METHOD

Kinetics of tRNA folding monitored by aminoacylation HARI BHASKARAN,1 ANNIA RODRIGUEZ-HERNANDEZ,1 and JOHN J. PERONA1,2,3,4 1 Department of Chemistry and Biochemistry, 2Interdepartmental Program in Biomolecular Science and Engineering, University of California, Santa Barbara, California 93106-9510, USA

ABSTRACT We describe a strategy for tracking Mg2+-initiated folding of 32P-labeled tRNA molecules to their native structures based on the capacity for aminoacylation by the cognate aminoacyl-tRNA synthetase enzyme. The approach directly links folding to function, paralleling a common strategy used to study the folding of catalytic RNAs. Incubation of unfolded tRNA with magnesium ions, followed by the addition of aminoacyl-tRNA synthetase and further incubation, yields a rapid burst of aminoacyl-tRNA formation corresponding to the prefolded tRNA fraction. A subsequent slower increase in product formation monitors continued folding in the presence of the enzyme. Further analysis reveals the presence of a parallel fraction of tRNA that folds more rapidly than the majority of the population. The application of the approach to study the influence of post-transcriptional modifications in folding of Escherichia coli tRNA1Gln reveals that the modified bases increase the folding rate but do not affect either the equilibrium between properly folded and misfolded states or the folding pathway. This assay allows the use of 32P-labeled tRNA in integrated studies combining folding, post-transcriptional processing, and aminoacylation reactions. Keywords: RNA folding; aminoacyl-tRNA synthetase; RNA stability; post-transcriptional modification

INTRODUCTION Folding of tRNA molecules to the canonical L-shaped tertiary structure is essential to translation, where specific interactions with aminoacyl-tRNA synthetases (aaRSs), initiation and elongation factors, and ribosomes are required (Agirrezabala and Frank 2009; Demeshkina et al. 2010; Kolitz and Lorsch 2010). Proper tRNA folding is likely to also be important for its noncanonical roles, such as participation in the cell wall, heme, and antibiotic biosynthesis (Banerjee et al. 2010; Francklyn and Minajigi 2010); tumorigenesis (Mei et al. 2010); and viral replication (Kleiman et al. 2010). A wealth of knowledge has been gained from almost five decades of biophysical investigation on the folding properties of tRNAs. Early studies of tRNA folding by thermal melting analysis and temperature-jump kinetics showed that formation of the native structure is highly dependent on temperature and ionic strength (Cole and Crothers 1972; Cole et al. 1972; Yang and Crothers 1972). Relaxation kinetics and nuclear magnetic resonance suggested that heat-denatured, inactive tRNAs possess an altered tertiary

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Present address: Department of Chemistry, Portland State University, Portland, OR 97207, USA; Department of Biochemistry and Molecular Biology, Oregon Health and Sciences University, Portland, OR 97239, USA. 4 Corresponding author. E-mail [email protected]. Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.030080.111.

conformation that is not aminoacylable by aaRS (Yang and Crothers 1972; Crothers et al. 1974; Bina-Stein et al. 1976; Hilbers et al. 1976). Temperature-dependent unfolding occurs through distinct, sequence-dependent ensembles in different tRNA species: The order of melting of the tertiary core and four helical arms of the cloverleaf is not fully conserved (Hilbers et al. 1973; Coutts et al. 1975). Magnesium ions play a key role in tRNA folding. The addition of Mg2+ cations at an elevated temperature converted inactive preparations of yeast tRNA3Leu to an aminoacylable form (Lindahl et al. 1966; Adams et al. 1967), and later work employing oligonucleotide binding revealed that the dihydrouracil (D) arm unravels in the denatured state (Uhlenbeck et al. 1974). In another example, nuclear magnetic resonance and relaxation kinetics were used to demonstrate that the native and denatured forms of Escherichia coli tRNA2Glu, prepared by incubation at a moderate temperature in the presence and absence of Mg2+ ions, respectively, also possess distinct structures in the D-arm and tertiary core (Eisinger and Gross 1975; Bina-Stein et al. 1976). Further experiments using chemical and enzymatic probes confirmed that the denatured and native forms of E. coli tRNA2Glu possess the same conformations in the acceptor and anticodon arms, but differ markedly in the central core region (Madore et al. 1999). Multiple delocalized Mg2+ ions appear to influence tRNA folding; there are few examples of well-defined sites in X-ray structures. A study of Mg2+-induced tRNAPhe folding kinetics

RNA (2012), 18:569–580. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2012 RNA Society.

