities were quantified and normalized to control bands. Lead cleavage efficiency of each site in vacant ribosomes was taken as 1.00 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 as one band. ND, not determined.

age signal in domain V of 23S rRNA appears periodically and can be followed throughout a whole elongation cycle. This indicates that the translocation-dependent structural rearrangements in 23S rRNA are reversible. Discussion Ribosomal protein synthesis requires a series of subsequent steps of initiation, tRNA binding, peptidyl transfer, elongation factor binding, GTP hydrolysis, translocation, and termination. Although much progress in elucidating the structure of vacant ribosomes (Frank et al., 1995; Stark et al., 1995) and subunits (Ban et al., 1999; Clemons et al., 1999; Gabashvili et al., 1999b), as well those complexed with functional ligands (tRNA [Agrawal et al., 1996; Malhotra et al., 1998; Cate et al., 1999], IF-3 [McCutcheon et al., 1999], EF-G [Agrawal et al., 1998, 1999; Stark et al., 2000], and EF-Tu-GTP-aatRNA [Stark et al., 1997b]) has recently been made by cryo-electron microscopic and X-ray crystallographic methods, the dynamics of elongation and the structural components of ribosomal peptidyl transferase are not yet characterized in molecular terms. In order to get insight into possible structural changes in tRNA and rRNA in well-defined functional states of the

ribosomal elongation cycle, we applied Pb2⫹ cleavage of the RNA backbone. The site specificity of Pb2⫹ cleavage dependent on a correct tertiary fold allows it to serve as a method to monitor fine structural differences in RNA molecules (Gornicki et al., 1989; Behlen et al., 1990; Michalowski et al., 1996; Dorner and Barta, 1999). Incubation of tRNAs with Pb2⫹ revealed that unbound deacylated tRNAPhe and AcPhe-tRNAPhe have indistinguishable cleavage patterns. This indicates that the structures of the tRNAs are similar in solution, at least in the elbow region, since all observed cuts clustered there in the tertiary structure (Figure 3B). The strongest cuts were mapped to positions D16 and G18 in the D loop, which is in agreement with previously published data (Marciniec et al., 1989). When bound to the ribosome, the Pb2⫹ cleavage patterns of deacylated tRNAPhe and AcPhe-tRNAPhe were different. In general, cleavage rates at all positions were diminished, but to differing degrees. Similar effects were observed in a recent study, where tRNA Pb2⫹ cleavage was inhibited due to binding to the nuclear tRNA export receptor of Xenopus oocytes (Arts et al., 1998). The authors concluded that this may reflect a structural change in either the D or the T loop of tRNA rather than a direct shielding against cleavage. In the case of ribosome-bound AcPhe-tRNAPhe, the most

Structural Dynamics of Translating Ribosomes 165

Figure 5. Primer Extension Analysis Pb2⫹-Cleaved Ribosomal Complexes

of

Mapping of Pb2⫹ cleavage sites in domain V (A) and domain VI (B) in 23S rRNA. Vacant ribosomes (lane 2) as well as Pi complexes (lane 3), PRE complexes (lane 4), and POST complexes (lane 5) were cleaved with 10 mM Pb2⫹ for 5 min at 25⬚C. Lane 1 shows the reverse transcription of uncleaved rRNA. A and C (lanes 6 and 7) denote dideoxy sequencing lanes. Asterisks (*) indicate sites of enhanced cleavage rate in the POST complex.

