Minimum requirements for substrates of mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes a 3' trailer after the discriminator nucleotide from precursor tRNA (pre-tRNA). To elucidate the minimum requirements for 3' tRNase substrates, we tested small pre-tRNA(Arg) substrates lacking the D and anticodon stem-loop domain for cleavage by purified pig 3' tRNase. A small pre-tRNA (R-ATW) composed of an acceptor stem, an extra loop, a T stem-loop domain, a discriminator nucleotide, and a 3' trailer was cleaved more efficiently than the full-length wild type. The catalytic efficiencies of three R-ATW derivatives, which were constructed to destroy the original T stem base pairs, were also higher than that of the full-length wild type. Pig 3' tRNase efficiently processed a "minihelix" (R-ATM5) that consists of a T stem-loop domain, an acceptor stem, a discriminator nucleotide, and a 3' trailer, while the enzyme never cleaved a "microhelix" that is composed of a T loop, an acceptor stem, a discriminator nucleotide, and a 3' trailer. Five R-ATM5 derivatives that have one to seven base substitutions in the T loop were all cleaved slightly more efficiently than the full-length wild type and slightly less efficiently than R-ATM5. A helix ("minihelixDelta1") one base pair smaller than minihelices was a good substrate, while small helices containing a continuous 10-base pair stem were poor substrates. The cleavage of these three small substrates occurred after the discriminator and one to three nucleotides downstream of the discriminator. From these results, we conclude that minimum substrates for efficient cleavage by mammalian 3' tRNase are minihelices or minihelicesDelta1, in which there seem to be no essential bases.     

Efficient 50S ribosome-catalyzed peptide bond synthesis with an aminoacyl minihelix.

RNA minihelices that recreate the amino acid acceptor domain of the two-domain L-shaped tRNA molecule are substrates for specific aminoacylation by tRNA synthetases. Some lines of evidence suggest that this domain arose independently of and predated the second, anticodon-containing domain. With puromycin and a minihelix charged with alanine, we show here efficient 50S ribosome catalyzed peptide synthesis. The aminoacyl minihelix is as active as aminoacyl tRNA in the synthetic reaction. The high efficiency of the charged minihelix is due to a relatively strong interaction with the 50S particle. In contrast, an aminoacyl RNA fragment that recreates the 3'-side of the tRNA acceptor stem has a much weaker interaction with the 50S particle. These results are consistent with the minihelix domain being the major loci for tRNA interactions with the 50S ribosome. They may also have implications for the historical development of RNA-based systems of peptide synthesis.     

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.     

Identity elements for specific aminoacylation of a tRNA by mammalian lysyl-tRNA synthetase bearing a nonspecific tRNA-interacting factor

Mammalian lysyl-tRNA synthetase (LysRS) has an N-terminal polypeptide chain extension appended to a prokaryotic-like synthetase domain. This extension, termed a tRNA-interacting factor (tIF), possesses a RNA-binding motif [KxxxK(K/R)xxK] that binds nonspecifically the acceptor TPsiC stem-loop domain of tRNA and provides a potent tRNA binding capacity to this enzyme. Consequently, native LysRS aminoacylates a RNA minihelix mimicking the amino acid acceptor stem-loop domain of tRNA(3)(Lys). Here, examination of minihelix recognition showed that mammalian LysRS aminoacylates RNA minihelices without specificity of sequence, revealing that none of the nucleotides from the acceptor TPsiC stem-loop domain are essential determinants of tRNA(Lys) acceptor identity. To test whether the tIF domain reduces the specificity of the synthetase with regard to complete tRNA molecules, aminoacylation of wild-type and mutant noncognate tRNAs by wild-type or N-terminally truncated LysRS was examined. The presence of the UUU anticodon of tRNA(Lys) appeared to be necessary and sufficient to transform yeast tRNA(Asp) or tRNA(i)(Met) into potent lysine acceptor tRNAs. Thus, nonspecific RNA-protein interactions between the acceptor stem of tRNA and the tIF domain do not relax the tRNA specificity of mammalian LysRS. The possibility that interaction of the full-length cognate tRNA with the synthetase is required to induce the catalytic center of the enzyme into a productive conformation is discussed.     

