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Originally published In Press as doi:10.1074/jbc.M512603200 on February 1, 2006

J. Biol. Chem., Vol. 281, Issue 14, 9801-9811, April 7, 2006
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A Model for C74 Addition by CCA-adding Enzymes

C74 ADDITION, LIKE C75 AND A76 ADDITION, DOES NOT INVOLVE tRNA TRANSLOCATION*Formula

HyunDae D. Cho{ddagger}, Yu Chen§, Gabriele Varani{ddagger}§, and Alan M. Weiner{ddagger}1

From the {ddagger}Department of Biochemistry, School of Medicine, University of Washington, Seattle, Washington 98195-7350 and the §Department of Chemistry, University of Washington, Seattle, Washington 98195-1700

Received for publication, November 28, 2005 , and in revised form, February 1, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The CCA-adding enzyme adds CCA to the 3'-end of tRNA one nucleotide at a time, using CTP and ATP as substrates. We found previously that tRNA does not rotate or translocate on the enzyme during the addition of C75 and A76. We therefore predicted that the growing 3'-end of tRNA must, upon addition of each nucleotide, refold to reposition the new 3'-hydroxyl equivalently relative to the solitary nucleotidyltransferase motif. Cocrystal structures of the class I archaeal Archaeoglobus fulgidus enzyme, poised for addition of C75 and A76, confirmed this prediction. We have also demonstrated that an evolutionarily flexible beta-turn facilitates progressive refolding of the 3'-terminal C74 and C75 residues during C75 and A76 addition. Although useful cocrystals corresponding to C74 addition have not yet been obtained, we now show experimentally that tRNA does not rotate or translocate during C74 addition. We therefore propose, based on the existing A. fulgidus cocrystal structures, that the same flexible beta-turn functions as a wedge between the discriminator base (N73) and the terminal base pair of the acceptor stem, unstacking and repositioning N73 to attack the incoming CTP. Thus a single flexible beta-turn would orchestrate consecutive addition of all three nucleotides without significant movement of the tRNA on the enzyme surface.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
CCA-adding enzymes (ATP(CTP):tRNA nucleotidyltransferases) catalyze the addition and regeneration of the 3'-terminal CCA sequence of tRNA (1). The activity is found in all three kingdoms of life (2) and is essential for viability (3) in those organisms, including all eukaryotes, many Archaea, and some eubacteria, where some or all of the tRNA genes do not encode the 3'-terminal CCA sequence. In organisms where all tRNA genes encode CCA, CCA-adding activity repairs the CCA terminus, and loss of activity impairs cell growth (4). Curiously, in some eubacteria, CCA-adding activity is the shared responsibility of closely related CC- and A-adding enzymes, which are themselves highly homologous to the CCA-adding enzymes of other eubacteria (5).

All CCA-adding enzymes belong to the nucleotidyltransferase superfamily (6), which can be divided into two classes based on sequence similarities (2). Class I nucleotidyltransferases include archaeal CCA-adding enzymes, as well as eukaryotic DNA polymerase beta, poly(A) polymerase, terminal deoxynucleotidyltransferase, and eubacterial kanamycin and streptomycin nucleotidyltransferases. Class II nucleotidyltransferases include both eubacterial and eukaryotic CCA-adding enzymes, as well as eubacterial poly(A) polymerase. As expected, the nucleotidyltransferase domains of both classes are structurally homologous, but elsewhere the structures diverge completely; class I structures are compact and rich in beta-sheet (7, 8), whereas class II structures are extended, rich in {alpha}-helix, and devoid of beta-sheet except in the nucleotidyltransferase domain (9, 10). Thus the two classes may have evolved independently from an ancestral nucleotidyltransferase core.

Unlike all other sequence-specific RNA and DNA polymerases, the CCA-adding enzyme does not use a nucleic acid template (2). On the basis of biochemical experiments demonstrating that tRNA does not translocate or rotate along the CCA-adding enzyme during addition of C75 and A76, we proposed a "collaborative templating" model (11). The growing 3'-end of tRNA and the active site of the enzyme would work together to refold the 3'-end after each step and to reposition the new 3'-hydroxyl for attack on the next incoming nucleotide. The emerging crystal structures confirmed this proposal.

Cocrystal structures of the eubacterial Bacillus stearothermophilus enzyme in complex with CTP and ATP revealed that class II enzymes use a pure protein template, which recognizes both CTP and ATP (9), although it is not yet clear how the addition of C75 switches the nucleotide binding specificity from CTP to ATP. A cocrystal structure for the class II eubacterial Aquifex aeolicus A-adding enzyme in complex with tRNA-NCC substrate and an ATP analog (5), although tantalizing, did not clarify this question.

Cocrystal structures of the Archaeoglobus fulgidus enzyme with oligonucleotide mimics of two different tRNA substrates (tRNA-NC + CTP and tRNA-NCC + ATP) as well as with mature tRNA-NCCA demonstrated that class I enzymes use a true ribonucleoprotein template. The growing 3'-end of tRNA works together with the active site to specify the incoming nucleotide, accommodate the growing 3'-end, and position the new 3'-hydroxyl for attack on the incoming nucleotide (12). Strict use of a ribonucleoprotein template explains why the Archaeoglobus enzyme does not bind nucleotides specifically in the absence of tRNA substrate (7, 8). The Archaeoglobus structures also revealed the key role of a highly conserved beta-turn in accommodating and positioning the growing 3'-end of the tRNA during C75 and A76 addition (12) as was subsequently confirmed by mutational analysis (13).