pronounced cleavage inhibition was seen at D16 (Figures 2B and 3). Interestingly, the cleavage pattern does not change significantly when AcPhe-tRNAPhe was bound at the A site of pretranslocational ribosomes (PRE state) or at the P site after EF-G-catalyzed translocation (POST state). On the other hand, the strongest Pb2⫹ cleavage inhibition of ribosome-bound deacylated tRNAPhe was identified at G18 (Figures 2C and 3). It is noteworthy that also in the case of deacylated tRNAPhe, the cleavage pattern hardly changed upon translocation from the P site (PRE state) to the E site (POST state). The slightly different Pb2⫹ cleavage pattern of AcPhetRNAPhe and deacylated tRNAPhe bound to ribosomes indicates that they adopt different conformations, which, however, do not change during translocation. Recent findings showed that the iodine cleavage patterns of phosphorothioated tRNA at the A and P sites, although different from each other, also hardly changed during translocation to the P and E sites (Dabrowski et al., 1998). Based on these findings, a movable ribosomal domain has been postulated to bind both tRNAs and carry them together from the A-P to the P-E sites, respectively (termed ␣⫺⑀ model). Our observations that tRNA Pb2⫹ cleavages are unaffected support this hypothesis. The agreement seems to be particularly significant, since the applied experimental methods are different. Iodine cleavage is suitable to probe for tRNA– ribosome contacts and gives evidence about the environment of bound tRNAs. Pb2⫹ cleavage, on the other

hand, probes from the interior of the tRNA rather than from the surface, therefore monitoring the tertiary fold of the molecule and not necessarily shedding light on its ribosomal environment. Since all characterized Pb2⫹ sites are clustered around the elbow region of the tRNA, we cannot extrapolate our interpretation to other parts of the molecule, such as the acceptor stem and the anticodon stem loop. As mentioned in the Introduction, the dynamic functional oscillation between the PRE and POST state has led to the hypothesis that the ribosome is a macromolecular machine (reviewed in Wilson and Noller, 1998). It is implicit in this metaphor, however, that the ribosome itself consists of dynamic and mobile features. This assumption, however, has not yet been demonstrated conclusively, though it is integral to all current models of the ribosomal elongation cycle. In the ␣⫺⑀ model, a movable ribosomal domain is predicted that carries the two tRNAs from the PRE to the POST state (Dabrowski et al., 1998). Also in the hybrid state model, in which the movement of the tRNA on the small subunit is uncoupled from the tRNA movement on the large subunit, a rearrangement of ribosomal features is to be expected during translocation (Moazed and Noller, 1989a; Green et al., 1998). Cryo-electron microscopy investigations on PRE and POST state ribosomes have not yet reached the necessary resolution to unequivocally answer this question on a molecular level (Stark et al., 1997a). However, in recent publications, conformational changes

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Figure 6. Summary of Pb2⫹ Cleavage Sites in the 3⬘ 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 cleavage rate at A1966 in the Pi complex and at C2347, C2626, U2694, and G2695 in the POST complex compared to vacant ribosomes are indicated by red arrows (see also insert).

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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. After the first translocation step, EF-G was removed by gel filtration. (B) Tracking the Pb2⫹ 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) Pb2⫹ cleavage efficiency at C2347 was quantified in the different ribosomal states whereby the relative cleavage rate in the starting PRE complex was taken as 1.00. Values shown here are averages from four independent PRE II and POST II complexes. Cleavage rates in PRE II/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 3 pmol in the cases of PRE II or POST II complexes, respectively. The specific activities were 1073 dpm/pmol for Ac[14C]Phe-tRNAPhe and 2000 dpm/pmol for [3H]Val-tRNAVal. The specificity of the investigated complexes was calculated from the puromycin reactivity of peptidyl-tRNA before and after EF-G-promoted translocation. Translocation efficiency was calculated as specified in the Experimental Procedures. nd, not determined.

mainly in the L7/L12 stalk and in the head of the small subunit have been proposed upon EF-G-driven translocation (Agrawal et al., 1999; Stark et al., 2000). We therefore set out to address this issue by applying Pb2⫹ cleavage of rRNA in defined functional ribosomal complexes (Figure 1). Pi, PRE, and POST complexes have been constructed, and subsequent to Pb2⫹ cleavage, the patterns on the rRNAs were identified by primer extension and compared to that of vacant ribosomes. All 11 cleavages on 16S rRNA showed indistinguishable

behavior in all of the investigated states (Figure 4 and Table 1). Since Pb2⫹ cleavage was shown to be a very sensitive tool to probe changes in tertiary structure, we conclude that the conformation of 16S rRNA near the sites of strand scission is very similar before and after translocation. This might be at least the case for those regions of 16S rRNA where Pb2⫹ cleavage occurred, namely the 5⬘ domain and 3⬘ major domain. The fact that no cleavages were observed in the central domain and the 3⬘ minor domain (including the decoding center)

