Structural Studies of the tRNA Domain of tmRNA

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doi:10.1006/jmbi.2001.4632 available online at on

J. Mol. Biol. (2001) 309, 727±735

Structural Studies of the tRNA Domain of tmRNA Scott M. Stagg1, Ashley A. Frazer-Abel2, Paul J. Hagerman3 and Stephen C. Harvey1* 1

Department of Biochemistry and Molecular Genetics University of Alabama at Birmingham, Birmingham AL 35294, USA 2 Cancer Causation and Prevention, AMC Cancer Research Center, 1600 Pierce St., Denver, CO 80214, USA 3

Department of Biological Chemistry, University of California, Davis, School of Medicine, One Shields Ave. Davis, CA 95616, USA

tmRNA is a small, stable prokaryotic RNA. It rescues ribosomes that have become stalled during the translation of mRNA fragments lacking stop codons, or during periods of tRNA scarcity. It derives its name from the presence of two separate domains, one that functions as a tRNA, and another that serves as an mRNA. We have carried out modeling and transient electric birefringence studies to determine the angle between the acceptor stem and anticodon stem of the tRNA domain of Eschericia coli tmRNA. The results of the modeling studies yielded an interstem angle of 110  , in agreement with the lower end of the range of angles (111  137  ) determined experimentally for various solution conditions. The range of experimental angles is greater than the angles observed for any of the tRNA crystal structures, in line with the presence of a shortened D stem. The secondary structure of the tRNA domain is conserved for all known tmRNA sequences, so we propose that the angle is also conserved. These results also suggest that the region of tmRNA between P2a and P2b may interact with the decoding site of the ribosome. # 2001 Academic Press

*Corresponding author

Keywords: tmRNA; ssrA RNA; transient electric birefringence; RNA structure; molecular modeling

Introduction When a ribosome translates an mRNA that lacks a stop codon, or during periods of tRNA scarcity, it becomes stalled, unable to release either the mRNA or the nascent peptide. Prokaryotes have evolved a molecule, tmRNA, that functions to recycle these ribosomes (Karzai et al., 2000). tmRNA has a tRNA-like domain that is aminoacylated with alanine before the molecule recognizes the stalled ribosome and enters the empty A-site. Peptidyl transfer attaches the growing peptide chain to tmRNA, which then moves to the P-site by translocation. The molecule also has a short mRNA domain that then enters the A-site and is translated as a normal message. The mRNA domain ends in a stop codon, thus allowing the ribosome to release the nascent peptide and mRNA, freeing the ribosome for recycling. Interestingly, the mRNA domain encodes an amino acid sequence recognized by proteases, so the resulting protein contains a C-terminal sequence that targets Abbreviations used: TEB, transient electric birefringence. E-mail address of the corresponding author: [email protected] 0022-2836/01/030727±9 $35.00/0

it for degradation. This prevents accumulation of incomplete translation products. To perform these complicated functions, tmRNA must make speci®c and unique interactions with the ribosome. During normal translation, the selection of the correct tRNA depends on Watson-Crick base-pairing between the tRNA and the mRNA at the ribosomal decoding site. With a damaged message, this mechanism cannot be used for selection of tmRNA during its initial binding to the ribosome. After binding, tmRNA must promote transpeptidation, and then translocation. Since it is much larger than tRNA (363 nucleotides for E. coli tmRNA, versus 76 for a typical tRNA), it must use a novel set of interactions to facilitate these transitions. Finally, tmRNA must move its mRNA domain into the proper position in the decoding site, and its tRNA domain must be ejected from the ribosomal P-site, so that the normal translation mechanisms can take over to ®nish synthesis of the protein's carboxy terminus. All of these steps require a unique set of interactions between tmRNA, the ribosome, and translational cofactors. Structural studies on tmRNA, in isolation and later in situ, are needed to elucidate structure-function relationships in this important molecule. No data have yet been reported for the threedimensional structure of tmRNA, but the second# 2001 Academic Press