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.     

Distinct kinetic determinants for the stepwise CCA addition to tRNA

The universally conserved CCA sequence is present at the 3′ terminal 74–76 positions of all active tRNA molecules as a functional tag to participate in ribosome protein synthesis. The CCA enzyme catalyzes CCA synthesis in three sequential steps of nucleotide addition at rapid and identical rates. However, the kinetic determinant of each addition is unknown, thus limiting the insights into the kinetic basis of CCA addition. Using our recently developed single turnover kinetics of Escherichia coli CCA enzyme as a model, we show here that the identical rate of the stepwise CCA addition is determined by distinct kinetic parameters. Specifically, the kinetics of C74 and C75 addition is controlled by the chemistry of nucleotidyl transfer, whereas the kinetics of A76 addition is controlled by a prechemistry conformational transition of the active site. In multiple turnover condition, all three steps are controlled by slow product release, indicating enzyme processivity from one addition to the next. However, the processivity decreases as the enzyme progresses to complete the CCA synthesis. Together, these results suggest the existence of a network of diverse kinetic parameters that determines the overall rate of CCA addition for tRNA maturation.     

Mechanism for the definition of elongation and termination by the class II CCA‐adding enzyme

The CCA‐adding enzyme synthesizes the CCA sequence at the 3′ end of tRNA without a nucleic acid template. The crystal structures of class II Thermotoga maritima CCA‐adding enzyme and its complexes with CTP or ATP were determined. The structure‐based replacement of both the catalytic heads and nucleobase‐interacting neck domains of the phylogenetically closely related Aquifex aeolicus A‐adding enzyme by the corresponding domains of the T. maritima CCA‐adding enzyme allowed the A‐adding enzyme to add CCA in vivo and in vitro. However, the replacement of only the catalytic head domain did not allow the A‐adding enzyme to add CCA, and the enzyme exhibited (A, C)‐adding activity. We identified the region in the neck domain that prevents (A, C)‐adding activity and defines the number of nucleotide incorporations and the specificity for correct CCA addition. We also identified the region in the head domain that defines the terminal A addition after CC addition. The results collectively suggest that, in the class II CCA‐adding enzyme, the head and neck domains collaboratively and dynamically define the number of nucleotide additions and the specificity of nucleotide selection.     

Cell growth inhibition by sequence-specific RNA minihelices.

RNA minihelices which reconstruct the 12 base pair acceptor-T psi C domains of transfer RNAs interact with their cognate tRNA synthetases. These substrates lack the anticodons of the genetic code and, therefore, cannot participate in steps of protein synthesis subsequent to aminoacylation. We report here that expression in Escherichia coli of either of two minihelices, each specific for a different amino acid, inhibited cell growth. Inhibition appears to be due to direct competition between the minihelix and its related tRNA for binding to their common synthetase. This competition, in turn, sharply lowers the pool of the specific charged tRNA for protein synthesis. Inhibition is relieved by single nucleotide changes which disrupt the minihelix-synthetase interaction. The results suggest that sequence-specific RNA minihelix substrates bind to cognate synthetases in vivo and can, in principle, act as cell growth regulators. Naturally occurring non-tRNA substrates for aminoacylation may serve a similar purpose.     