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of 16S rRNA limits our interpretation of the possible structural dynamics of the 30S subunit during translocation. Since no Pb2⫹ cleavages could be mapped to 5S rRNA, we have no information about possible structural changes during translocation. However, in a recent probing study, an unaltered contact pattern of the phosphate residues of 5S rRNA in PRE and POST states has been reported (Shpanchenko et al., 1998). 23S rRNA was shown to be frequently targeted by site-specific metal ion hydrolysis (Polacek and Barta, 1998), which is mirrored by the identification of Pb2⫹ cleavage sites dispersed over the whole molecule. This enabled us to investigate the effects of translocation on rRNA structure in all domains of 23S rRNA. Interestingly, the Pb2⫹ cleavage rate at position C2347 in domain V increases significantly, on average about 3-fold, in the POST state (Figure 5A and Table 1). This site was also targeted by Pb2⫹ in human ribosomes, implying that this nucleotide resides in a structurally highly conserved part of domain V of 23S rRNA (Polacek and Barta, 1998). Recent cross-linking studies have placed C2347 of domain V and adjacent regions in close proximity to 5S rRNA (Osswald and Brimacombe, 1999). An E site– specific tRNA footprint on 23S rRNA (C2394) (Moazed and Noller, 1989b) also mapped close to the affected Pb2⫹ cleavage site of domain V in the POST state. However, control experiments revealed that filling the E site directly with tRNAPhe or binding EF-G alone did not show this enhanced cleavage pattern. Also, the Pi state with an AcPhe-tRNAPhe at the P site and a free A site did not show the enhanced cleavage at C2347, thus rendering it unlikely that the reduced cleavage rate seen in the PRE state is due to shielding the Pb2⫹ access by A site–bound tRNA. Therefore, we conclude that the translocation step accounts for the observed alterations in the Pb2⫹ cleavage rates and hence indicates structural differences in 23S rRNA between PRE and POST complexes. Additionally, three new cleavages occurred in the POST complex at C2626, G2694, and U2695 in domain VI (Figure 5B). These sites flank the evolutionarily highly constrained ␣-sarcin loop (C2646-G2674), which is a major component of the elongation factor binding site. The identified Pb2⫹ cleavage enhancements in the POST state could be due to a conformational change that either leads to a higher affinity metal ion binding pocket for the attacking Pb2⫹, or to a more suitable geometry and positioning of the Pb(OH)⫹ relative to the 2⬘-hydroxyl of the ribose and the targeted phosphodiester bond. Other sites of strand scission in domain V and domain VI are unaffected by translocation, implying that the proposed conformational rearrangement on 23S rRNA in the POST state is localized. Is this indeed a moving element of 23S rRNA that has the potential of adopting different conformations before and after translocation? And if so, does this 23S rRNA element flip back when the ribosome reenters the PRE state on its way through the elongation cycle? To test this hypothesis, the rRNA fold was probed with Pb2⫹ during a complete elongation cycle (PRE→POST→PRE II→POST II). To this end, ribosomal complexes with a heteropolymeric mRNA that contains three unique codons for Met, Phe, and Val were constructed. After construction of the first POST complex, the POST-specific