728 ary structure of E. coli tmRNA has been well established through phylogenetic analyses (Williams, 1999; Williams & Bartel, 1996; Zwieb et al., 1999) and chemical probing (Felden et al., 1997; Hickerson et al., 1998). These reveal a highly folded structure of 363 nucleotides with a tRNA domain, an mRNA domain, and four conserved pseudoknots (Figure 1). The secondary structure of the tRNA domain is similar to that of canonical tRNAs, with a normal acceptor stem, hairpin elements that resemble the D and T stems, and an extended anticodon stem. There is nothing that resembles an anticodon loop, although there are two interesting internal loops in the extended anticodon stem (Figure 1). tmRNA also has modi®ed bases in positions analogous to those of canonical tRNAs (Felden et al., 1998). Functionally, the tRNA domain resembles tRNA, since it has a 30 CCA terminus that is aminoacylated (Komine et al., 1994), and since it is known to interact with elongation factor Tu in vitro (Rudinger-Thirion et al., 1999). The only major difference between the tRNA domain and normal tRNAs is the shortened D stem, with two base-pairs rather than the canonical four base-pairs. tRNAs with shortened D-stems are common in mitochondria, and these have unusual tertiary structures. These truncated tRNAs have a greater angle between their acceptor and anticodon arms than do tRNAs with normal D stems. It was ®rst argued on theoretical grounds that the interarm angle would have to be increased in order to maintain the distance from the end of the acceptor stem to the anticodon loop, since the ``primary axis

Structural Studies of the tRNA Domain of tmRNA

length'' is necessary for the tRNAs to interact simultaneously with the ribosomal transpeptidation and decoding sites (Steinberg & Cedergren, 1994; Steinberg et al., 1994). This proposal was later con®rmed experimentally (Frazer-Abel & Hagerman, 1999), and it provided the basis for a broader set of rules about structural compensation in tRNAs with unusual secondary structures (Steinberg et al., 1997). If the angle between the acceptor stem and anticodon stem were known for the tRNA domain of tmRNA, one might use the conserved primary axis length to estimate the position on the extended tmRNA anticodon stem that corresponds to the location of the anticodon, thereby identifying the region of tmRNA that is likely to interact with the ribosomal decoding site. Angles between double helices in RNAs can be quanti®ed using the method of transient electric birefringence (TEB) (Friederich et al., 1995; Vacano & Hagerman, 1997; Zacharias & Hagerman, 1995). In the TEB experiment, a transient electric ®eld is used to align the molecules, yielding solution birefringence. When the ®eld is turned off, the birefringence decay re¯ects the free rotational diffusion of the molecules. One compares the rotational diffusion of a test molecule containing a putative bend with that of a fully base-paired control. The angle between the adjacent helices of the bent molecule can be determined from the ratio of the terminal decay times of these molecules. This technique has been used to measure the interarm angle in tRNAPhe, and the result, 89(4)  , compares favorably with the crystallographic value of 82  (Friederich et al., 1995). The angles have been

Figure 1. Secondary structure of E. coli tmRNA featuring the tRNA domain. Helices are in red, residues 8 and 9 are in yellow, the D loop is in blue, residues 333-335 are in orange, and the T loop is in green. The mRNA domain is shown in purple.

Structural Studies of the tRNA Domain of tmRNA

measured for various mitochondrial tRNAs (Frazer-Abel & Hagerman, 1999; Leehey et al., 1995). Here, we report both TEB measurements and molecular modeling studies on tmRNA, with the goal of determining the angle between the tmRNA acceptor arm and the arm corresponding to the stacked D stem/anticodon stem in tRNAs.