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using attached fluorophores revealed four conformational transitions, with two Mg2+ ions binding at micromolar affinities and two more at millimolar affinities (Serebrov et al. 2001). These and other studies by single molecule (Sorin et al. 2004) and computational approaches (Ding et al. 2008) suggest that tRNA folding features a dynamic landscape with multiple energy minima, as has been proposed for the folding of larger RNAs (Zhuang et al. 2000; Russell et al. 2002a). Like larger RNAs, tRNAs appear to also fold via parallel pathways with discrete intermediates, some acting as long-lived kinetic traps (Treiber and Williamson 1999; Kent et al. 2000; Brooks and Hampel 2009). tRNAs also serve as substrates for a wide variety of processing and modification enzymes (Phizicky and Hopper 2010): Over 100 distinct modifications of varying complexity are known (Jackman et al. 2003; Grosjean 2009). Modified tRNAs extracted from cells are more stable than their unmodified counterparts obtained by in vitro transcription, as judged by thermal melting analysis and chemical and enzymatic probing (Sampson and Uhlenbeck 1988). Indeed, rapid tRNA decay has been demonstrated in vivo in yeast when the molecule is incompletely modified (Alexandrov et al. 2006). Unmodified tRNAs also exhibit a greater requirement for Mg2+ ions to maintain the active conformation (Hall et al. 1989; Yue et al. 1994; Maglott et al. 1998). Many unmodified tRNAs can be efficiently aminoacylated, however, leading to the general view that the modifications contribute more subtly to structure-function relationships. An extraordinary exception to this principle exists for human mitochondrial tRNALys: The unmodified form folds not into a cloverleaf but instead into an extended bulged hairpin, and the key determinant of native folding has been identified as a single post-transcriptionally added methyl group at the m1A9 position in the tertiary core (Helm et al. 1999b). Studies of tRNA folding and stability have benefited from the development of a variety of techniques, including high-resolution footprinting and single-molecule FRET (fluorescence resonance energy transfer) to study equilibrium structures (Chakshusmathi et al. 2003; Rangan et al. 2004; Wang et al. 2008; Messmer et al. 2009; Dammertz et al. 2011), nuclear magnetic resonance to provide shortrange dynamics information (Vermeulen et al. 2005; Farjon et al. 2009), and birefringence decay and phased t ratio analysis to address long-range flexibility (Friederich et al. 1998). The folding kinetics of fluorescently labeled tRNA has been followed directly (Serebrov et al. 2001). Timeresolved footprinting and small-angle X-ray scattering methods, although not yet applied directly to tRNA folding, have been used to follow the folding kinetics of large RNAs on a millisecond time scale (Sclavi et al. 1998; Russell et al. 2002b; Das et al. 2003). Although these methods can quantify the energetics of interconversion among different conformational states and can measure the degree of structure formation in tRNAs, they do not provide a direct link to function. Because the 570

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canonical function of tRNA in translation begins with aminoacylation, the aaRS enzyme is the key cellular factor that interrogates the tRNA to determine whether native structure is present or not. In the field of ribozyme catalysis, folding studies have been greatly facilitated by the robust enzymatic activity of the molecule, which allows straightforward distinction of native from non-native conformations (Wan et al. 2009). Here, we offer a parallel approach by demonstrating a facile kinetic folding assay that utilizes the potent activities of aaRSs to monitor tRNA folding. This method is highly complementary to the aforementioned approaches and provides a generally applicable tool to integrate studies of tRNA stability, folding, modification, and aminoacylation. RESULTS To monitor tRNA folding kinetics, we took advantage of the approach to measuring aminoacylation developed by Uhlenbeck and colleagues, which relies on the preparation of tRNA substrates that are selectively [32P]-labeled at the 39 internucleotide linkage using tRNA nucleotidyltransferase (Wolfson et al. 1998; Wolfson and Uhlenbeck 2002; Ledoux and Uhlenbeck 2008). This assay possesses significant advantages over the conventional method that relies instead on radiolabeled amino acid: (1) The fraction of aminoacylable tRNA (plateau level) can be directly monitored by the ratio of substrate and product intensities on a thin-layer chromatography (TLC) plate; (2) the sensitivity of the assay is much higher, enabling the detection of even very weak aminoacylation levels; and (3) the use of unlabeled amino acid allows high and saturating concentrations of this substrate to be used. This assay is generally applicable to many and perhaps all aaRSs, including those that utilize nonstandard amino acid substrates, and yields precise measurements of steady-state and elementary rate constants for both cognate and misacylation reactions (Uter and Perona 2004; Hauenstein et al. 2008; Ledoux and Uhlenbeck 2008; Dulic et al. 2010). We describe now the further use of this assay to monitor precatalytic folding of the tRNA substrate, including the influence of posttranscriptional modifications on this process. Previously, we determined kcat, Km(tRNAGln(CUG)), Km(Glu), and Km(ATP) for Glu-tRNAGln synthesis by the nondiscriminating glutamyl-tRNA synthetase (GluRSND) from Methanothermobacter thermautotrophicus (MT) (Rodriguez-Hernandez et al. 2010). Maximal plateau levels for aminoacylation were obtained by folding the unmodified 32P-labeled tRNAGln transcript by heating to 80°C, adding MgCl2 to a final concentration of 10 mM, and slow-cooling to the ambient temperature of 21°C (Fig. 1). Under conditions of enzyme molar excess such that single turnovers are monitored and at saturating levels of ATP and glutamate, the plateau aminoacylation level in reactions performed at 21°C, 37°C, or 45°C following this prefolding procedure is 60%–65% (Fig. 1B,C).