Pb2⫹ cleavage enhancement at C2347 in domain V of 23S rRNA was seen again (Figures 7B and 7C). As soon as the A site was refilled with ternary complex containing Val-tRNAVal, the ribosome flipped back into the PRE state (PRE II complex in Figure 7A) with a simultaneous loss of the cleavage enhancement at C2347. Since in this state peptide bond formation has already occurred, one can exclude peptidyl transfer as the cause for the observed structural changes in 23S rRNA. Forcing this PRE II complex to undergo another round of translocation (POST II state in Figure 7A) led to the reappearance of the POST-specific Pb2⫹ cleavage enhancement at C2347 (Figures 7B and 7C). The observations presented here show that parts of 23S rRNA can adopt two different conformations that are specific for the functional state of the ribosome during the elongation cycle. It appears that the functional oscillation between PRE and POST state ribosomes is mirrored by a recurrent structural rearrangement of the large subunit rRNA. It is of note that the Pb2⫹ cleavages in or near the central multibranched loop of domain V were shown to be very similar in all tested functional complexes (Table 1). This indicates that the structure of this domain, which is known to be an important part of the peptidyl transferase center, is not significantly affected in any of the functional states. This is somehow unexpected, since tRNA footprinting (Moazed and Noller, 1989b) and photoaffinity labeling studies (Steiner et al., 1988) indicate interaction of some of the highly conserved residues of this loop with the aminoacyl end of tRNA. Focusing our structural investigations with Pb2⫹ simultaneously on tRNA and rRNA in defined functional states of the ribosomal elongation cycle led us to conclude that: (1) ribosome-bound tRNA adopts a different conformation compared to tRNA free in solution; (2) the tertiary fold of bound AcPhe-tRNAPhe and tRNAPhe is distinguishable; (3) translocation does not change tRNA structure; (4) most of the Pb2⫹-sensitive regions of 16S and 23S rRNA are unaffected by translocation; (5) some parts of domain V and VI of 23S rRNA exist in different structural conformations in the PRE and the POST state; and (6) this flexible 23S rRNA element can periodically shift between the two different states during elongation. The latter finding provides evidence for a flexible and possibly mobile 23S rRNA element that is involved in translocation and hints at a pulsing structural feature that is beating in the catalytic heart of translating ribosomes. Our findings add motion and structural flexibility to the RNA components of the hitherto rather rigid protein-synthesizing “ribosome machine”. Experimental Procedures Preparation of Reassociated Ribosomes Ribosomal subunits from 400 g of frozen E. coli Can 20 cells were prepared as described (Bommer et al., 1997). 4460 A260 units of 30S and 7550 A260 units of 50S were reassociated in reassociation buffer (20 mM HEPES/KOH [pH 7.6] [0⬚C], 20 mM MgCl2, 30 mM KCl, and 4 mM ␤-mercaptoethanol) for 60 min at 40⬚C in a volume of 150 ml. Portions of 4000 A260 units were subjected to a zonal centrifugation at 20,000 rpm (Beckman Ti 15 rotor) for 16 hr at 4⬚C on a 6%–38% (w/v) sucrose gradient made up in reassociation buffer. Fractions of the 70S peak were pooled and ribosomes pelleted by ultracentrifugation at 50,000 ⫻ g for 24 hr at 4⬚C. Ribosome pellets were resuspended and pooled in reassociation buffer and allowed to