Results Construction of RNA molecules with extended helices In order to increase the sensitivity of TEB analysis to the interarm angles of RNA molecules, the two arms are normally extended. For the tRNA domain of tmRNA, this was accomplished by the method of Friedrich et al. (1995). DNA encoding the sequence for the 50 -half of the molecule (nucleotides 1-27) was cloned into plasmid pGJ122A, while DNA encoding the sequence for the 30 -half (nucleotides 326-359) was cloned into plasmid pGJ122B. The sequences ¯anking the cloning site of pGJ122A are complementary to the sequences ¯anking the cloning site of pGJ122B. When RNAs are transcribed from these plasmids, they can be annealed together to form a bimolecular complex encompassing the tRNA domain with both the

729 acceptor and anticodon stems extended by 68 basepairs (Figure 2). A full duplex control must be constructed with the same contour length as the RNA molecule to be studied. The accuracy of the TEB measurement is sensitive to the length of the linear control, because the interstem angle is determined from the ratio of the rotational diffusion times of the bent and linear RNAs, which vary roughly as the cube of the length (VanHolde, 1971). For the tRNA domain of tmRNA, the contour length is the sum of the distances from the end of the acceptor and anticodon stems to the projected vertex in the tRNA elbow. From modeling studies, the appropriate length was found to be 37 bp (see Materials and Methods). A DNA oligonucleotide of this length with the appropriate restriction sites at the ends was cloned into plasmids pGJ122A and pGJ122B, and transcription produced a control RNA of 173 bp (37 bp plus two arms each containing 68 bp). Comparison of gel electrophoretic mobilities in response to Mg2‡ Some general features of RNA structure can be determined by relative electrophoretic mobilities in native gels. The extended linear control and tRNA domain RNAs were run on 8 % polyacrylamide

Figure 2. Schematic of the method for synthesizing the RNA analogous to the tRNA domain of tmRNA but with extended helices. DNA oligonucleotides were cloned into the HindIII site of plasmids pGJ122A and pGJ122B. The regions between the T7 promoter and SmaI site in these plasmids are complementary and antiparallel to each other so that when RNA is transcribed from these plasmids the ends are complementary and will anneal. The resultant RNA is extended by 68 bp on either end.

730 gels in varying concentrations of MgCl2 (Figure 3). The tmRNA construct has a lower relative mobility than the linear control, indicating that the tRNA domain contains a bend. Furthermore, the relative mobility of the tmRNA construct decreases with Mg2‡ concentrations greater than 1 mM, indicating that the tRNA-like structure is more bent in the presence of Mg2‡. Transient electric birefringence measurements of RNAs with extended helices General features of TEB have been described (Hagerman, 2000). In brief, the RNA to be analyzed is placed in a cell between two crossed polarizers. The solution is subjected to a brief (1 ms) electric ®eld pulse that partially orients the molecules and creates a birefringence signal. When the ®eld is turned off, rotational Brownian motion causes randomization of RNA orientations, leading to birefringence decay. With extended RNA molecules like those used in the current investigation, the rotational diffusion coef®cient depends strongly on the angle between the two arms of the molecule. Decay pro®les are recorded for two molecules, one being the test RNA (possessing a central non-helix element), and the other being a linear control (a full duplex RNA of the same total contour length as the test RNA). The ratio of the

Structural Studies of the tRNA Domain of tmRNA

terminal decay times for the two molecules yields the apparent angle between the helix arms of the test RNA (Vacano & Hagerman, 1997). In the current work, TEB experiments were performed in a variety of buffers and temperatures. In the absence of Mg2‡, measurements were taken in TEB and 2  TEB both at 4  C and 20  C. For all of these conditions, the angle measured was approximately 128  (Figure 4(a) and Table 1). For TEB with 1 mM MgCl2 at 4  C, the angle measured was 111  , while at 20  C the angle was 137  (Figure 4(b) and Table 1). TEB analysis is very sensitive to RNA aggregation. In some of the gel-mobility assays with 2 mM MgCl2 a minor band was observed in the tRNA domain lane at approximately twice the molecular mass of the RNA, suggesting association (data not shown). To avoid aggregation, TEB measurements were not taken with MgCl2 concentrations above 1 mM. Modeling of the tRNA domain To date, there are 11 published crystal structures of tRNAs, some free and some complexed to proteins. These tRNAs have similar tertiary interactions, including several layers of stacked bases. The tRNA domain of tmRNA has a secondary structure that closely resembles that of canonical tRNAs, except for some differences in the D stem and loop, so we based the tertiary structure of the model on known tRNA tertiary structures, primarily tRNAPhe (Hingerty et al., 1978) and tRNAAsp (Westhof et al., 1988) (Figure 5). Models were developed using both of these as prototype structures, with the subtle differences between them providing different options for solving stereochemical problems. tmRNA residues were initially placed by superimposing them on the analogous residues of the tRNA crystal structures. Since the core of all

