Archaeal CCA-adding enzymes: central role of a highly conserved beta-turn motif in RNA polymerization without translocation

The CCA-adding enzyme (tRNA nucleotidyltransferase) builds and repairs the 3' end of tRNA. A single active site adds both CTP and ATP, but the enzyme has no nucleic acid template, and tRNA does not translocate or rotate during C75 and A76 addition. We modeled the structure of the class I archaeal Sulfolobus shibatae CCA-adding enzyme on eukaryotic poly(A) polymerase and mutated residues in the vicinity of the active site. We found mutations that specifically affected C74, C75, or A76 addition, as well as mutations that progressively impaired addition of CCA. Many of these mutations clustered in an evolutionarily versatile beta-turn located between strands 3 and 4 of the nucleotidyltransferase domain. Our mutational analysis confirms and extends recent crystallographic studies of the highly homologous Archaeoglobus fulgidus enzyme. We suggest that the unusual phenotypes of the beta-turn mutants reflect the consecutive conformations assumed by the beta-turn as it presents the discriminator base N73, then C74, and finally C75 to the active site without translocation or rotation of the tRNA acceptor stem. We also suggest that beta-turn mutants can affect nucleotide selection because the growing 3' end of tRNA must be properly positioned to serve as part of the ribonucleoprotein template that selects the incoming nucleotide.     

Unusual synthesis by the Escherichia coli CCA-adding enzyme

The tRNA 3' end contains the conserved CCA sequence at the 74-76 positions. The CCA sequence is synthesized and maintained by the CCA-adding enzymes. The specificity of the Escherichia coli enzyme at each of the 74-76 positions was investigated using synthetic minihelix substrates that contain permuted 3' ends. Results here indicate that the enzyme has the ability to synthesize unusual 3' ends. When incubated with CTP alone, the enzyme catalyzed the addition of C74, C75, C76, and multiple Cs. Although the addition of C74 and C75 was as expected, that of C76 and multiple Cs was not. In particular, the addition of C76 generated CCC, which would have conflicted with the biological role of the enzyme. However, the presence of ATP prevented the synthesis of CCC and completely switched the specificity to CCA. The presence of ATP also had an inhibitory effect on the synthesis of multiple Cs. Thus, the E. coli CCA enzyme can be a poly(C) polymerase but its synthesis of poly(C) is regulated by the presence of ATP. These features led to a model of CCA synthesis that is independent of a nucleic acid template. The synthesis of poly(C) by the CCA-adding enzyme is reminiscent of that of poly(A) by poly(A) polymerase and it provides a functional rationale for the close sequence relationship between these two enzymes in the family of nucleotidyltransferases.     

A single catalytically active subunit in the multimeric Sulfolobus shibatae CCA-adding enzyme can carry out all three steps of CCA addition

The CCA-adding enzyme ATP(CTP):tRNA nucleotidyltransferase builds and repairs the 3'-terminal CCA sequence of tRNA. Although this unusual RNA polymerase has no nucleic acid template, it can construct the CCA sequence one nucleotide at a time using CTP and ATP as substrates. We found previously that tRNA does not translocate along the enzyme during CCA addition (Yue, D., Weiner, A. M., and Maizels, N. (1998) J. Biol. Chem. 273, 29693-29700) and that a single nucleotidyltransferase motif adds all three nucleotides (Shi, P.-Y., Maizels, N., and Weiner, A. M. (1998) EMBO J. 17, 3197-3206). Intriguingly, the CCA-adding enzyme from the archaeon Sulfolobus shibatae is a homodimer that forms a tetramer upon binding two tRNAs. We therefore asked whether the active form of the S. shibatae enzyme might have two quasi-equivalent active sites, one adding CTP and the other ATP. Using an intersubunit complementation approach, we demonstrate that the dimer is active and that a single catalytically active subunit can carry out all three steps of CCA addition. We also locate one UV light-induced tRNA cross-link on the enzyme structure and provide evidence suggesting the location of another. Our data rule out shuttling models in which the 3'-end of the tRNA shuttles from one quasi-equivalent active site to another, demonstrate that tRNA-induced tetramerization is not required for CCA addition, and support a role for the tail domain of the enzyme in tRNA binding.     