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reassociate again for 20 min at 40⬚C to undo possible partial dissociation that can occur during the zonal run. Ribosomes were then dialyzed against buffer containing 20 mM HEPES/KOH (pH 7.6) (0⬚C), 6 mM MgCl2, 30 mM KCl, and 4 mM ␤-mercaptoethanol, divided into small aliquots, quick frozen, and stored at –80⬚C. tRNAs E. coli tRNAPhe, [32P]-5⬘-end-labeled tRNAPhe, or tRNAVal was charged, N-acetylated, and purified by reverse-phase HPLC as described (Rheinberger et al., 1988). tRNA/Ribosome Complexes for tRNA Cleavage Binding buffer conditions in all steps were constant 20 mM HEPES/ KOH (pH 7.6) (0⬚C), 6 mM MgCl2, 150 mM NH4Cl, 2 mM spermidine, 0.05 mM spermine, and 4 mM ␤-mercaptoethanol. PRE complexes were constructed by incubating 175 pmol of 70S ribosomes with 350 pmol of deacylated tRNAPhe and 350 ␮g of poly(U) in 87.5 ␮l of binding buffer for 15 min at 37⬚C. In the second step, the A site was filled by adding 140 pmol of double-labeled Ac[14C]Phe-[32P]tRNAPhe (14C:996.6 dpm/pmol; 32P: 9100 dpm/pmol) and incubated at 37⬚C for 30 min in a total volume of 175 ␮l. Nonbound tRNA was removed by gel filtration over a Sephacryl-S300 cDNA spun column (Pharmacia). To establish the POST state, aliquots of the elute were incubated with EF-G (EF-G:70S ⫽ 0.1, 115 ␮M GTP) for 10 min at 37⬚C. The puromycin reaction was performed overnight at 0⬚C in the presence of 0.8 mM puromycin. The reaction was stopped by addition of 1 vol 0.3 M sodium acetate saturated with MgSO4 (pH 5.5), followed by extraction with ethyl acetate. Thirty percent of ribosomes bound Ac[14C]Phe-[32P]tRNAPhe as measured by membrane filtration, and the site specificity of bound Ac[14C]Phe[32P]tRNAPhe was better than 85% according to the puromycin reaction. To probe deacylated tRNAPhe structure at the P site in PRE and at the E site in POST complexes, 135 pmol of [32P]-5⬘-labeled tRNA (6800 dpm/pmol) was incubated in the first step with 270 pmol of ribosomes and 450 ␮g of poly(U) in a total volume of 112.5 ␮l. In the second step, 324 pmol of Ac[14C]Phe-tRNA (1008 dpm/pmol) was added (total volume 225 ␮l). Ribosomes (25%–30%) bound deacylated [32P]tRNAPhe, and 40%–70% carried Ac[14C]Phe-tRNAPhe. Since under the applied conditions the P site is not completely blocked with deacylated tRNA, AcPhe-tRNAPhe also binds to the P site, which results in a mixed population of Pi and PRE complexes. However, an increase in Ac[14C]-Phe-puromycin counts of 31%–60% after EF-G addition indicates quantitative translocation of PRE state ribosomes. Under the near in vivo conditions applied, deacylated tRNAPhe remains stongly bound to the E site after translocation as indicated by membrane filtration (32P counts: 25,082 dpm before, and 28,042 dpm after translocation). Pi complexes were constructed by incubating 20–40 pmol of double-labeled Ac[14C]Phe-[32P]tRNAPhe with 100 pmol of 70S ribosomes and 250 ␮g of poly(U) for 15 min at 37⬚C in 62.5 ␮l of binding buffer. Input AcPhe-tRNAPhe (90%–100%) was bound and 73%–80% of it was puromycin reactive, reflecting the efficiency of the puromycin reaction under the applied conditions (overnight incubation at 0⬚C).