Cocrystals corresponding to C74 addition have remained elusive, however, and insufficient information was available to model this step in the reaction pathway. We have now used an ethylnitrosourea (ENU)2 interference protocol (11) and synthetic tRNA-like minihelices (14-16), to ask whether tRNA translocates or rotates on the enzyme following C74 addition. To ensure the generality of our results, we tested the archaeal class I enzymes from A. fulgidus and Sulfolobus shibatae and the eubacterial class II enzyme from B. stearothermophilus. We had previously demonstrated that tRNA does not rotate or translocate during C75 and A76 addition (11); our new data allows us to conclude that tRNA remains fixed on the enzyme through all three steps of CCA addition. Moreover, these new data, in combination with the C75 addition structure for the Archaeoglobus enzyme (12) and our mutational analysis of the S. shibatae beta-turn (13), enabled us to model C74 addition by the Archaeoglobus enzyme. We propose that the highly conserved beta-turn adopts four consecutive conformations, the first of which functions as a wedge to disrupt stacking of the discriminator base on the terminal base pair of the acceptor stem, driving the 3'-hydroxyl of the ribose upwards toward the incoming CTP.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
CCA-adding Enzyme Expression and Assay—Hexahistidine-tagged A. fulgidus, S. shibatae, and B. stearothermophilus CCA-adding enzymes were prepared by affinity chromatography on nickel-nitrilotriacetic acid resin (Qiagen), dialyzed into buffer B (20 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM MgCl2, 10 mM beta-mercaptoethanol, 5% glycerol, and 0.1 mM phenylmethylsulfonyl fluoride), and stored at -70 °C in buffer B containing 50% glycerol (16, 17).

The two tRNAIle minihelices, tRNAIle-N32 and tRNAIle-NC33, were based on Mycoplasma pneumoniae tRNA (see Fig. 1A). Crude hairpin minihelices (Dharmacon) were purified by denaturing 15% polyacrylamide gel electrophoresis, visualized by UV light shadowing, carefully excised to exclude aberrant products, and renatured by heating to 80 °C followed by snap cooling. As shown Fig. 1A, the minihelix sequences are tRNAIle-N32, 5'-GGAAGUAGAUGGUUCAAGUCCAUUUACUUCCA-3', and tRNAIle-NC33, 5'-GGAAGUAGAUGGUUCAAGUCCAUUUACUUCCAC-3'.

The 10-µl CCA addition assays contained 100 mM glycine/NaOH (pH 9.0), 10 mM MgCl2, 1 mM dithiothreitol, 5 µM CTP, 50 µM ATP, 2 µM tRNA minihelix, 100 nM [{alpha}-32P]CTP or [{alpha}-32P]ATP (3,000 Ci/mmol, Amersham Biosciences), and 10 nM purified protein at 60 °C (A. fulgidus), 70 °C (S. shibatae), or 55 °C (B. stearothermophilus) for 5 min. Reactions were terminated by the addition of 5 µl of 95% deionized formamide containing 20 mM sodium EDTA (pH 8.0), xylene cyanol (0.2%), and bromphenol blue (0.2%). Products were resolved by 12% denaturing PAGE and quantified using a Storm PhosphorImager (Amersham Biosciences).

Ethylnitrosourea Interference Assay—Alkylation of backbone phosphates by ENU was done as described (11, 18). Briefly, 3 µg of unlabeled tRNAIle-N32 and tRNAIle-NC33 were alkylated by incubation for 3 h at room temperature in 20 µl containing 300 mM sodium cacodylate (pH 8.0), 2 mM EDTA, 20 mM MgCl2, 50 mM NaCl, and 20% v/v of a saturated solution of ENU (Sigma) in ethanol. The reactions were stopped by the addition of 100 µl of 0.3 M sodium acetate (pH 6.0) and ethanol precipitated with glycogen carrier (Roche Applied Science). Following three consecutive ethanol precipitations to remove all traces of ENU, aliquots of the alkylated tRNA minihelices (1 µg) were incubated with A. fulgidus, S. shibatae, and B. stearothermophilus CCA-adding enzymes (5, 15, 30 ng) in 15-µl addition reactions containing 1 µM [{alpha}-32P]CTP. The assays were stopped after 5 min by addition of 10 µl of RNA loading buffer. The 3'-end-labeled tRNA-NC* and tRNA-NCC* were purified by denaturing 15% PAGE, eluted, and ethanol-precipitated. Alkylated tRNAs were taken up in 10 µl of 100 mM Tris-HCl (pH 9.0), and phosphotriester bonds were cleaved by incubation at 50 °C for 5 min. In a control reaction to map alkylation sites on free tRNA-N and tRNA-NC, tRNA substrates were first 3'-end-labeled with [{alpha}-32P]CTP, purified by 15% PAGE, then treated with ENU, cleaved in alkali, and resolved by 20% PAGE. Markers were generated by partial digestion with RNase T1 (Ambion), which specifically cleaves single-stranded RNA after G residues producing 3'-phosphorylated ends (19).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
tRNA Minihelices
We previously performed ethylation protection and interference experiments using ENU to identify tRNA backbone phosphates that are critical for addition of C75 and A76 to tRNA-NC and tRNA-NCC, respectively (11). These experiments established that tRNA does not rotate or translocate on the surface of the CCA-adding enzyme during C75 and A76 addition; however, we were unable to examine C74 addition because short tRNA cleavage fragments corresponding to phosphates nearest the labeled 3'-end were very inefficiently recovered (11, 18). To overcome this problem, we have now used A+U-rich tRNA minihelices as substrates (Fig. 1A), which compensate for the low yield of short cleavage fragments by providing improved resolution during the gel electrophoresis step.

CCA-adding enzymes recognize universally conserved features of tRNA located primarily in the top half of the molecule (7, 11, 14), and tDNA minihelices corresponding to the top half of Escherichia coli tRNAval are good substrates for CCA addition in regard to catalytic efficiency and specificity (14-16). We designed two minihelix substrates corresponding to the top half of the A+U-rich M. pneumoniae tRNAIle: tRNAIle-N32, and tRNAIle-NC33, lacking 3'-terminal CCA and CA, respectively (Fig. 1A). The hairpin minihelices were purified by denaturing 15% PAGE to remove any aberrant synthetic products and were renatured by snap cooling. As expected, both minihelices were good substrates for the class I archaeal CCA-adding enzymes from A. fulgidus and S. shibatae, as well as for the class II enzyme from B. stearothermophilus (Fig. 1B) with the reaction in the linear range (Fig. 1C).