A model for C74 addition by CCA-adding enzymes: C74 addition, like C75 and A76 addition, does not involve tRNA translocation

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.     

U2 small nuclear RNA is a substrate for the CCA-adding enzyme (tRNA nucleotidyltransferase).

The CCA-adding enzyme builds and repairs the 3' terminus of tRNA. Approximately 65% of mature human U2 small nuclear RNA (snRNA) ends in 3'-terminal CCA, as do all mature tRNAs; the other 35% ends in 3' CC or possibly 3' C. The 3'-terminal A of U2 snRNA cannot be encoded because the 3' end of the U2 snRNA coding region is CC/CC, where the slash indicates the last encoded nucleotide. The first detectable U2 snRNA precursor contains 10-16 extra 3' nucleotides that are removed by one or more 3' exonucleases. Thus, if 3' exonuclease activity removes the encoded 3' CC during U2 snRNA maturation, as appears to be the case in vitro, the cell may need to build or rebuild the 3'-terminal A, CA, or CCA of U2 snRNA. We asked whether homologous and heterologous class I and class II CCA-adding enzymes could add 3'-terminal A, CA, or CCA to human U2 snRNA lacking 3'-terminal A, CA, or CCA. The naked U2 snRNAs were good substrates for the human CCA-adding enzyme but were inactive with the Escherichia coli enzyme; activity was also observed on native U2 snRNPs. We suggest that the 3' stem/loop of U2 snRNA resembles a tRNA minihelix, the smallest efficient substrate for class I and II CCA-adding enzymes, and that CCA addition to U2 snRNA may take place in vivo after snRNP assembly has begun.     

Poly(C) synthesis by class I and class II CCA-adding enzymes

The CCA-adding enzymes [ATP(CTP):tRNA nucleotidyl transferases], which catalyze synthesis of the conserved CCA sequence to the tRNA 3' end, are divided into two classes. Recent studies show that the class II Escherichia coli CCA-adding enzyme synthesizes poly(C) when incubated with CTP alone, but switches to synthesize CCA when incubated with both CTP and ATP. Because the poly(C) activity can shed important light on the mechanism of the untemplated synthesis of CCA, it is important to determine if this activity is also present in the class I CCA enzymes, which differ from the class II enzymes by significant sequence divergence. We show here that two members of the class I family, the archaeal Sulfolobus shibatae and Methanococcus jannaschii CCA-adding enzymes, are also capable of poly(C) synthesis. These two class I enzymes catalyze poly(C) synthesis and display a response of kinetic parameters to the presence of ATP similar to that of the class II E. coli enzyme. Thus, despite extensive sequence diversification, members of both classes employ common strategies of nucleotide addition, suggesting conservation of a mechanism in the development of specificity for CCA. For the E. coli enzyme, discrimination of poly(C) from CCA synthesis in the intact tRNA and in the acceptor-TPsiC domain is achieved by the same kinetic strategy, and a mutation that preferentially affects addition of A76 but not poly(C) has been identified. Additionally, we show that enzymes of both classes exhibit a processing activity that removes nucleotides in the 3' to 5' direction to as far as position 74.     

