A Novel Endonucleolytic Mechanism to Generate the CCA 3 (cid:1) Termini of tRNA Molecules in Thermotoga maritima *

The tRNA 3 (cid:1) -terminal CCA sequence is essential for aminoacylation of the tRNAs and for translation on the ribosome. The tRNAs are transcribed as larger precursor molecules containing 5 (cid:1) and 3 (cid:1) extra sequences. In the tRNAs that do not have the encoded CCA, the 3 (cid:1) extra sequence after the discriminator nucleotide is usually cleaved off by the tRNA 3 (cid:1) processing endoribonuclease (3 (cid:1) tRNase, or RNase Z), and the 3 (cid:1) -terminal CCA residues are added thereto. Here we analyzed Thermotoga maritima 3 (cid:1) tRNase for enzymatic properties using various pre-tRNAs from T. maritima , in which all 46 tRNA genes encode CCA with only one exception. We found that the enzyme has the unprecedented activity that cleaves CCA-containing pre-tRNAs precisely after the CCA sequence, not after the discriminator. The assays for pre-tRNA variants suggest that the CA residues at nucleotides 75 and 76 are required for the enzyme to cleave pre-tRNAs after A at nucleotide 76 and that the cleavage occurs after nucleotide 75 if the sequence is not CA. Intriguingly, the pre-tRNA Met that is the only T. maritima pre-tRNA without the encoded CCA was cleaved after the discriminator. The kinetics

In contrast, the CCA sequences of all Escherichia coli tRNAs are encoded in its genome, and the six exoribonucleases RNase BN, RNase II, polynucleotide phosphorylase, RNase PH, RNase D, and RNase T are involved in the removal of 3Ј trailers to generate the CCA termini (17,18). RNase II and polynucleotide phosphorylase, however, prefer unstructured RNAs such as mRNAs as substrates, so that their roles in tRNA maturation would probably be limited. In the other eubacteria and archaea, percentages of the CCA-coding tRNA genes vary with species from 0 to 100% (Table I). From the above precedents, it was assumed that prokaryotes containing 0 and 100% CCAcoding tRNA genes would primarily utilize the eukaryote-type and E. coli-type systems, respectively, to generate the CCA termini, and that the other prokaryotes would make use of both systems depending on the presence or absence of the encoded CCA sequence.
In the course of compilation of tRNA, 3Ј tRNase, and exoribonuclease genes from available prokaryote genome data, however, we became aware that the Thermotoga maritima genome (19) encodes orthologues to 3Ј tRNase, RNase II, and polynucleotide phosphorylase but no orthologues to RNase BN, RNase PH, RNase D, and RNase T, although its 46 tRNA genes encode CCA with only one exception (Table I). If T. maritima 3Ј tRNase is responsible for removal of 3Ј extra sequences from the pre-tRNAs containing the CCA residues, the reason why the genome preserves CCA in the tRNA genes is a mystery, because 3Ј tRNases so far characterized are believed to cleave pre-tRNAs immediately after the discriminator (6 -14). It should be noted, however, that in some cases additional cleavages were observed in vitro 1-nt upstream, or 1-or 2-nt downstream.
To solve this enigma, we analyzed T. maritima 3Ј tRNase for enzymatic properties using various pre-tRNAs. Here we show that T. maritima 3Ј tRNase has the unprecedented activity that cleaves CCA-containing pre-tRNAs precisely after the CCA sequence, not after the discriminator. We also identify essential residues in substrate and enzyme for the cleavage site selection and the catalysis. The mechanism for 3Ј tRNase to select the cleavage site must have co-evolved with the gain or loss of the CCA sequence in tRNA genes.