Identification of Phosphates Essential for C74 Addition by CCA-adding Enzymes
To determine whether tRNA translocates or rotates after C74 addition, we used ENU interference to identify tRNA backbone phosphates, which are critical for addition of 3'-terminal C74; addition of C75, which we had assayed previously (11), served as the control (Fig. 2). The tRNAIle-N32 and tRNAIle-NC33 minihelix substrates were first alkylated with ENU, then incubated with the Archaeoglobus, Sulfolobus, or Bacillus enzymes under standard assay conditions in the presence of 1 µM [{alpha}-32P]CTP (11). As additional controls, tRNAIle-N32 and tRNAIle-NC33 were 3'-end-labeled with [{alpha}-32P]CTP before alkylation with ENU. The essential phosphates were identified as bands that were absent or greatly reduced when alkylation preceded end-labeling. Lest high levels of enzyme activity obscure the effects of alkylation, ENU treatment was carried out at several enzyme concentrations (5, 15, and 30 ng/15 µl) (Fig. 1C).

We identified 14 phosphates in the tRNAIle-N32 substrate that are essential for C74 addition by the class I Archaeoglobus and Sulfolobus enzymes (Fig. 2, A and B, left panels): phosphates 6 (minihelix 6), 48-52 (minihelix 7-11), 61-65 (minihelix 20-24), and 67-69 (minihelix 26-28). A very similar phosphate interference pattern, except for phosphates 53 (minihelix 12) and 66 (minihelix 25), was observed for C75 addition to tRNAIle-NC33 by these enzymes (Fig. 2, A and B, right panels). Although the alkylation interference patterns differ in detail between the intact B. subtilis tRNAAsp substrate used previously (11) and the M. pneumoniae minihelix substrates used here (Fig. 2), the new data agree with our previous observation that all alkylated phosphates that interfere with activity are located within the top half of tRNA.

Intriguingly, the efficiency of C74 and C75 addition was dramatically enhanced by alkylation of minihelix phosphate 14 within the T-loop of the M. pneumoniae tRNAIle minihelix (Fig. 2, arrowheads), whereas alkylation of phosphate 55 (equivalent to minihelix phosphate 14) inhibited C75 and A76 addition when full-length B. subtilis tRNAAsp was used as substrate (11). We can only speculate based on the available cocrystal structures (12), but elimination of unfavorable negative charge by alkylation may enhance minihelix binding, perhaps because the T-loop is unable to fold correctly in the absence of a companion D-loop (but see Ref. 20), whereas alkylation of phosphate 55 in full-length tRNA would prevent interaction with R344 and R361 (discussed below; also see Fig. 6). Consistent with these explanations, binding of full-length tRNA to the enzyme protects phosphate 55 from ENU alkylation, and alkylation of phosphates 48 and 49, which are located where the base of the T-stem interacts with the adjacent variable loop, enhances the reaction in intact B. subtilis tRNAAsp (11).


Figure 1
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  		FIGURE 1.
Mycoplasma pneumoniae tRNAIle minihelices are good substrates for both class I and class II CCA-adding enzymes. A, secondary structure of M. pneumoniae tRNAIle and the corresponding tRNAIle-N32, and tRNAIle-NC33 minihelices. B, CCA addition to minihelix substrates by class I and class II CCA-adding enzymes. C, CTP addition to minihelices is proportional to added A. fulgidus enzyme. Af, A. fulgidus; Ss, S. shibatae; Bst, B. stearothermophilus; CTP*, [{alpha}-32P]CTP. Assays as in "Experimental Procedures"; products resolved by denaturing 15% PAGE.

 
The phosphate interference patterns observed for C74 and C75 addition by the class II Bacillus enzyme were nearly identical (Figs. 2C and 3), indicating that tRNA does not rotate or translocate between C74 and C75 addition. Moreover, as shown in Fig. 4, the phosphate interference patterns observed for C74 and C75 addition by class I and class II enzymes are also nearly identical (compare Fig. 2, A-C) except for phosphates 53 and 54 (minihelix phosphates 12 and 13). This remarkable observation is consistent with evidence that tRNA/protein interactions are nearly identical for class I and class II enzymes (5, 7, 12), although the two classes have unrelated protein structures (5, 7, 9, 12) and exhibit no similarity outside the immediate vicinity of the nucleotidyltransferase domain (2, 6).

Nearly all alkylation interference sites lie on same face of the tRNA minihelix (Fig. 4) as observed previously (11), and almost every essential phosphate identified by alkylation interference is in direct contact with the enzyme (Fig. 4) as seen in the cocrystal structures of both the class I A. fulgidus CCA-adding enzyme (12) (Fig. 4A) and the class II A. aeolicus A-adding enzyme (5) (Fig. 4B). Loss of activity caused by alkylation of the variable loop (phosphates 49 and 50) may reflect a structural alteration caused by alkylation of the tRNA backbone, rather than direct interference of these alkylated phosphates with activity (11). None of the interference data appear to be explained by tRNA interactions with the other subunit of the enzyme dimer (not shown in Fig. 4), although the T- and D-loops of the bound tRNA may interact with {alpha}-helix H and beta-strand 8 of the nonbinding monomer (Arg-197 is 5.6Å from O-4 of U17) (8). Admittedly, the phosphate alkylations in the C74 and C75 minihelix T-loops which interfere with B. stearothermophilus activity are shifted by one nucleotide (Fig. 3), but it would be premature to interpret this because we do not know whether the solitary T-loop is "preorganized" and can fold independently of the D-loop (20); the shift could mean that the minihelices rotate and/or translocate very subtly, or (as discussed below) minihelices lacking a companion D-loop may tend to slip toward the tail domain. Taken together with previous evidence that tRNA does not rotate or translocate during C75 and A76 addition (11), we conclude that the tRNA minihelix remains fixed on the surface of the enzyme throughout CCA addition.