On the role of a conserved, potentially helix-breaking residue in the tRNA-binding alpha-helix of archaeal CCA-adding enzymes

Archaeal class I CCA-adding enzymes use a ribonucleoprotein template to build and repair the universally conserved 3'-terminal CCA sequence of the acceptor stem of all tRNAs. A wealth of structural and biochemical data indicate that the Archaeoglobus fulgidus CCA-adding enzyme binds primarily to the tRNA acceptor stem through a long, highly conserved alpha-helix that lies nearly parallel to the acceptor stem and makes many contacts with its sugar-phosphate backbone. Although the geometry of this alpha-helix is nearly ideal in all available cocrystal structures, the helix contains a highly conserved, potentially helix-breaking proline or glycine near the N terminus. We performed a mutational analysis to dissect the role of this residue in CCA-addition activity. We found that the phylogenetically permissible P295G mutant and the phylogenetically absent P295T had little effect on CCA addition, whereas P295A and P295S progressively interfered with CCA addition (C74>C75>A76 addition). We also examined the effects of these mutations on tRNA binding and the kinetics of CCA addition, and performed a computational analysis using Rosetta Design to better understand the role of P295 in nucleotide transfer. Our data indicate that CCA-adding activity does not correlate with the stability of the pre-addition cocrystal structures visualized by X-ray crystallography. Rather, the data are consistent with a transient conformational change involving P295 of the tRNA-binding alpha-helix during or between one or more steps in CCA addition.     

A story with a good ending: tRNA 3'-end maturation by CCA-adding enzymes

CCA-adding enzymes (tRNA nucleotidyltransferases) are responsible for the maturation or repair of the functional 3' end of tRNAs. These enzymes are remarkable because they polymerize the essential nucleotides CCA onto the 3' terminus of tRNA precursors without using a nucleic acid template. Recent crystal structures, plus three decades of enzymology, have revealed the elegant mechanisms by which CCA-adding enzymes achieve their substrate specificity in a nucleic acid template independent fashion. The class I CCA-adding enzyme employs both an arginine sidechain and backbone phosphates of the bound tRNA to recognize incoming nucleotides. It switches from C to A addition through changes in the size and shape of the nucleotide-binding pocket, which is progressively altered by the elongating 3' terminus of the tRNA. By contrast, the class II CCA-adding enzyme uses only amino acid sidechains, which form a protein template for incoming nucleotide selection.     

Sulfolobus shibatae CCA-adding enzyme forms a tetramer upon binding two tRNA molecules: A scrunching-shuttling model of CCA specificity

The tRNA CCA-adding enzyme adds CCA stepwise to immature transfer RNA molecules untemplated, but with high specificity. We examined the oligomerization state of the enzyme from Sulfolobus shibatae and its binding to transfer RNA molecules, using various biophysical and biochemical methods including size exclusion chromatography, multi-angle laser light scattering, small-angle X-ray scattering, and gel electrophoresis band mobility shift assay. The 48 kDa monomer forms a stable salt- resistant dimer in solution. Further dimerization of the dimeric enzyme to form a tetramer is induced by the binding of two tRNA molecules. The formation of a tetramer with only two bound tRNA molecules leads us to suggest that one pair of active sites may be specific for adding two C bases, which results in scrunching of the primer strand. An adjacent second pair of active sites may be specific for adding A after addition of two C bases which makes the 3' terminus long enough to reach the second pair of active sites.     

Crystal structures of an archaeal class I CCA-adding enzyme and its nucleotide complexes

CCA-adding enzymes catalyze the addition of CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template and have been divided into two classes based on their amino acid sequences. We have determined the crystal structures of a class I CCA-adding enzyme from Archeoglobus fulgidus (AfCCA) and its complexes with ATP, CTP, or UTP. Although it and the class II bacterial Bacillus stearothermophilus CCA enzyme (BstCCA) have similar dimensions and domain architectures (head, neck, body, and tail), only the polymerase domain is structurally homologous. Moreover, the relative orientation of the head domain with respect to the body and tail domains, which appear likely to bind tRNA, differs significantly between the two enzyme classes. Unlike the class II BstCCA, this enzyme binds nucleotides nonspecifically in the absence of bound tRNA. The shape and electrostatic charge distribution of the AfCCA enzyme suggests a model for tRNA binding that accounts for the phosphates that are protected from chemical modification by tRNA binding to AfCCA. The structures of the AfCCA enzyme and the eukaryotic poly(A) polymerase are very similar, implying a close evolutionary relationship between them.     