90% and was calculated as follows: % puromycin-reactive peptidyltRNA after EF-G addition/puromycin efficiency (ⵑ80%) ⫻ 100. To investigate rRNA structure during a complete elongation cycle, 300 pmol of ribosomes was programmed with 2400 pmol of MetPhe-Val-mRNA. This 49-nucleotide-long heteropolymeric mRNA carries the three unique codons AUG-UUC-GUU in the middle and was prepared as described for the MF-mRNA (Triana-Alonso et al., 1995). In the first step, the P site was blocked by incubating with 750 pmol of deacylated tRNAfMet in 312.6 ␮l of binding buffer for 15 min at 37⬚C. To construct PRE complexes, the A site was filled by adding 540 pmol of Ac[14C]Phe-tRNAPhe (1073 dpm/pmol), and the incubation continued for 30 min at 37⬚C in a total volume of 625.2 ␮l. POST complexes were established by incubation with EF-G as described above. Ribosomes (75%–82%) bound Ac[14C]PhetRNAPhe, and the homogeneity (ratio of puromycin reactivity before and after EF-G addition) of the complexes was 91%. After formation of the POST state ribosomes, EF-G was removed by gel filtration (spun columns) before ternary complex binding. Ternary complex ([3H]Val-tRNAVal•EF-Tu•GTP:70S ⫽ 1.5:1; EF-Tu:aa-tRNA ⫽ 1:8.1) was preformed in binding buffer for 2 min at 37⬚C before incubation with an equal volume of the ribosomal complexes for 2–5 min at 20⬚C in order to refill the A site (PRE II complex). Subsequently, an aliquot of this PRE II complex was incubated with EF-G to form POST II complexes. Membrane filtration reveals that 41%–51% of ribosomes bound [3H]Val-tRNAVal (2000 dpm/pmol), and the homogeneity of PRE II and POST II complexes was from 70%–87% according to the puromycin reaction. Translocation efficiency was better than 78% in the second step (calculated as above). The fact that more than 70% of POST complexes bound [3H]Val-tRNA that subsequently participates quantitatively in dipeptide bond formation indicates that also the preceding translocation (PRE→POST) must have been better than 70%. Indeed, a translocation efficiency of 85% was calculated (% puromycin-reactive tRNA after EF-G/puromycin efficiency (ⵑ30%–40%) ⫻ 100), as the control experiment with this mRNA resulted in a reduced puromycin efficiency of about 30%. Note that puromycin values are kinetic values and that under the conditions employed, the plus EF-G values reflect an almost quantitative translocation (Beyer et al., 1994). Pb2ⴙ Cleavage Procedure Pb2⫹ cleavage was initiated by adding freshly prepared Pb(OAc)2 to the desired ribosomal complex to a final concentration of 2 mM (for tRNA cleavage) or 10 mM (for rRNA cleavage). Cleavage was performed at 25⬚C for 15 min (tRNA) or 5 min (rRNA), respectively. The reaction was stopped by the addition of excess EDTA with respect to divalent metal ions. Cleaved tRNAs were purified by phenol extraction, precipitated with ethanol/0.3 M sodium acetate (pH 5.5), and resuspended in loading buffer, and 20,000–30,000 dpm was applied to a 13% denaturing polyacrylamide gel. Cleaved rRNAs were purified as described (Winter et al., 1997), and 0.2–0.4 pmol of rRNA was used as template for reverse transcription primed by DNA oligos (Winter et al., 1997), which was performed as described (Polacek and Barta, 1998). Gels were scanned and quantified using a Molecular Dynamics PhosphorImager. Acknowledgments

tRNA/Ribosome Complexes for rRNA Cleavage Pi complexes were prepared by incubating 45 pmol of Ac[14C]PhetRNAPhe (1008 dpm/pmol) with 30 pmol of poly(U)-programmed (150 ␮g) ribosomes for 15 min at 37⬚C in a volume of 37.5 ␮l. Binding controls revealed that 80%–100% of ribosomes bound AcPhetRNAPhe whereas 80% reacted with puromycin under the applied conditions indicating P site location. The PRE state was established by first blocking the P site of 60 pmol of 70S ribosomes with 72 pmol of deacylated tRNAPhe in 75 ␮l of binding buffer in the presence of 300 ␮g of poly(U) for 15 min at 37⬚C. Subsequently, 138 pmol of Ac[14C]Phe-tRNAPhe was added, and the incubation continued at 37⬚C for 30 min in a reaction volume of 150 ␮l. For translocation, an aliquot of the PRE complex was incubated with EF-G (EF-G:70S ⫽ 0.1, 115 ␮M GTP) for 10 min at 37⬚C. Ribosomes (60%–88%) bound Ac[14C]Phe-tRNAPhe, and the homogeneity of PRE and POST complexes was from 67%–75% as judged by the puromycin reaction. Translocation efficiency was around

We are grateful to Gregor Blaha for the help of preparing reassociated ribosomes and to O¨zlem Tastan for the Met-Phe-Val-mRNA clone and for help with tRNA charging. Our thanks are extended to Silke Dorner, Uwe von Ahsen, Sean Connell, and Uli Stelzl for critical review of the manuscript and to Viter Marquez and Uli Stelzl for stimulating discussions. This work was supported by grant NI 176/ 11-1 from the Deutsche Forschungsgemeinschaft to K. H. N. and by grant P13651-GEN from the Austrian Science Foundation (FWF) to A. B. Received December 27, 1999; revised May 16, 2000. References Agrawal, R.K., Penczek, P., Grassucci, R.A., Li, Y., Leith, A., Nierhaus, K.H., and Frank, J. (1996). Direct visualization of A-, P-, and

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