A new approach to tRNA folding kinetics

possibility is that this fraction adopts a misfolded conformation that cannot be renatured under any refolding protocol attempted (Johnson et al. 2005). The concentration of aminoacylated tRNA was normalized to 65% levels to more accurately reflect the true fraction of glutamylated tRNAGln in single turnover reactions. Gel electrophoresis confirmed that there is no degradation of the tRNAs (data not shown). To use the 32P-labeled tRNAGln in folding assays, we trapped a conformation of the molecule that could not be aminoacylated to high levels by altering the refolding protocol: After heating to 80°C, the tRNA sample was rapidly chilled in an ice-water bath at 0°C with the addition of 10 mM Mg2+. Under these conditions, the plateau level for aminoacylation is reduced to 20% (Fig. 1C). To confirm that this treatment does not irreversibly inactivate the tRNA, after chilling at 0°C we removed and incubated a portion of the material at 37°C for several hours before performing the aminoacylation reaction at 21°C. The aminoacylation level of 60% was recovered, demonstrating that the low level of aminoacylation after rapid chilling is due to the formation of kinetic traps and does not represent an irreversible inactivation of tRNAGln (Fig. 1C). Monitoring the folding of MT tRNAGln to the functional native state

FIGURE 1. (A) Proposed secondary structure of the M. thermautotrophicus (MT) tRNAGln(CUG) transcript. (B) Representative timecourse for formation of Glu-tRNAGln by MT GluRSND. Ap indicates the position where the nonaminoacylated 39-terminal monophosphorylated A76 nucleotide migrates. Glu-Ap indicates the position of migration for the glutamylated A76 nucleotide. (C) Glutamylation of MT tRNAGln(CUG). Open symbols represent reactions in which the tRNA substrate was prefolded by heating to 80°C, followed by addition of Mg2+ to 10 mM final concentration and slow cooling to 21°C. These glutamylation reactions were performed at 21°C (open circles), 37°C (open inverted triangles), or 45°C (open squares). The solid circles represent a reaction in which the tRNA substrate was heated to 80°C, followed by addition of Mg2+ to a 10 mM final concentration, rapid cooling to 0°C, and subsequent glutamylation performed at 21°C. The curve depicted by solid diamonds represents the aminoacylation timecourse at 21°C for a tRNA sample that was subjected to the rapidcooling protocol and then subsequently incubated for 300 min at 37°C.

This plateau level is insensitive to the concentration of Mg2+ ions and ATP-Mg2+ in the 2- to 10-mM range and to pH in the range from 6–8 (data not shown). Because the lability of the aminoacyl ester bond increases significantly at higher pH values, the insensitivity of the plateau level to this parameter suggests that deacylation is not a major factor preventing higher levels of aminoacylation. Thus, 35%–40% of the tRNA likely represents a portion of the substrate pool that is not aminoacylable because of 59- or 39-end heterogeneity from the transcription or 39-labeling reactions (Helm et al. 1999a; Sherlin et al. 2001), although a formal alternative

We next set up a folding reaction to track Mg2+-induced formation of the functional tRNAGln(CUG) beginning from the trapped, inactive conformation (Fig. 2). A low concentration of 32P-labeled MT tRNAGln (2 h. The digested tRNAs were precipitated by centrifugation at 14,000 rpm for 30 min, pellets washed with 70% ethanol and dried, and the tRNA samples loaded on a 15% 8M urea denaturing polyacrylamide sequencing gel that was prerun for >3 h at 50 W. The samples were run at 55 W for 7–8 h. The gel was then exposed to a phosphorimager screen overnight and analyzed with ImageQuant (version 5.2). Bands were assigned based on the T1 nuclease digestion ladder.