A top-half tDNA minihelix is a good substrate for the eubacterial CCA-adding enzyme.

The CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase] catalyzes the addition and regeneration of the 3'-terminal CCA sequence of tRNAs. We show that the CCA-adding enzyme will specifically add a CCA terminus to synthetic full-length tDNA and to DNA oligonucleotides corresponding to the "top half" of tRNA-the acceptor stem and TpsiC stem-loop of tRNA. CCA addition to the top half tDNA minihelices requires a 2' as well as a 3' OH at the 3' terminus of the tDNA. Addition also depends on the length of the base paired stem, and is facilitated by, but is not dependent upon, the presence of a TpsiC loop. These results provide further evidence for independent functions of the top and bottom halves of tRNA, and support the hypothesis that these two structurally distinct and functionally independent domains evolved independently.     

Crystal structures of the Bacillus stearothermophilus CCA-adding enzyme and its complexes with ATP or CTP

CCA-adding enzymes polymerize CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template. The 3.0 A resolution crystal structures of the CCA-adding enzyme from Bacillus stearothermophilus and its complexes with ATP or CTP reveal a seahorse-shaped subunit consisting of four domains: head, neck, body, and tail. The head is structurally homologous to the palm domain of DNA polymerase beta but has additional structural features and functions. The neck, body, and tail represent new protein folding motifs. The neck provides a specific template for the incoming ATP or CTP, whereas the body and tail may bind tRNA. Each subunit has one active site capable of switching its base specificity between ATP and CTP, an important component of the CCA-adding mechanism.     

Use of nucleotide analogs by class I and class II CCA-adding enzymes (tRNA nucleotidyltransferase): deciphering the basis for nucleotide selection

We explored the specificity and nature of the nucleotide-binding pocket of the CCA-adding enzyme (tRNA nucleotidyltransferase) by using CTP and ATP analogs as substrates for a panel of class I and class II enzymes. Overall, class I and class II enzymes displayed remarkably similar substrate requirements, implying that the mechanism of CCA addition is conserved between enzyme classes despite the absence of obvious sequence homology outside the active site signature sequence. CTP substrates are more tolerant of base modifications than ATP substrates, but sugar modifications prevent incorporation of both CTP and ATP analogs by class I and class II enzymes. Use of CTP analogs (zebularine, pseudoisocytidine, 6-azacytidine, but not 6-azauridine) suggests that base modifications generally do not interfere with recognition or incorporation of CTP analogs by either class I or class II enzymes, and that UTP is excluded because N-3 is a positive determinant and/or O-4 is an antideterminant. Use of ATP analogs (N6-methyladenosine, diaminopurine, purine, 2-aminopurine, and 7-deaza-adenosine, but not guanosine, deoxyadenosine, 2'-O-methyladenosine, 2'-deoxy-2'-fluoroadenosine, or inosine) suggests that base modifications generally do not interfere with recognition or incorporation of ATP analogs by either class I or class II enzymes, and that GTP is excluded because N-1 is a positive determinant and/or the 2-amino and 6-keto groups are antideterminants. We also found that the 3'-terminal sequence of the growing tRNA substrate can affect the efficiency or specificity of subsequent nucleotide addition. Our data set should allow rigorous evaluation of structural hypotheses for nucleotide selection based on existing and future crystal structures.     

CCA-adding enzymes and poly(A) polymerases are all members of the same nucleotidyltransferase superfamily: characterization of the CCA-adding enzyme from the archaeal hyperthermophile Sulfolobus shibatae.