Table 1. Angles observed for different buffer conditions in the TEB analysis of the tRNA domain of tmRMA Conditions TEB, 4  C TEB, 20  C 2  TEB, 4  C 2  TEB, 20  C TEB, 1 mM MgCl2, 4  C TEB, 1 mM MgCl2, 20  C

Figure 3. Gel electrophoresis of RNAs with extended helices. The 8 % polyacrylamide gels were run in TBE with (a) no MgCl2 and (b) 4 mM MgCl2. Lanes contain 1, Gel Marker I2 (Research Genetics); 2, extended tRNA domain RNA; 3, 173 bp linear control. (c) Relative electrophoretic mobility of the tRNA domain RNA to the linear control RNA at the indicated MgCl2 concentrations.

Angle (deg.)

Std. err.a

127.8 127.5 120.5 130.0 111.4 136.7

2.0 3.9 3.1 4.1 2.0 4.0

a Standard errors. The variance arising from the uncertainty in the length of the linear control (s12) was estimated by applying an interpolation function derived from a series of experiments on linear controls ranging in size from 170 to 192 bp to the data for each experimental condition, assuming that 2 bp corresponds to one standard deviation in length. The error arising from statistical scatter in measured decay times was calculated directly from the data, expressed as the variance of the mean (s22). The total standard error is the square-root of the sum of the variances, SE ˆ (s12 ‡ s22)0.5. Note that s12 is the dominant term.

Structural Studies of the tRNA Domain of tmRNA

Figure 4. Birefringence decay curves for tRNA domain and linear control RNAs. Graphs are shown for TEB in the (a) absence and (b) presence of 1 mM MgCl2. Measurements were made at 4  C (blue) and 20  C (red). Curves for the linear control are shown as diamonds while those for the tRNA domain are shown as circles.

known tRNA structures is comprised of several layers of stacked and coplanar bases, this assured the proper stacking and planarity for analogous residues. To assure that all the sequential 50 -phosphate to 30 -hydroxyl connections could be made, small adjustments were made to base positions and to backbone and glycosidic torsion angles. While adjusting base positions, efforts were made to maximize hydrogen bonding. The elements in the tRNA domain most similar in secondary structure to those of canonical tRNAs were modeled ®rst. These included the acceptor stem, the T stem and loop, and resi-