Figure 2
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  		FIGURE 2.
Alkylation interference to identify tRNA phosphates essential for C74 and C75 addition. CTP addition was assayed using ENU-treated tRNAIle-N32 (left panels) or tRNAIle-NC33 (right panels) with the A. fulgidus (A), S. shibatae (B), and B. stearothermophilus (C) enzymes. Control reactions were performed with minihelices that were 3'-labeled before ENU treatment. Markers with 3'-phosphorylated ends were generated by partial digestion of 3'-labeled minihelices with RNase T1. Small arrowheads indicate that alkylation of phosphate 55 (minihelix phosphate 14) enhances the reaction.

 


Figure 3
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  		FIGURE 3.
Summary of alkylation interference experiments using the M. pneumoniae tRNAIle-N32 and tRNAIle-NC34 minihelix substrates. Solid squares, phosphates that interfere with activity of the A. fulgidus, S. shibatae, and B. stearothermophilus enzymes when alkylated. Solid circles, phosphates that interfere with activity of the B. stearothermophilus enzyme when alkylated. Unfilled squares, phosphates that enhance activity of the A. fulgidus, S.shibatae, and B.stearothermophilusenzymeswhenalkylated.Largersymbolsindicatestronger effects.

 


Figure 4
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  		FIGURE 4.
Comparison of critical phosphates in the tRNA substrate identified by alkylation interference experiments using class I and class II CCA-adding enzymes. A, a ribbon representation of the class I archaeal A. fulgidus CCA-adding enzyme complexed with mature tRNA. Only a monomer is shown, but the dimeric enzyme can bind two tRNAs (12). B, a ribbon representation of the class II A. aeolicus A-adding enzyme complexed with tRNA-NCC; only the acceptor and T-arms were visible in the cocrystal structure (5). The head, neck, body, and tail domains are colored magenta, green, blue, and cyan, respectively. Incoming nucleotide and bound divalent metal ions are omitted for clarity. tRNA is shown in a stick representation with the backbone path highlighted in yellow. Red spheres, phosphates that interfere with CCA-adding activity when alkylated; blue sphere, phosphate at which alkylation dramatically enhances CCA-adding activity. These and all other images were drawn in PyMOL (35).

 
Molecular Basis of tRNA/Protein Interactions
To better understand how the tRNA substrate can remain bound without rotation or translocation during the three consecutive steps of CCA addition, we examined the interactions between tRNA and the class I A. fulgidus enzyme (Fig. 5). The A. fulgidus enzyme is a dimer that can bind two tRNAs (12); however, tRNA binding does not appear to be cooperative3 and other highly homologous archaeal CCA-adding enzymes appear to bind only a single tRNA (13, 21).4 These data suggest that the dimeric enzyme normally binds only a single tRNA in vivo. The enzyme monomer unit can be divided into head, neck, body, and tail domains; the head contains the nucleotidyltransferase active site, the nucleotide binding site, and the flexible beta-turn that progressively refolds to accommodate the growing 3'-end of the tRNA (12, 13). The 3'-end of the tRNA nestles in the head domain, the acceptor ("top half") minihelix is nearly aligned with {alpha}-helix L of the body domain, and the corner formed by the T- and D-loops fits snugly against the tail domain (Fig. 4A, also see Fig. 6). Sequence alignment of the helix L region from 20 archaeal CCA-adding enzymes representing 5 phylogenetically distinct groups (Fig. 5A) revealed 3 apparently invariant residues (Asp-291, Gln-296, and Arg-299 in the A. fulgidus numbering system), one nearly invariant residue (Pro-295, sometimes Gly, rarely Ala), and a number of distal, more weakly conserved basic or polar residues (Arg-302, Lys-303, and Arg-310 in A. fulgidus).

Remarkably, all three invariant residues interact specifically with the tRNA acceptor stem nearest the growing CCA terminus: Asp-291 interacts the 2'-ribose hydroxyl of the discriminator base G73, Gln-296 with the C2 backbone phosphate, and Arg-299 with the G3, G4, and A64 backbone phosphates (Fig. 5B). The Gln-296-G3 phosphate interaction may also be reinforced by interaction of the C2 phosphate with highly conserved Lys-402 (Fig. S1, A. fulgidus numbering) in helix P (Fig. 5C). The more weakly conserved residues in helix L emanate from two or three consecutive turns facing the tRNA minihelix (Fig. 5A, highlighted in yellow) and interact with backbone phosphates further from the growing 3'-end (e.g. Arg-302, Lys-303, and Arg-310 in A. fulgidus; Lys-291, Arg-294, Lys-297 in S. shibatae; Arg-315, Arg-318 in H. halobium; and Lys-214, Lys-218 in P. furiosus). A similar pattern of RNA/protein interactions is seen for minihelix binding, except that the minihelices (at least in the cocrystals) are shifted one base pair toward the tail domain (see Figs. 8 and 9, and discussion below).