ACKNOWLEDGMENTS We thank Benjamin Rauch and Jeremie Lever for providing purified E. coli tRNA nucleotidyltransferase, and Rick Russell for critical reading and comments on the manuscript. This work was supported by a grant from the National Institutes of Health (GM63713) to J.J.P. Received August 25, 2011; accepted November 23, 2011.

REFERENCES Adams A, Lindahl T, Fresco JR. 1967. Conformational differences between the biologically active and inactive forms of a transfer ribonucleic acid. Proc Natl Acad Sci 57: 1684–1691. Agirrezabala X, Frank J. 2009. Elongation in translation as a dynamic interaction among the ribosome, tRNA, and elongation factors EF-G and EF-Tu. Q Rev Biophys 42: 159–200. Alexandrov A, Chernyakov I, Gu W, Hiley SL, Hughes TR, Grayhack EJ, Phizicky EM. 2006. Rapid tRNA decay can result from lack of nonessential modifications. Mol Cell 21: 87–96. Arnez JG, Steitz TA. 1994. Crystal structure of unmodified tRNA(Gln) complexed with glutaminyl-tRNA synthetase and ATP suggests a possible role for pseudo-uridines in stabilization of RNA structure. Biochemistry 33: 7560–7567. Banerjee R, Chen S, Dare K, Gilreath M, Praetorius-Ibba M, Raina M, Reynolds NM, Rogers T, Roy H, Yadavalli SS, et al. 2010. tRNAs: cellular barcodes for amino acids. FEBS Lett 584: 387–395. Bina-Stein M, Crothers DM, Hilbers CW, Shulman RG. 1976. Physical studies of denatured tRNA2Glu from Escherichia coli. Proc Natl Acad Sci 73: 2216–2220. Brooks KM, Hampel KJ. 2009. A rate-limiting conformational step in the catalytic pathway of the glmS ribozyme. Biochemistry 48: 5669– 5678. Bullock TL, Uter N, Nissan TA, Perona JJ. 2003. Amino acid discrimination by a class I aminoacyl-tRNA synthetase specified by negative determinants. J Mol Biol 328: 395–408. Chadalavada DM, Senchak SE, Bevilacqua PC. 2002. The folding pathway of the genomic hepatitis delta virus ribozyme is dominated by slow folding of the pseudoknots. J Mol Biol 317: 559– 575.