731 dues analogous to 8-9 and 45-48 of canonical tRNAs. The sequences of the acceptor/T stem and loop are almost identical with those of tRNAPhe, so these elements were modeled by superimposing the tmRNA sequences onto the tRNAPhe crystal structure. Residues A8, U9, G333, A334, and C335 were modeled by superimposing them on tRNA residues 8, 9, 45, 46, and 48. (This was done twice, once using a tRNAPhe template, and once using tRNAAsp, to explore slightly different orientations.) These bases are important, because, in tRNAs, they form a core of stacked bases connecting the coaxially stacked T and acceptor stems to the coaxially stacked D and anticodon stems. The positions of some of these bases had to be slightly adjusted in order to make the sequential 50 to 30 connections, but overall they remain stacked in positions similar to those of the tRNA crystal structures. The next elements to be modeled were the D loop and stem, followed by the anticodon stem. G13 and G14 are highly conserved in tmRNAs (Williams, 1999), suggesting that they interact with the T loop in a manner similar to that observed for G18 and G19 in normal tRNAs (Ushida et al., 1994). It is important to note that G13 and G14 are on the 50 -side of the D loop in the tmRNA secondary structure map, whereas the analogous G18 and G19 are on the 30 -side in canonical tRNAs. Therefore, residues G13 and G14 were superimposed on residues G18 and G19 of tRNAPhe. This placement is supported by the fact that these residues are protected in chemical probing experiments (Hickerson et al., 1998). The remaining residues of the D loop and stem were then placed by positioning them so that all of the sequential 50 to 30 connections could be made. The D stem is modeled as two base-pairs based on probing data (Felden et al., 1997; Hickerson et al., 1998). Sequence analysis neither con®rms nor disproves these base-pairs, because G19 and A20 are 100 % conserved and the bases at position 10 and 11 vary but can pair with G19 and A20 most of the time. There is only one base, U12, to bridge the gap between the D stem and G13. Although this is a large gap, U12 can bridge it adequately. In building the D loop and stem, small adjustments had to be made to the positions of previously placed bases in order to make the connections, but stacking and coplanarity were conserved in every case. Finally, the structure was subjected to gentle energy minimization to correct any unfavorable bond lengths and angles, and to eliminate unacceptable steric clashes. Figure 5 compares the ®nal tRNA domain model with the structure of tRNAAsp. The similarity in the folding of the two structures is clear. The angle between the acceptor and anticodon stems in the model is 110  . This angle is in good agreement with the TEB data for the tRNA domain at 4  C in the presence of Mg2‡, adding support to the model.


Structural Studies of the tRNA Domain of tmRNA

Figure 5. Stereo diagrams of the (a) tRNA domain model and (b) tRNAAsp. The tRNAs are shown in similar orientations to show the similarities in stacking and other tertiary interactions. The coloring scheme is the same as that used in Figure 1.

Possible interaction of the tRNA domain with the ribosome The model offers a direct way of predicting what region of the tmRNA might interact with the ribosomal decoding site, under the assumption that this region must lie at the same distance from the end of the acceptor stem as does the center of the anticodon loop in tRNA. This assumption is based on the conservation of that distance (the primary axis) in tRNAs with shortened D stems and other unusual secondary structure features (Steinberg & Cedergren, 1994; Steinberg et al., 1997). We de®ne the primary axis as the distance from the phosphate group of residue 1 to the phosphate group of residue 35 (the second residue in the anticodon). In the crystal structures of tRNAPhe (Hingerty et al., (Basavappa, 1991), and tRNAAsp 1978), tRNAMet f (Westhof et al., 1988), the primary axes measure Ê , 68 A Ê , and 66 A Ê , respectively. These dis65 A tances closely match those of tRNAs modeled Ê Thermus thermophilus ribosome into the 7.8 A Ê and crystal structure (Cate et al., 1999) (65 A Ê for the A and P-site tRNAs, respectively), 68 A Ê cryo-electron microscopy and into the 11.5 A reconstruction of the E. coli ribosome (Gabashvili

Ê for the P-site tRNA). Using et al., 2000) (65 A Ê , as a the average of these distances, 66 A measuring rule, we ®nd that base-pair U27-A326 is at the appropriate distance from residue 1 (Figure 6). This suggests that the region between P2a and P2b of tmRNA may interact with the ribosomal decoding site. The exact nature of that interaction is not known, and it probably changes as the tmRNA moves from the A-site to the P-site, but the presence of two unstructured loops between P2a and P2b (Figure 1) is almost certainly related to the dynamic nature of tmRNA's interactions with the decoding site.