The observed pattern of tRNA/enzyme interactions, highly conserved nearest the nucleotidyltransferase motif and the critical beta-turn that accommodates the growing CCA end (13) but more variable approaching the T- and D-loops, is consistent with the experimental observation that neither tRNA-N (this work) nor tRNA-NC and tRNA-NCC (11) move during CCA addition. In particular, interaction of the invariant Asp-291 (Fig. 5A) with the 2'-ribose hydroxyl of the discriminator base G73 (Fig. 5B) explains why tDNA substrates require a 2'-hydroxyl group (14), and strongly suggests that recognition of the ribose moiety of the discriminator base is largely responsible for holding tRNA and tRNA oligonucleotide mimics in the correct register for faithful CCA addition. This would explain why tDNA minihelices of 11, 12, and 13 bp (14, 16), as well as tRNA-NC and tRNA-NCC minihelix mimics, which lack the T- and D-loops (12), can all function as substrates for faithful CCA addition; although the T- and D-loops of a full-length tRNA substrate fit snugly into the tail domain of the enzyme (Ref. 12 and Fig. 6), these interactions are evidently not essential. We conclude that the proper tRNA register is mainly determined by conserved binding of the CCA end of the acceptor stem to helix L, not by binding of T- and D-loops to the tail domain (Figs. 4A and 7); contrary to expectation, the enzyme does not "measure" the length of the tRNA minihelix from the tail domain, but rather, relies on binding of the unpaired discriminator base and the 9 base pairs closest to the growing 3'-end to identify bona fide substrates.


Figure 5
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  		FIGURE 5.
Highly conserved interactions between the tRNA minihelix and helix L of archaeal CCA-adding enzymes. A, alignment of helix L sequences from archaeal CCA-adding enzymes. Class I archaeal CCA-adding enzymes. Af, A. fulgidus (NC_000917 [GenBank] ); Ap, Aeropyrum pernix (NP_148168 [GenBank] ); Ss, S. shibatae (U66004 [GenBank] ); Ssol, Sulfolobus solfataricus (NP_342511 [GenBank] ); Stok, Sulfolobus tokodaii (NP_376857 [GenBank] ); Ne, N. equitans (NP_963446 [GenBank] ); Hal, Halobacterium sp. NRC-1 (NP_279277 [GenBank] ); Mkan, Methanopyrus kandleri (NP_614649 [GenBank] ); Mbar, Methanosarcina barkeri (ZP_00295845); Mb, Methanococcoides burtonii (ZP_00148440); Mm, Methanosarcina mazei (NP_ 632493); Mt, Methanobacterium thermoautotrophicus (NP_275727 [GenBank] ); Mj, Methanococcus jannaschii (NP_248104 [GenBank] ); Mmar, Methanococcus maripaludis (NP_988069 [GenBank] ); Fa, Ferroplasma acidarmanus (ZP_00307239); Ptor, Picrophilus torridus (NC_005877 [GenBank] .1); Ta, Thermoplasma acidophilum (NP_394900 [GenBank] ); Pf, Pyrococcus furiosus (NP_577755 [GenBank] ); Ph, Pyrococcus horikoshii (NP_142115 [GenBank] ); Pa, Pyrococcus abyssi (NP_125796 [GenBank] ). Red, highly conserved residues interacting with tRNA; black, variable residues potentially interacting with tRNA; green, conserved helix-distorting residue; overbar, helix L region; yellow, residues facing tRNA minihelix on 3 consecutive turns of helix L. B, interactions between the tRNA minihelix and helix L as seen in cocrystal of mature tRNA with the A. fulgidus CCA-adding enzyme (12). Note that Arg-299, the invariant residue most distant from the active site in helix L, contacts tRNA base pair A64:G4; more distant residues, which contact the tRNA minihelix, such as Lys-303 and Arg-310 in the A. fulgidus enzyme, are far less conserved (A) suggesting a weaker interaction with the enzyme. Light blue, nucleotides; dashed gray lines, hydrogen bonds with distances in Angstroms; other colors as in A. C, another view of interactions between the tRNA minihelix and helix L, illustrating potential interaction of the G3 phosphate with a conserved Lys-402 on helix P (also see Fig. S1).

 


Figure 6
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  		FIGURE 6.
The tail domain recognizes the T-loop of tRNA. Note hydrogen bonding of {psi}55 phosphate to Arg-344, and both {psi}55 and C56 phosphates to Arg-361. Key conserved residues (red) and less conserved residues (black) are shown. Lys-357 interacts with 6-keto group of G19, and Asn-354 with the 2-amino group of G19. Pro-347 stacks on C56, and Phe-358 provides part of the hydrophobic core binding {alpha}-helix to antiparallel beta-strands 13 and 14 (see inset).

 
Although side-by-side binding of the tRNA minihelix to {alpha}-helix L helps to explain why the two helix axes are nearly parallel (estimated divergence 17 °; see Fig. S2, A and B), helix L is interrupted by a highly conserved, potentially helix-breaking residue (Fig. 5, A and B); this residue is Pro-295 in A. fulgidus, a proline in the majority of archaeal enzymes, glycine at the equivalent position in a minority of enzymes, and alanine in Nanoarchaeum equitans. Proline and glycine are frequently considered helix breakers, but proline is readily accommodated within an {alpha}-helix and can form C-H···O hydrogen bonds through the {alpha} and {delta} carbons (22, 23). Indeed, C{alpha} of Pro-295 is close to the Glu-298 carbonyl and well within the distance for a C-H···O hydrogen bond according to Chakrabarti and co-workers (22, 23) (3.48Å in the C75 addition complex, and 3.55Å in A76 addition complex, but curiously, 5.25Å in the complex with mature tRNA; Fig. S3A); however, although proline can redirect a transmembrane {alpha}-helix by as much as 30 °, Pro-295 does not appear to cause a significant bend or kink in helix L (Fig. S3B). A fascinating possibility currently being explored is that helix L may break or partially dislocate at Pro-295 (or the equivalent glycine in homologous enzymes) during one or more steps in CCA addition.