A new approach to tRNA folding kinetics

Chakshusmathi G, Kim SD, Rubinson DA, Wolin SL. 2003. A La protein requirement for efficient pre-tRNA folding. EMBO J 22: 6562–6572. Cole PE, Crothers DM. 1972. Conformational changes of transfer ribonucleic acid. Relaxation kinetics of the early melting transition of methionine transfer ribonucleic acid (Escherichia coli). Biochemistry 11: 4368–4374. Cole PE, Yang SK, Crothers DM. 1972. Conformational changes of transfer ribonucleic acid. Equilibrium phase diagrams. Biochemistry 11: 4358–4368. Coutts SM, Riesner D, Romer R, Rabl CR, Maass G. 1975. Kinetics of conformational changes in tRNA Phe (yeast) as studied by the fluorescence of the Y-base and of formycin substituted for the 39-terminal adenine. Biophys Chem 3: 275–289. Crothers DM, Cole PE, Hilbers CW, Shulman RG. 1974. The molecular mechanism of thermal unfolding of Escherichia coli formylmethionine transfer RNA. J Mol Biol 87: 63–88. Dammertz K, Hengesbach M, Helm M, Nienhaus GU, Kobitski AY. 2011. Single-molecule FRET studies of counterion effects on the free energy landscape of human mitochondrial lysine tRNA. Biochemistry 50: 3107–3115. Das R, Kwok LW, Millett IS, Bai Y, Mills TT, Jacob J, Maskel GS, Seifert S, Mochrie SG, Thiyagarajan P, et al. 2003. The fastest global events in RNA folding: electrostatic relaxation and tertiary collapse of the Tetrahymena ribozyme. J Mol Biol 332: 311–319. Demeshkina N, Jenner L, Yusupova G, Yusupov M. 2010. Interactions of the ribosome with mRNA and tRNA. Curr Opin Struct Biol 20: 325–332. Ding F, Sharma S, Chalasani P, Demidov VV, Broude NE, Dokholyan NV. 2008. Ab initio RNA folding by discrete molecular dynamics: from structure prediction to folding mechanisms. RNA 14: 1164– 1173. Dulic M, Cvetesic N, Perona JJ, Gruic-Sovulj I. 2010. Partitioning of tRNA-dependent editing between pre- and post-transfer pathways in class I aminoacyl-tRNA synthetases. J Biol Chem 285: 23799– 23809. Ehresmann C, Baudin F, Mougel M, Romby P, Ebel JP, Ehresmann B. 1987. Probing the structure of RNAs in solution. Nucleic Acids Res 15: 9109–9128. Eisinger J, Gross N. 1975. Conformers, dimers, and anticodon complexes of tRNAGlu2 (Escherichia coli). Biochemistry 14: 4031– 4041. Farjon J, Boisbouvier J, Schanda P, Pardi A, Simorre JP, Brutscher B. 2009. Longitudinal-relaxation-enhanced NMR experiments for the study of nucleic acids in solution. J Am Chem Soc 131: 8571–8577. Francklyn CS, Minajigi A. 2010. tRNA as an active chemical scaffold for diverse chemical transformations. FEBS Lett 584: 366–375. Friederich MW, Vacano E, Hagerman PJ. 1998. Global flexibility of tertiary structure in RNA: yeast tRNAPhe as a model system. Proc Natl Acad Sci 95: 3572–3577. Grodberg J, Dunn JJ. 1988. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J Bacteriol 170: 1245–1253. Grosjean H. 2009. Nucleic acids are not boring long polymers of only four types of nucleotides: a guided tour. In DNA and RNA modification enzymes: Structure, mechanism, function and evolution (ed. H Grosjean), pp. 1–18. Landes Bioscience, Austin. Hall KB, Sampson JR, Uhlenbeck OC, Redfield AG. 1989. Structure of an unmodified tRNA molecule. Biochemistry 28: 5794–5801. Hauenstein SI, Hou YM, Perona JJ. 2008. The homotetrameric phosphoseryl-tRNA synthetase from Methanosarcina mazei exhibits half-of-the-sites activity. J Biol Chem 283: 21997–22006. Helm M. 2006. Post-transcriptional nucleotide modification and alternative folding of RNA. Nucleic Acids Res 34: 721–733. Helm M, Brule H, Giege R, Florentz C. 1999a. More mistakes by T7 RNA polymerase at the 59 ends of in vitro-transcribed RNAs. RNA 5: 618–621. Helm M, Giege R, Florentz C. 1999b. A Watson–Crick base-pairdisrupting methyl group (m1A9) is sufficient for cloverleaf folding