We describe the purification, cloning, and characterization of the CCA-adding enzyme [ATP(CTP):tRNA nucleotidyl transferase] from the thermophilic archaebacterium, Sulfolobus shibatae. Characterization of an archaeal CCA-adding enzyme provides formal proof that the CCA-adding activity is present in all three contemporary kingdoms. Antibodies raised against recombinant, expressed Sulfolobus CCA-adding enzyme reacted specifically with the 48-kDa protein and fully depleted all CCA-adding activity from S. shibatae crude extract. Thus, the cloned cca gene encodes the only CCA-adding activity in S. shibatae. Remarkably, the archaeal CCA-adding enzyme exhibits no strong homology to either the eubacterial or eukaryotic CCA-adding enzymes. Nonetheless, it does possess the active site signature G[SG][LIVMFY]xR[GQ]x5,6D[LIVM][CLIVMFY]3-5 of the nucleotidyltransferase superfamily identified by Holm and Sander (1995, Trends Biochem Sci 20:345-347) and sequence comparisons show that all known CCA-adding enzymes and poly(A) polymerases are contained within this superfamily. Moreover, we propose that the superfamily can now be divided into two (and possibly three) subfamilies: class I, which contains the archaeal CCA-adding enzyme, eukaryotic poly(A) polymerases, and DNA polymerase beta; class II, which contains eubacterial and eukaryotic CCA-adding enzymes, and eubacterial poly(A) polymerases; and possibly a third class containing eubacterial polynucleotide phosphorylases. One implication of these data is that there may have been intraconversion of CCA-adding and poly(A) polymerase activities early in evolution.     

Divergent evolutions of trinucleotide polymerization revealed by an archaeal CCA-adding enzyme structure

CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase], a template-independent RNA polymerase, adds the defined 'cytidine-cytidine-adenosine' sequence onto the 3' end of tRNA. The archaeal CCA-adding enzyme (class I) and eubacterial/eukaryotic CCA-adding enzyme (class II) show little amino acid sequence homology, but catalyze the same reaction in a defined fashion. Here, we present the crystal structures of the class I archaeal CCA-adding enzyme from Archaeoglobus fulgidus, and its complexes with CTP and ATP at 2.0, 2.0 and 2.7 A resolutions, respectively. The geometry of the catalytic carboxylates and the relative positions of CTP and ATP to a single catalytic site are well conserved in both classes of CCA-adding enzymes, whereas the overall architectures, except for the catalytic core, of the class I and class II CCA-adding enzymes are fundamentally different. Furthermore, the recognition mechanisms of substrate nucleotides and tRNA molecules are distinct between these two classes, suggesting that the catalytic domains of class I and class II enzymes share a common origin, and distinct substrate recognition domains have been appended to form the two presently divergent classes.     

Metal-ion-dependent catalysis and specificity of CCA-adding enzymes: a comparison of two classes

The CCA-adding enzymes [ATP(CTP):tRNA nucleotidyl transferases] catalyze synthesis of the conserved and essential CCA sequence to the tRNA 3' end. These enzymes are divided into two classes of distinct structures that differ in the overall orientation of the head to tail domains. However, the catalytic core of the two classes is conserved and contains three carboxylates in a geometry commonly found in DNA and RNA polymerases that use the two-metal-ion mechanism for phosphoryl transfer. Two important aspects of the two-metal-ion mechanism are tested here for CCA enzymes: the dependence on metal ions for catalysis and for specificity of nucleotide addition. Using the archaeal Sulfolobus shibabae enzyme as an example of the class I, and the bacterial Escherichia coli enzyme as an example of the class II, we show that both enzymes depend on metal ions for catalysis, and that both use primarily Mg2+ and Mn2+ as the "productive" metal ions, but several other metal ions such as Ca2+ as the "nonproductive" metal ions. Of the two productive metal ions, Mg2+ specifically promotes synthesis of the correct CCA, whereas Mn2+ preferentially accelerates synthesis of the noncognate CCC and poly(C). Thus, despite evolution of structural diversity of two classes, both classes use metal ions to determine catalysis and specificity. These results provide critical insights into the catalytic mechanism of CCA synthesis to allow the two classes to be related to each other, and to members of the larger family of DNA and RNA polymerases.