Figure 7
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  		FIGURE 7.
The flexible beta-turn may wedge A73 apart from the terminal C72:G1 base pair during C74 addition: a model in the minihelix (-1) register. A, C74 addition by the Archaeoglobus enzyme (left) was modeled on the actual cocrystal structure for C75 addition (right) using the biopolymer module in InsightII (Acclerys) (36). Structures are shown in a stick representation using the same colors for both the modeled and actual complexes. Wheat, tRNA acceptor stem; magenta, A73; blue, C74; orange, C75; green,beta-turncarbonatoms;yellow, otherproteincarbon atoms; blue, protein nitrogen atoms; red, protein oxygen atoms; cyan, metals; dashed lines, hydrogen bonds. Only A73 was allowed to move in the modeled C74 addition structure; the coordinates of all other atoms correspond to the actual C75 addition complex PDB 1TFY [PDB] (12). B, modeled C74 and actual C75 addition complexes showing only tRNA and incoming nucleotide. A dashed line indicates the distance between the 3'-hydroxyl of the primer nucleotide (A73 or C74) and the {alpha}-phosphate of the incoming CTP. All colors as in A; violet, phosphorus; wheat, the path of the tRNA backbone.

 
Modeling the C74 Addition Complex
To explain CCA addition without tRNA translocation, we proposed that the growing 3' terminus of the tRNA progressively refolds, allowing the solitary active site to reuse a single active site (11, 24). Cocrystal structures of the archaeal class I enzyme from A. fulgidus in complex with an oligonucleotide mimic of tRNA-NC74 and CTP (corresponding to C75 addition), and an oligonucleotide mimic of tRNA-NCC75 and ATP (corresponding to A76 addition), not only confirmed this hypothesis for C75 and A76 addition, but also revealed an important role for a key beta-turn in positioning and refolding the growing 3'-end (12, 13). This beta-turn, which is highly conserved among archaeal CCA-adding enzymes, can be seen in the cocrystal structures (12, 13) to adopt three consecutive conformations during CCA addition, one for C75 addition, one for A76 addition, and a final conformation following completion of CCA. These consecutive conformations accommodate the growing 3'-end, position each new 3'-hydroxyl group identically for attack on the next incoming nucleotide, and ultimately terminate the reaction by moving A76 beyond the reach of the active site (Fig. 7). Whether the beta-turn adopts another conformation, corresponding to C74 addition, is a matter of conjecture in the absence of a cocrystal structure. However, our earlier observation that a Y95V mutation in the S. shibatae beta-turn (equivalent to Archaeoglobus Y90V; see Fig. 7A, right panel) blocks addition of C74, but not C75 or A76 (13), strongly supports our working hypothesis that the beta-turn adopts a fourth distinct conformation during C74 addition.


Figure 8
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  		FIGURE 8.
Oligonucleotide mimics and mature tRNA bind to the enzyme in different registers: views of the RNAs. A, when the L helices in the cocrystal structures for A76 addition complex (magenta) and mature tRNA (gray) are superposed, the tRNA minihelix appear shifted ~1 base pair toward the tail domain. B, when the L helices are superposed for the C75 addition (magenta) and A76 addition (orange) complexes, the minihelices are in almost identical registers. C, an enlarged view of A showing 1-bp shift of minihelix toward tail domain.

 
We decided to model the C74 addition complex based on the three known cocrystal structures (12). The structure of the C75 addition complex (Fig. 7A, right panel) had revealed that the flexible beta-turn forms a wedge between C74 and the discriminator base A73, simultaneously pushing C74 up toward the incoming CTP (enabling CTP to stack on C74) while pinning A73 down (enabling A73 to stack on the terminal G1:C72 base pair of the acceptor stem). We therefore asked whether the flexible beta-turn might also facilitate C74 addition by forming an analogous wedge between A73 and the terminal base pair of the acceptor stem, pushing A73 up toward the incoming CTP (so that CTP can stack on A73) while replacing the stacking of A73 on the terminal base pair by a functionally equivalent protein interaction (see Fig. 7A, left panel, for one of two modeled complexes). This protein wedge would be a more elaborate and versatile version of the intercalated tyrosine and phenylalanine side chains observed at other sharp turns of the nucleic acid backbone in RNA and DNA polymerases (25-27).

Simplifying assumptions are necessary to model a structure of this complexity. First, we assumed that the overall conformation of the Archaeoglobus CCA-adding enzyme in the C74 addition complex (except for the flexible beta-turn) would be the same as in the C75 and A76 addition complexes. We based this assumption on the observations that 1) the head and neck domains move away from the body and tail only after CCA addition is complete (12) and 2) the beta-turn appears to be conformationally independent of the rest of the protein because (a) the turn is firmly anchored to the ends of beta-strands 3 and 4 in the highly structured nucleotidyltransferase motif; (b) it projects into a large superficial cleft which accommodates the growing 3'-end of the tRNA; and (c) distinct conformations of the beta-turn in the C75 and A76 addition complexes are compatible with the same protein structure (12). Second, we chose not to model the beta-turn itself because 1) this highly conserved turn is known to adopt at least three different conformations in the course of the reaction (12), so it seemed unlikely that current modeling techniques could generate a credible fourth conformation without very precise constraints, and 2) the wedge-shaped beta-turn in the C75 addition complex is already close to a plausible position for the modeled C74 addition complexes (see for example Fig. 7A).

Choosing the Register—With these simplifying assumptions, we still had to decide which register to use for tRNA interactions with the protein during C74 addition. In all three existing structures (12), the RNA helix nestles nearly parallel to protein helix L and is held in place by ionic and polar interactions with many conserved side chains as shown for mature tRNA in Fig. 5B. However, when the L helices of the three protein structures are superposed (Fig. 8A), the two minihelix RNAs superpose well (Fig. 8B), but the acceptor stem of mature tRNA appears shifted by 1 base pair toward the head domain (Fig. 8C). Indeed, consistent with a bona fide translocation involving a screw rotation of the RNA helix, neither the disposition of conserved side chains emanating from protein helix L, nor the corresponding interactions of these side chains with the RNA helix, change significantly when the C75 and A76 addition structures are compared (Fig. 9A), the A76 addition and mature tRNA structures are compared (Fig. 9B), or all three compared with each other (Fig. 9C).