Leucyl-tRNA synthetase from the hyperthermophilic bacterium Aquifex aeolicus recognizes minihelices

Aminoacylation of the minihelix mimicking the amino acid acceptor arm of tRNA has been demonstrated in more than 10 aminoacyl-tRNA synthetase systems. Although Escherichia coli or Homo sapiens cytoplasmic leucyl-tRNA synthetase (LeuRS) is unable to charge the cognate minihelix or microhelix, we show here that minihelix(Leu) is efficiently charged by Aquifex aeolicus synthetase, the only known heterodimeric LeuRS (alpha beta-LeuRS). Aminoacylation of minihelices is strongly dependent on the presence of the A73 identity nucleotide and greatly stimulated by destabilization of the first base pair as reported for the E. coli isoleucyl-tRNA synthetase and methionyl-tRNA synthetase systems. In the E. coli LeuRS system, the anticodon of tRNA(Leu) is not important for recognition by the synthetase. However, the addition of RNA helices that mimic the anticodon domain stimulates minihelix(Leu) charging by alpha beta-LeuRS, indicating possible domain-domain communication within alpha beta-LeuRS. The leucine-specific domain of alpha beta-LeuRS is responsible for minihelix recognition. To ensure accurate translation of the genetic code, LeuRS functions to hydrolyze misactivated amino acids (pretransfer editing) and misaminoacylated tRNA (posttransfer editing). In contrast to tRNA(Leu), minihelix(Leu) is unable to induce posttransfer editing even upon the addition of the anticodon domain of tRNA. Therefore, the context of tRNA is crucial for the editing of mischarged products. However, the minihelix(Leu) cannot be misaminoacylated, perhaps because of the tRNA-independent pretransfer editing activity of alpha beta-LeuRS.     

RNA determinants for translational editing. Mischarging a minihelix substrate by a tRNA synthetase

The fidelity of protein synthesis requires efficient discrimination of amino acid substrates by aminoacyl-tRNA synthetases. Accurate discrimination of the structurally similar amino acids, valine and isoleucine, by isoleucyl-tRNA synthetase (IleRS) results, in part, from a hydrolytic editing reaction, which prevents misactivated valine from being stably joined to tRNAIle. The editing reaction is dependent on the presence of tRNAIle, which contains discrete D-loop nucleotides that are necessary to promote editing of misactivated valine. RNA minihelices comprised of just the acceptor-TPsiC helix of tRNAIle are substrates for specific aminoacylation by IleRS. These substrates lack the aforementioned D-loop nucleotides. Because minihelices contain determinants for aminoacylation, we thought that they might also play a role in editing that has not previously been recognized. Here we show that, in contrast to tRNAIle, minihelixIle is unable to trigger the hydrolysis of misactivated valine and, in fact, is mischarged with valine. In addition, mutations in minihelixIle that enhance or suppress charging with isoleucine do the same with valine. Thus, minihelixIle contains signals for charging (by IleRS) that are independent of the amino acid and, by itself, minihelixIle provides no determinants for editing. An RNA hairpin that mimics the D-stem/loop of tRNAIle is also unable to induce the hydrolysis of misactivated valine, both by itself and in combination with minihelixIle. Thus, the native tertiary fold of tRNAIle is required to promote efficient editing. Considering that the minihelix is thought to be the more ancestral part of the tRNA structure, these results are consistent with the idea that, during the development of the genetic code, RNA determinants for editing were added after the establishment of an aminoacylation system.     

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: essential elements for recognition of tRNA substrates within the anticodon stem-loop

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes the alkylation of the exocyclic amine of A37 by a dimethylallyl unit in tRNAs with an adenosine in the third anticodon position (position 36). By use of purified recombinant enzyme, steady- state kinetic studies were conducted with chemically synthesized RNA oligoribonucleotides to determine the essential elements within the tRNA anticodon stem-loop structure required for recognition by the enzyme. A 17-base oligoribonucleotide corresponding to the anticodon stem-loop of E. coli tRNA(Phe) formed a stem-loop minihelix (minihelix(Phe)) when annealed rapidly on ice, while the same molecule formed a duplex structure with a central loop when annealed slowly at higher concentrations. Both the minihelix and duplex structures gave k(cat)s similar to that for the normal substrate (full-length tRNA(Phe) unmodified at A37), although the K(m) for minihelix(Phe) was approximately 180-fold higher than full-length tRNA. The A36-A37-A38 motif, which is completely conserved in tRNAs modified by the enzyme, was found to be important for modification. Changing A36 to G in the minihelix resulted in a 260-fold reduction in k(cat) compared to minihelix(Phe) and a 13-fold increase in K(m). An A38G variant was modified with a 9-fold reduction in k(cat) and a 5-fold increase in K(m). A random coil 17-base oligoribonucleotide in which the loop sequence of E. coli tRNA(Phe) was preserved, but the 5 base pair helix stem was completely disrupted and showed no measurable activity, indicating that a helix-loop structure is essential for recognition. Finally, altering the identity of several base pairs in the helical stem did not have a major effect on catalytic efficiency, suggesting that the enzyme does not make base-specific contacts important for binding or catalysis in this region.     