of human mitochondrial tRNALys. Biochemistry 38: 13338– 13346. Hilbers CW, Shulman RG, Kim SH. 1973. High resolution NMR study of the melting of yeast tRNA Phe. Biochem Biophys Res Commun 55: 953–960. Hilbers CW, Robillard GT, Shulamn RG, Blake RD, Webb PK, Fresco R, Riesner D. 1976. Thermal unfolding of yeast glycine transfer RNA. Biochemistry 15: 1874–1882. Ibba M, Hong KW, Sherman JM, Sever S, Soll D. 1996. Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinity of the enzyme. Proc Natl Acad Sci 93: 6953–6958. Jackman JE, Montange RK, Malik HS, Phizicky EM. 2003. Identification of the yeast gene encoding the tRNA m1G methyltransferase responsible for modification at position 9. RNA 9: 574–585. Johnson TH, Tijerina P, Chadee AB, Herschlag D, Russell R. 2005. Structural specificity conferred by a group I RNA peripheral element. Proc Natl Acad Sci 102: 10176–10181. Juhling F, Morl M, Hartmann RK, Sprinzl M, Stadler PF, Putz J. 2009. tRNAdb 2009: compilation of tRNA sequences and tRNA genes. Nucleic Acids Res 37: D159–D162. Kent O, Chaulk SG, MacMillan AM. 2000. Kinetic analysis of the M1 RNA folding pathway. J Mol Biol 304: 699–705. Kleiman L, Jones CP, Musier-Forsyth K. 2010. Formation of the tRNALys packaging complex in HIV-1. FEBS Lett 584: 359–365. Kolitz SE, Lorsch JR. 2010. Eukaryotic initiator tRNA: finely tuned and ready for action. FEBS Lett 584: 396–404. Ledoux S, Uhlenbeck OC. 2008. [39-32P]-labeling tRNA with nucleotidyltransferase for assaying aminoacylation and peptide bond formation. Methods 44: 74–80. Lindahl T, Adams A, Fresco JR. 1966. Renaturation of transfer ribonucleic acids through site binding of magnesium. Proc Natl Acad Sci 55: 941–948. Lyakhov DL, He B, Zhang X, Studier FW, Dunn JJ, McAllister WT. 1997. Mutant bacteriophage T7 RNA polymerases with altered termination properties. J Mol Biol 269: 28–40. Madore E, Florentz C, Giege R, Lapointe J. 1999. Magnesiumdependent alternative foldings of active and inactive Escherichia coli tRNA(Glu) revealed by chemical probing. Nucleic Acids Res 27: 3583–3588. Maglott EJ, Deo SS, Przykorska A, Glick GD. 1998. Conformational transitions of an unmodified tRNA: implications for RNA folding. Biochemistry 37: 16349–16359. Maloy SR, Stewart VJ, Taylor RK. 1996. Genetic analysis of pathogenic bacteria: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Mei Y, Stonestrom A, Hou YM, Yang X. 2010. Apoptotic regulation and tRNA. Protein Cell 1: 795–801. Mertz JA, Chadee AB, Byun H, Russell R, Dudley JP. 2009. Mapping of the functional boundaries and secondary structure of the mouse mammary tumor virus Rem-responsive element. J Biol Chem 284: 25642–25652. Messmer M, Putz J, Suzuki T, Sauter C, Sissler M, Catherine F. 2009. Tertiary network in mammalian mitochondrial tRNAAsp revealed by solution probing and phylogeny. Nucleic Acids Res 37: 6881–6895. Motorin Y, Helm M. 2010. tRNA stabilization by modified nucleotides. Biochemistry 49: 4934–4944. Perona JJ, Swanson R, Steitz TA, Soll D. 1988. Overproduction and purification of Escherichia coli tRNA2Gln and its use in crystallization of the glutaminyl-tRNA synthetase-tRNAGln complex. J Mol Biol 202: 121–126. Phizicky EM, Hopper AK. 2010. tRNA biology charges to the front. Genes Dev 24: 1832–1860. Rangan P, Masquida B, Westhof E, Woodson SA. 2004. Architecture and folding mechanism of the Azoarcus Group I Pre-tRNA. J Mol Biol 339: 41–51. Rodriguez-Hernandez A, Perona JJ. 2011. Heat maps for intramolecular communication in an RNP enzyme encoding glutamine. Structure 19: 386–396.

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579

Bhaskaran et al.