Discussion In the current study, we determined the angle between the acceptor stem and anticodon stem of the tRNA domain of tmRNA through independent experimentation and modeling. Several different angles were observed in the TEB analysis. In the absence of Mg‡2 the observed angles clustered around 127  . In the presence of Mg‡2, two angles were observed, 111  at 4  C, and 137  at 20  C. This temperature-dependence in the presence of Mg‡2 is probably due to loss of Mg‡2 binding at the higher temperature. This is supported by the

Structural Studies of the tRNA Domain of tmRNA


Ê distance from the end Figure 6. Structures of tRNAPhe (left) and the tRNA domain model (right) showing the 66 A of the acceptor stem to the beginning of the decoding site. For the tRNA domain model, this distance falls on the base-pair between U27 and A326, suggesting that the region around these bases interacts with the decoding site.

relative electrophoretic mobility studies, where the relative mobility of the tRNA domain RNA increases slightly at 1 mM Mg‡2 compared to that in the absence of Mg‡2, then decreases with Mg‡2 concentrations greater than 1 mM. This shows that the angle becomes more acute in the presence of greater than 1 mM Mg‡2. Together these data support a conclusion that in the absence of Mg‡2 the tRNA domain has an angle of 127  , but in the presence of greater than 1 mM Mg‡2 the tRNA domain takes on a more folded structure with an angle of 111  . Independently of the TEB data, the tRNA domain of tmRNA was modeled by similarity to the crystal structures of various tRNAs, particularly tRNAPhe and tRNAAsp. The secondary structure of the tRNA domain is very similar to that of canonical tRNAs with differences only in the length of the D stem and the sequential placement of the G18 and G19 analogs in the D loop. Though the tRNA domain was modeled manually, the method used is similar to that of automated homology modeling for proteins. Residues were assigned three-dimensional coordinates by superimposing them on analogous residues in the tRNA cystal structures. The resulting structure maintained the conserved tertiary interactions of the known tRNA structures but had an angle of 110  . The modeling results are in good agreement with the TEB data. The 110  angle calculated in the modeled tRNA domain structure matches very closely the 111  angle seen in the TEB experiment with 1 mM Mg‡2 at 4  C. To make a model where the angle matched the larger observed angles (127  -137  ), several of the tertiary interactions (speci®cally those involving residues 8, 9, and 333-

335) in the tRNA-like core would have to be broken. Thus, a model where the tRNA domain has an angle of 110  is most similar to canonical tRNAs and is the most likely conformation for the tRNA domain at physiological conditions. It is likely that other tmRNAs have a similar angle for their tRNA domains. Sequence analysis of numerous tmRNA sequences (Williams, 1999; Zwieb et al., 1999) has shown that the tRNA domain consists of a normal acceptor stem and T stem and loop, but a shortened or absent D stem. Here, we have found that a shortened D stem in the tRNA domain of tmRNA coincides with a 110  angle between the acceptor stem and the anticodon stem. Since the non-canonical D stem is phylogenetically conserved, tmRNAs from other species probably have similar angles. It is likely that the non-canonical D stem probably has some importance for tmRNA function, perhaps in the way tmRNA interacts with the ribosome. In normal translation, an mRNA codon pairs with a tRNA anticodon at the decoding site of the ribosome, and cognate pairing provides the signal for subsequent steps leading to peptidyl transfer. Since tmRNA has evolved to interact with the ribosome when the mRNA is damaged, the tRNA domain of tmRNA does not have an anticodon. The primary axis length from the end of the acceptor stem to the anticodon can be used to determine the region of the tmRNA that is closest to the decoding site of the ribosome. This distance is Ê in normal tRNAs. In our model for the about 66 A Ê tRNA domain of tmRNA, the region that lies 66 A from the acceptor terminus falls in the extended anticodon stem, between P2a and P2b. This region consists of a series of bulges and non-canonical base-pairs, according to the tmRNA secondary

734 structure (Figure 1). These could form a structure that interacts with the decoding site and mimics the codon/anticodon of a normal mRNA/tRNA complex, signaling the ribosome to proceed with peptidyl transfer and begin synthesizing the carboxy-terminal region of the incomplete protein.