Because the C75 and A76 addition structures both appear properly poised for catalysis (12), a formal possibility is that all three steps of CCA addition occur in the minihelix ("-1") register, after which the mature tRNA translocates toward the head domain by 1 bp ("0" register). We consider this scenario unlikely because the T- and D-loops of mature tRNA rest snugly against the tail domain in the cocrystal structure (Ref. 12, also see Fig. 6), making it difficult to imagine how immature tRNA (as opposed to a minihelix lacking these loops) could avoid a stereochemical clash with the tail domain when bound in the -1 register. Thus a more plausible explanation for the two binding registers observed in the cocrystal structures may be that the minihelices, lacking the T- and D-loops, artifactually shift 1 bp toward the tail domain. To compensate for translocation of the RNA minihelices relative to mature tRNA, the 3'-end of the minihelix acceptor stem would deform, allowing the backbone and discriminator base to extend further toward the catalytic head domain (Fig. 8, note that the smooth curve of the mature tRNA backbone cannot be unambiguously superposed on the more irregular backbone of the minihelix). The 3'-end of the deformed tRNA backbone would still be correctly poised for nucleotide addition because it is pinned into place by a network of interactions including hydrogen bonding of invariant residue Asp-291 (Fig. 5A) to the discriminator 2'-ribose hydroxyl (Fig. 5B), and ionic interactions of the discriminator phosphate with the side chains of His-133 and Arg-224. Although much less extreme, this 3'-deformation would functionally resemble melting of the terminal base pair of tRNAGln in complex with GlnRS and ATP (28). The ability of the enzyme to bind the tRNA helix in at least two registers, while still correctly positioning the 3'-end for faithful CCA addition, may also reflect accommodations required to recognize >50 distinguishable tRNA species as typically found in prokaryotic or eukaryotic organisms.


Figure 9
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  		FIGURE 9.
Oligonucleotide mimics and mature tRNA bind to the enzyme in different registers: views of the protein side chains. A, when the L helices are superposed, the C75 addition minihelix (salmon) and A76 addition minihelix (light blue) are in almost perfect register, and most key side chains on helix L are nearly superimposable. B, when the L helices are superposed, the A76 addition minihelix (light blue) and mature tRNA (yellow) are 1 base pair out of register (mature tRNA appears shifted toward the head domain) but most key side chains on helix L remain nearly superimposable. C, when L helices are superposed for the C75 addition minihelix (salmon), the A76 addition minihelix (light blue), and mature tRNA (yellow), most key side chains on helix L remain nearly superimposable.

 
Modeling C74 Addition in the Minihelix Register—Instead of arbitrarily choosing to model C74 addition using the -1 minihelix register or 0 tRNA register, we tried both. First, we used the minihelix register. C74 was deleted from the C75 addition structure; only A73 and the adjacent {alpha}-phosphate were allowed to move, and the conformation of the A73 ribose was assumed to be C3'-endo as seen in the C75 and A76 addition structures (12). For simplicity, we assumed that the incoming CTP, the tRNA (including the terminal base pair of the acceptor stem), the nucleotidyltransferase motif, and the beta-turn that accommodates the growing the 3'-end of the tRNA, were all fixed. Any or all of these components of the reaction center could in principle move significantly, but the subtlety of the RNA and protein movements during C75 and A76 addition (12), and the apparent immobility of the tRNA minihelix between C74 and A76 addition as judged experimentally (Figs. 2 and 3), suggested that any conformational changes during C74 addition (except for the beta-turn) were likely to be subtle. Yet even without modeling an interaction between A73 and the beta-turn, some of the forces that could hold A73 in place for C74 addition were apparent. The 4-NH2 of CTP hydrogen bonds to the phosphate backbone of A73 in both the actual C75 addition complex and the first model for C74 addition (Fig. 7A). Hydrogen bonding of the 6-NH2 group of A73 to the peptide carbonyl of A126 in nearby protein helix E may help to position A73 for C74 addition; this bond measures 3.17 Å when C74 addition is modeled in the minihelix (-1) register, 4.38 Å for the actual C75 addition structure (1TFY), and 3.62 Å for C74 addition modeled in the mature tRNA (0) register (see below). The 4-NH2 of the incoming CTP also appears able to hydrogen bond with the 3'-OH of A73; this bond measures 2.46 Å for C74 addition modeled in the minihelix register, 7.12 Å for the actual C75 addition structure (1TFY), and 4.85 Å for C74 addition modeled in the mature tRNA register (see below). Hydrogen bonding of the 4-NH2 of CTP to the 3'-OH of A73 would, however, interfere with attack of the 3'-OH on the {alpha}-phosphate of CTP. Finally, it is important to keep in mind that in the C75 addition structure (Fig. 7A, right panel), A73 stacks over the middle of the terminal G1:C72 base pair of the acceptor stem in the expected lowest energy conformation (29), and this is likely to be where A73 resides until the beta-turn wedges the base away from the acceptor stem.

With these constraints, the 3'-OH of the minihelix substrate in the first model could not be maneuvered any closer than 5.4 Å to the {alpha}-phosphate of the incoming CTP (Fig. 7, A and B). Although the modeled 5.4 Å falls >2 Å shy of the 3.2 Å observed in the actual C75 addition structure, this 2-Å gap could be narrowed, and A73 stacking with the incoming CTP improved, if partial melting of the C72:G1 base pair were allowed; however, this would not necessarily bring the 3'-O close enough to metal A in the crystal structure for catalysis (Fig. 7A). Alternatively, if addition of C75 and A76 to immature tRNA (as opposed to loopless minihelices) occurs in the 0 rather than the -1 register (see discussion above), apparent outward movement of the head domain may be an artifact of comparing mature tRNA to loopless minihelix substrates, and inward movement of the head domain during C74 addition may take place (as seen for the C75 and A76 minihelices relative to mature tRNA).