A recurrent general RNA binding domain appended to plant methionyl-tRNA synthetase acts as a cis-acting cofactor for aminoacylation

The cDNA encoding rice methionyl-tRNA synthetase was isolated. The protein exhibited a C-terminal polypeptide appended to a classical MetRS domain. This supplementary domain is related to endothelial monocyte activating polypeptide II (EMAPII), a cytokine produced in mammals after cleavage of p43, a component of the multisynthetase complex. It is also related to Arc1p and Trbp111, two tRNA binding proteins. We expressed rice MetRS and a derivative with a deletion of its EMAPII-like domain. Band-shift analysis showed that this extra-domain provides MetRS with non-specific tRNA binding properties. The EMAPII-like domain contributed a 10-fold decrease in K:(M) for tRNA in the aminoacylation reaction catalyzed by the native enzyme, as compared with the C-terminally truncated MetRS. Consequently, the EMAPII domain provides MetRS with a better catalytic efficiency at the free tRNA concentration prevailing in vivo. This domain binds the acceptor minihelix of tRNA(Met) and facilitates its aminoacylation. These results suggest that the EMAPII module could be a relic of an ancient tRNA binding domain that was incorporated into primordial synthetases for aminoacylation of RNA minihelices taken as the ancestor of modern tRNA.     

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.     

The C-terminal end of the Trypanosoma brucei editing deaminase plays a critical role in tRNA binding

Adenosine to inosine editing at the wobble position allows decoding of multiple codons by a single tRNA. This reaction is catalyzed by adenosine deaminases acting on tRNA (ADATs) and is essential for viability. In bacteria, the anticodon-specific enzyme is a homodimer that recognizes a single tRNA substrate (tRNA(Arg)(ACG)) and can efficiently deaminate short anticodon stem-loop mimics of this tRNA in vitro. The eukaryal enzyme is composed of two nonidentical subunits, ADAT2 and ADAT3, which upon heterodimerization, recognize seven to eight different tRNAs as substrates, depending on the organism, and require a full-length tRNA for activity. Although crystallographic data have provided clues to why the bacterial deaminase can utilize short substrates, residues that provide substrate binding and recognition with the eukaryotic enzymes are not currently known. In the present study, we have used a combination of mutagenesis, binding studies, and kinetic analysis to explore the contribution of individual residues in Trypanosoma brucei ADAT2 (TbADAT2) to tRNA recognition. We show that deletion of the last 10 amino acids at the C terminus of TbADAT2 abolishes tRNA binding. In addition, single alanine replacements of a string of positively charged amino acids (KRKRK) lead to binding defects that correlate with losses in enzyme activity. This region, which we have termed the KR-domain, provides a first glance at key residues involved in tRNA binding by eukaryotic tRNA editing deaminases.     