Materials and Methods Preparation of plasmids Construction of the plasmids used for the in vitro transcription of the RNAs used in this study was as described (Friederich et al., 1995). Duplex DNAs containing the sequence of residues 1-25 and 326-359 of the tRNA domain of tmRNA were synthesized by Research Genetics. These were cloned into the HindIII site of plasmids pGJ122A and pGJ122B, respectively. For the linear control, DNA oligonucleotides were synthesized with sequences close to those of the tRNA domain, thus reducing the chance of sequence-dependent differences in the rotational decay time. These were likewise inserted into the HindIII sites of the pGJ122A and pGJ122B. In vitro transcription and annealing of RNA molecules For the in vitro transcription of the RNAs used in this study, reactions were set up in 1 ml volumes with the following reagents: 20 mg of template plasmid, 2.5 mM each NTP, 5 units of pyrophosphatase, 400 units of RNasin, and 100 units of phage T7 RNA polymerase. RNA pairs were annealed by mixing equimolar amounts of each RNA in 100 mM Tris-HCl (pH 6.5), 100 mM NaCl, 10 mM EDTA (pH 8.0), 5 mM EGTA. The solutions were then heated to 95  C and allowed to cool to room temperature. For puri®cation, the annealed RNAs were run out on 6 % (w/v) polyacrylamide gels, and bands were cut out and eluted. Determination of the length of the linear control For determining the length of the linear control, a reduced representation model of the tRNA domain was made with YAMMP (Tan & Harvey, 1993). For this model, normal A-form helix parameters were used as constraints for Watson-Crick base-pairs. Also the acceptor stem was stacked on the T stem, and the D stem was stacked on the anticodon stem. Finally, residues 13 and Ê of residues 51 and 14 were constrained to be within 18 A 52, respectively, which is analogous to the interactions of G13 and G14 with U342 and C343. The structure with these constraints was subjected to 10,000 rounds of steepest descents minimization. From the resulting structure, the distances from the end of the acceptor stem and anticodon stem to the intersection where they meet was Ê . This length measured, giving a total length of 104 A Ê , the rise per base of a standard was divided by 2.8 A A-form helix, to give 37 bp, which is the length of the insert used for the linear control. Gel electrophoresis The 8 % polyacrylamide gels (19:1 (w/w) monomer to bisacrylamide) were run at room temperature with recirculating buffer. TBE running buffer was used with the

Structural Studies of the tRNA Domain of tmRNA speci®ed concentration of MgCl2. Gels were run at 100V for 2.5 hours. The relative mobilities were determined from the ratio of the mobility of the tRNA domain RNA to the linear control. Transient electric birefringence The application of TEB to the study of angles between adjacent helices in RNA has been described (Hagerman, 1996, 2000; Vacano & Hagerman, 1997). The speci®c conditions used in this study are as follows: 1.0 ms pulse width, 1 Hz pulse frequency, and 10 kV/cm orienting ®eld strength. RNAs to be measured were placed in either TEB (5 mM NaPi, 0.125 mM Na2EDTA) or 2  TEB (10 mM NaPi, 0.25 mM Na2EDTA) buffer as speci®ed. Birefringence decay pro®les were collected by taking ®ve sets of averaged 512 pulses for the 20  C, 1 mM MgCl2 data set. For all other cases, ®ve sets of averaged 125 pulses were taken. The interarm angle, y, is given by: y ˆ 180 ÿ f1:46 cosÿ1 …R† ‡ 0:005‰sinÿ1 …1 ÿ R†Š2:3 g where R is the ratio of the terminal decay time of the test RNA to that of the linear control (Frazer-Abel & Hagerman, 1999). Modeling The tRNA domain model was constructed as described in Results using InsightII (Molecular Simulations, Inc) and MANIP (Massire & Westhof, 1998). In order to relieve any unfavorable bond lengths, angles, or steric interactions, the resultant model was subjected to 200 rounds of steepest descents minimization using the cvff force-®eld in InsightII. Coordinates for the model are available at

Acknowledgments We thank Stephen Hajduk and his group for the use of laboratory space and reagents, and for numerous helpful discussions. This work was supported by grants from the NIH (GM53827 to S.C.H. and GM35305 to P.J.H.).

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Edited by I. Tinoco (Received 8 January 2001; received in revised form 16 March 2001; accepted 16 March 2001)

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