Rodriguez-Hernandez A, Bhaskaran H, Hadd A, Perona JJ. 2010. Synthesis of Glu-tRNAGln by engineered and natural aminoacyltRNA synthetases. Biochemistry 49: 6727–6736. Rogers KC, Soll D. 1993. Discrimination among tRNAs intermediate in glutamate and glutamine acceptor identity. Biochemistry 32: 14210–14219. Rogers KC, Crescenzo AT, Soll D. 1995. Aminoacylation of transfer RNAs with 2-thiouridine derivatives in the wobble position of the anticodon. Biochimie 77: 66–74. Russell R, Zhuang X, Babcock HP, Millett IS, Doniach S, Chu S, Herschlag D. 2002a. Exploring the folding landscape of a structured RNA. Proc Natl Acad Sci 99: 155–160. Russell R, Millett IS, Tate MW, Kwok LW, Nakatani B, Gruner SM, Mochrie SG, Pande V, Doniach S, Herschlag D, et al. 2002b. Rapid compaction during RNA folding. Proc Natl Acad Sci 99: 4266–4271. Sampson JR, Uhlenbeck OC. 1988. Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro. Proc Natl Acad Sci 85: 1033–1037. Savochkina L, Alekseenkova V, Belyanko T, Dobrynina N, Beabealashvilli R. 2008. RNase footprinting demonstrates antigenomic hepatitis delta virus ribozyme structural rearrangement as a result of self-cleavage reaction. BMC Res Notes 1: 15. doi: 10.1186/1756-0500-1-15. Sclavi B, Sullivan M, Chance MR, Brenowitz M, Woodson SA. 1998. RNA folding at millisecond intervals by synchrotron hydroxyl radical footprinting. Science 279: 1940–1943. Serebrov V, Clarke RJ, Gross HJ, Kisselev L. 2001. Mg2+-induced tRNA folding. Biochemistry 40: 6688–6698. Sherlin LD, Bullock TL, Nissan TA, Perona JJ, Lariviere FJ, Uhlenbeck OC, Scaringe SA. 2001. Chemical and enzymatic synthesis of tRNAs for high-throughput crystallization. RNA 7: 1671–1678. Sohm B, Sissler M, Park H, King MP, Florentz C. 2004. Recognition of human mitochondrial tRNALeu(UUR) by its cognate leucyl-tRNA synthetase. J Mol Biol 339: 17–29. Sorin EJ, Nakatani BJ, Rhee YM, Jayachandran G, Vishal V, Pande VS. 2004. Does native state topology determine the RNA folding mechanism? J Mol Biol 337: 789–797. Taylor RW, Turnbull DM. 2005. Mitochondrial DNA mutations in human disease. Nat Rev Genet 6: 389–402. Treiber DK, Williamson JR. 1999. Exposing the kinetic traps in RNA folding. Curr Opin Struct Biol 9: 339–345.

580

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Uhlenbeck OC. 1995. Keeping RNA happy. RNA 1: 4–6. Uhlenbeck OC, Chirikjian JG, Fresco JR. 1974. Oligonucleotide binding to the native and denatured conformers of yeast transfer RNA-3 Lea. J Mol Biol 89: 495–504. Uter NT, Perona JJ. 2004. Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics. Proc Natl Acad Sci 101: 14396–14401. Vermeulen A, McCallum SA, Pardi A. 2005. Comparison of the global structure and dynamics of native and unmodified tRNAval. Biochemistry 44: 6024–6033. Voigts-Hoffmann F, Hengesbach M, Kobitski AY, van Aerschot A, Herdewijn P, Nienhaus GU, Helm M. 2007. A methyl group controls conformational equilibrium in human mitochondrial tRNALys. J Am Chem Soc 129: 13382–13383. Wan Y, Mitchell D 3rd, Russell R. 2009. Catalytic activity as a probe of native RNA folding. Methods Enzymol 468: 195–218. Wang B, Wilkinson KA, Weeks KM. 2008. Complex ligand-induced conformational changes in tRNAAsp revealed by single-nucleotide resolution SHAPE chemistry. Biochemistry 47: 3454–3461. Wolfson AD, Uhlenbeck OC. 2002. Modulation of tRNAAla identity by inorganic pyrophosphatase. Proc Natl Acad Sci 99: 5965– 5970. Wolfson AD, Pleiss JA, Uhlenbeck OC. 1998. A new assay for tRNA aminoacylation kinetics. RNA 4: 1019–1023. Yang SK, Crothers DM. 1972. Conformational changes of transfer ribonucleic acid. Comparison of the early melting transition of two tyrosine-specific transfer ribonucleic acids. Biochemistry 11: 4375–4381. Yokogawa T, Kitamura Y, Nakamura D, Ohno S, Nishikawa K. 2010. Optimization of the hybridization-based method for purification of thermostable tRNAs in the presence of tetraalkylammonium salts. Nucleic Acids Res 38: e89. doi: 10.1093/nar/gkp1182. Yue D, Kintanar A, Horowitz J. 1994. Nucleoside modifications stabilize Mg2+ binding in Escherichia coli tRNA(Val): an imino proton NMR investigation. Biochemistry 33: 8905–8911. Zhang CM, Perona JJ, Ryu K, Francklyn C, Hou YM. 2006. Distinct kinetic mechanisms of the two classes of aminoacyl-tRNA synthetases. J Mol Biol 361: 300–311. Zhuang X, Bartley LE, Babcock HP, Russell R, Ha T, Herschlag D, Chu S. 2000. A single-molecule study of RNA catalysis and folding. Science 288: 2048–2051.