Figure 10
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  		FIGURE 10.
The flexible beta-turn may wedge A73 apart from the terminal C72:G1 base pair during C74 addition: a model in the mature tRNA (0) register. A, model for C74 addition in mature tRNA register (left) preserves network of hydrogen bonds between incoming CTP and ribonucleoprotein template composed of protein side chains and backbone phosphates of A73 and C72 (right). B, proximity of the 3'-hydroxyl of A73 to the {alpha}-phosphate of the incoming CTP in the modeled C74 addition (left) and the actual C75 addition structure (right). The model was built by combining the mature tRNA from structure 1SZ1 with the protein and CTP from 1TFY [PDB] , using the Biopolymer module in InsightII as in Fig. 7. The 1SZ1 and 1TFY structures were aligned by superposing {alpha}-helices L; the 3'-terminal CCA was deleted from the mature tRNA; and the O3' of A73 was positioned for attack on the {alpha}-phosphate of CTP. To facilitate movement of A73, C72 was translated ~0.5 Å perpendicular to the previous base plane without disrupting the C72:G1 base pair. To optimize hydrogen bonding interactions with A73, the surrounding protein residues (Arg-224, Glu-164, Arg-129, Thr-130) were subjected to local energy minimization. Despite considerable movement of the side chains, backbone motions were minimal. In the final model, the distance between O3' and the phosphate is 3.26 Å, nearly identical to what is observed in the 1TFY structure for the C75 addition complex.

 
Modeling C74 Addition in the Mature tRNA Register—Next we used the mature tRNA register to model C74 addition by combining the catalytically correct protein conformation of the C74 addition structure with tRNA in the 0 register as observed in the mature tRNA structure. In this second model, the 3'-hydroxyl of the discriminator A73 in the C3'-endo conformation was only 3.2 Å from the {alpha}-phosphate of the incoming CTP, very close to the corresponding distance for C75 addition (3.2 Å) and A76 addition (3.8 Å) (12). Moreover, as in the first model, C74 addition would involve the same ribonucleoprotein template as C75 addition (the C72 and A73 phosphates, and the His-129, Arg-125, and Arg-221 side chains; see Fig. 7A).

The proximity of the 3'-hydroxyl of A73 to the {alpha}-phosphate in the second model is encouraging (Fig. 10), but what of geometry? The "inline fitness" of a phosphoester bond transfer reaction plays only a minor role in favoring, but a major role in disfavoring, the transfer reaction (Refs. 30 and 31, but see Ref. 32 for a different point of view). A perfectly positioned in-line attack may confer as little as 10-20-fold acceleration relative to the uncatalyzed rate, whereas deprotonation of the attacking hydroxyl can accelerate the reaction by as much as 2-million-fold (33). Moreover, although the R-OH hydroxyl should ideally be positioned in-line for attack on the phosphate, the disposition of the R substituent in space varies widely, one extreme being base-catalyzed hydrolysis of an RNA chain where the highly constrained 2'-hydroxyl group attacks an adjacent phosphoester bond to yield a 2',3'-cyclic phosphate (31). An improperly positioned R substituent (the A73 ribose in this case) can, however, prevent the hydroxyl from assuming a tolerable position for in-line attack. We calculated the in-line fitness factor F as described (30) for the models in the minihelix (-1) and mature tRNA (0) registers (Fig. S4, A and B, respectively) and compared these to the actual structures for C75 and A76 addition (C and D, respectively). F > 0.4 is considered to be near in-line configuration; C75 and A76 addition have factors of 0.79 and 0.45, respectively, F is 0.64 for C74 addition modeled in the mature tRNA (0) register but 0.09 for C74 addition modeled in the minihelix (-1) register. We conclude that the tRNA register is more likely to be correct.

Implications for Class II Enzymes
Our new experimental data for C74 addition (Fig. 2), together with earlier work on C75 and A76 addition (11), demonstrate that tRNA does not translocate or rotate during CCA addition by either class I or class II enzymes. However, the mechanism of CCA addition by class II enzymes remains elusive. We know that class I and class II enzymes differ dramatically in structure and sequence outside of the conserved nucleotidyltransferase ("head") domain (7-10). We also know that class II enzymes use a pure protein template, which can recognize both CTP and ATP in the absence of tRNA substrate (9, 10), whereas class I enzymes use a ribonucleoprotein template (for example, see Fig. 7A, right) and thus recognize CTP and ATP only in the presence of tRNA substrate (7, 8, 12). Whether different templates for CCA addition and different modes of tRNA binding are compatible with similar mechanisms of CCA addition is not known. The structure of the A. aeolicus class II A-adding enzyme in complex with ATP and tRNA-NCC (5) provides few clues. The equivalent beta-turn between strands 3 and 4 is short and far less highly conserved in class II enzymes (34) but might conceivably work in concert with a long proline-rich loop between strand 5 and helix D that is disordered in both class II apoenzyme structures (9, 10) and the A76 addition structure of the Aquifex A-adding enzyme (5). Clear answers await additional cocrystal structures.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Award GM59804 (to A. M. W.) and National Institutes of Health Grant GM064440 (to G. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental figures. Back

1 To whom correspondence should be addressed. Tel.: 206-543-1768; Fax: 206-685-9231; E-mail: amweiner{at}u.washington.edu.

2 The abbreviations used are: ENU, ethylnitrosourea; tRNA-NCCA, mature tRNA where N is the discriminator base. Back

3 H.-D. Cho, unpublished data. Back

4 J. Kwak, H.-D. Cho, and A. M. Weiner, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Yong Xiong and Tom Steitz for ongoing discussions throughout the course of this work and communicating results in advance of publication.



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 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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