The intron of tRNA-TrpCCA is dispensable for growth and translation of Saccharomyces cerevisiae

A part of eukaryotic tRNA genes harbor an intron at one nucleotide 3' to the anticodon, so that removal of the intron is an essential processing step for tRNA maturation. While some tRNA introns have important roles in modification of certain nucleotides, essentiality of the tRNA intron in eukaryotes has not been tested extensively. This is partly because most of the eukaryotic genomes have multiple genes encoding an isoacceptor tRNA. Here, we examined whether the intron of tRNA-Trp(CCA) genes, six copies of which are scattered on the genome of yeast, Saccharomyces cerevisiae, is essential for growth or translation of the yeast in vivo. We devised a procedure to remove all of the tRNA introns from the yeast genome iteratively with marker cassettes containing both positive and negative markers. Using this procedure, we removed all the introns from the six tRNA-Trp(CCA) genes, and found that the intronless strain grew normally and expressed tRNA-Trp(CCA) in an amount similar to that of the wild-type genes. Neither incorporation of (35)S-labeled amino acids into a TCA-insoluble fraction nor the major protein pattern on SDS-PAGE/2D gel were affected by complete removal of the intron, while expression levels of some proteins were marginally affected. Therefore, the tRNA-Trp(CCA) intron is dispensable for growth and bulk translation of the yeast. This raises the possibility that some mechanism other than selective pressure from translational efficiency maintains the tRNA intron on the yeast genome.     

Major anticodon-binding region missing from an archaebacterial tRNA synthetase

The small size of the archaebacterial Methanococcus jannaschii tyrosyl-tRNA synthetase may give insights into the historical development of tRNAs and tRNA synthetases. The L-shaped tRNA has two major arms-the acceptor.TpsiC minihelix with the amino acid attachment site and the anticodon-containing arm. The structural organization of the tRNA synthetases parallels that of tRNAs. The more ancient synthetase domain contains the active site and insertions that interact with the minihelix portion of the tRNA. A second, presumably more recent, domain interacts with the anticodon-containing section of tRNA. The small size of the M. jannaschii enzyme is due to the absence of most of the second domain, including a segment thought to bind to the anticodon. Consistent with the absence of an anticodon-binding motif, a mutation of the central base of the anticodon had a relatively small effect on the aminoacylation efficiency of the M. jannaschii enzyme. In contrast, others showed earlier that the same mutation severely reduced charging by a normal-sized bacterial enzyme that has the aforementioned anticodon-binding motif. However, the M. jannaschii enzyme has a peptide insertion into its catalytic domain. This insertion is shared with all other tyrosyl-tRNA synthetases and is needed for a critical minihelix interaction. We show that the M. jannaschii enzyme is active on minihelix substrates over a wide temperature range and has preserved the same peptide-dependent minihelix specificity seen in other tyrosine enzymes. These findings are consistent with the concept that anticodon interactions of tRNA synthetases were later adaptations to the emerging synthetase-tRNA complex that was originally framed around the minihelix.     

Mode of Action of RNase BN/RNase Z on tRNA Precursors: RNase BN DOES NOT REMOVE THE CCA SEQUENCE FROM tRNA*

RNase BN, the Escherichia coli homolog of RNase Z, was previously shown to act as both a distributive exoribonuclease and an endoribonuclease on model RNA substrates and to be inhibited by the presence of a 3′-terminal CCA sequence. Here, we examined the mode of action of RNase BN on bacteriophage and bacterial tRNA precursors, particularly in light of a recent report suggesting that RNase BN removes CCA sequences (Takaku, H., and Nashimoto, M. (2008) Genes Cells 13, 1087–1097). We show that purified RNase BN can process both CCA-less and CCA-containing tRNA precursors. On CCA-less precursors, RNase BN cleaved endonucleolytically after the discriminator nucleotide to allow subsequent CCA addition. On CCA-containing precursors, RNase BN acted as either an exoribonuclease or endoribonuclease depending on the nature of the added divalent cation. Addition of Co²⁺ resulted in higher activity and predominantly exoribonucleolytic activity, whereas in the presence of Mg²⁺, RNase BN was primarily an endoribonuclease. In no case was any evidence obtained for removal of the CCA sequence. Certain tRNA precursors were extremely poor substrates under any conditions tested. These findings provide important information on the ability of RNase BN to process tRNA precursors and help explain the known physiological properties of this enzyme. In addition, they call into question the removal of CCA sequences by RNase BN.