EXPERIMENTAL PROCEDURES
Construction of Expression Plasmids for 3Ј tRNases from T. maritima, Thermoplasma acidophilum, and Pyrobaculum aerophilum-Twelve DNA fragments were chemically synthesized to produce the double-stranded DNA encoding T. maritima 3Ј tRNase, in which the codons are optimized for translation in E. coli. The full-length DNA for 3Ј tRNase (data not shown) was created by PCR with the primer pair * 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.
The full-length 3Ј tRNase coding regions of E. coli (915 bp), T. acidophilum (921 bp), and P. aerophilum (861 bp) were PCR-amplified from their genomes. The primer pairs 5ЈEc/3ЈEc, 5ЈTa/3ЈTa, and 5ЈPa/ 3ЈPa (Table II) were used for the amplification of E. coli, T. acidophilum, and P. aerophilum genes, respectively. Each amplified gene was cloned between the BamHI and SalI sites of pQE-80L (Qiagen). We confirmed that the insert regions of pQE/Ec, pQE/Ta, and pQE/Pa are the same as the sequences previously published (GenBank TM accession numbers Q47012, NP_394611, and NP_560582, respectively).
Construction of Expression Plasmids for T. maritima 3Ј tRNase Variants-Based upon pQE/Tm(WT), we constructed its 15 derivatives to express in E. coli 15 different T. maritima 3Ј tRNase variants: pQE/ Tm(D25A) for the enzyme containing a D25A substitution, pQE/Tm-(G27A) for the enzyme containing a G27A substitution, pQE/Tm(V30T) for the enzyme containing a V30T substitution, pQE/Tm(S31Q) for the enzyme containing a S31Q substitution, pQE/Tm(T33Q) for the enzyme containing a T33Q substitution, pQE/Tm(V38A) for the enzyme containing a V38A substitution, pQE/Tm(H48A) for the enzyme containing a H48A substitution, pQE/Tm(G49L) for the enzyme containing a G49L substitution, pQE/Tm(H50A) for the enzyme containing a H50A substitution, pQE/Tm(V51G) for the enzyme containing a V51G substitution, pQE/Tm(D52A) for the enzyme containing a D52A substitution, pQE/Tm(H53A) for the enzyme containing a H53A substitution, pQE/ Tm(A55L) for the enzyme containing an A55L substitution, pQE/Tm-(G49L/V51G) for the enzyme containing G49L and V51G substitutions, and pQE/Tm(43LTR44) for the enzyme containing LTR insertions between residues 43 and 44. These pQE/Tm(WT) derivatives were generated by site-directed mutagenesis by overlap extension using PCR (13,20,21) and by the conventional DNA recombination technique with DNA restriction enzymes and DNA ligase. The primer pairs used for the mutagenesis are listed in Table II. We confirmed that the insert sequences are changed correctly by DNA sequencing.
Expression and Purification of Recombinant Proteins-E. coli strain DH5␣ that harbors a pQE expression plasmid derivative (Qiagen) for prokaryotic 3Ј tRNase was incubated at 37°C in a 250-ml LB medium containing 50 g/ml ampicillin until the A 600 of the culture reached 0.6. At this point, the histidine-tagged protein was induced by adding 0.1 mM isopropyl-␤-D-thiogalactopyranoside. After further incubation at 37°C for 1 h, the cells were harvested by centrifugation. Cell pellets were resuspended in a 10-ml lysis buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5% glycerol, 5 mM ␤-mercaptoethanol) containing 1 mM imidazole. The cells were sonicated and centrifuged at 9500 ϫ g for 30 min. The cleared lysate was incubated with 0.2-ml nickel-agarose beads at 4°C for 1 h. After exhaustive washing, the retained proteins were eluted from the beads with 1 ml of the lysis buffer containing 200 mM imidazole. All of the purification steps were carried out at 4°C.
Pre-tRNA Synthesis-The pre-tRNAs were synthesized in vitro with T7 RNA polymerase (Takara Shuzo) from the synthetic pre-tDNAs containing its promoter. The transcription reactions were carried out in the presence or absence of [␣-32 P]UTP (Amersham Biosciences) under the conditions recommended by the manufacturer (Takara Shuzo), and the transcribed pre-tRNAs were gel-purified.
The unlabeled pre-tRNAs were subsequently labeled with fluorescein according to the manufacturer's protocol (Amersham Biosciences). Briefly, after the removal of the 5Ј-phosphates of the transcripts with bacterial alkaline phosphatase (Takara Shuzo), the transcripts were phosphorylated with ATP␥S using T4 polynucleotide kinase (Takara Shuzo). Then a single fluorescein moiety was appended onto the 5Јphosphorothioate site. The resulting pre-tRNAs with fluorescein were gel-purified before assays.
In Vitro tRNA 3Ј Processing Assay-The 3Ј processing reactions for 32 P-labeled or fluorescein-labeled pre-tRNA were performed with 3Ј The number of CCA-containing tRNA genes versus the number of the total tRNA genes.
tRNases of various origins in a mixture (6 l) containing 10 mM Tris-HCl (pH 8), 1.5 mM dithiothreitol, 25 mM NaCl, and 10 mM MgCl 2 (or 0.2 mM MnCl 2 in kinetic assays) at 60°C. The assays for 3Ј tRNases from other species than T. maritima were carried out at 50°C. After resolution of the reaction products on a 10% polyacrylamide-8 M urea gel, the gel was autoradiographed using an intensifying screen at Ϫ80°C, or analyzed with a Typhoon 9210 (Amersham Biosciences). RNA Sequencing-Unlabeled pre-tRNA (2 pmol) was reacted with T. maritima 3Ј tRNase (50 ng) under the standard assay conditions at 60°C for 10 min, extracted with phenol/chloroform, and precipitated with ethanol. The reaction products dissolved in water were 3Ј-endlabeled with T4 ligase (Takara Shuzo) and [5Ј-32 P]pCp (Amersham Biosciences) at 4°C for 10 h. The 5Ј cleavage product was gel-purified, and its 3Ј-terminal sequence was determined by the chemical RNA sequencing method (22). The gel was autoradiographed using an intensifying screen at Ϫ80°C.

T. maritima 3Ј tRNase Cleaves Pre-tRNAs after CCA-First
of all, we chemically synthesized DNA fragments to produce the double-stranded DNA encoding T. maritima 3Ј tRNase (GenBank TM accession number NP_228673), in which the codons are optimized for translation in E. coli. The full-length DNA for 3Ј tRNase (data not shown) was created by PCR using proof-readable DNA polymerase and cloned into the expression vector pQE-80L. The resulting plasmid pQE/Tm(WT), which is designed to produce 3Ј tRNase containing N-terminal histidines, was introduced into E. coli DH5␣ cells. The enzyme was overexpressed in the cells and purified with nickel-agarose.
We examined the recombinant 3Ј tRNase for in vitro processing of arginine, methionine, and phenylalanine pre-tRNAs from T. maritima, which contain both 5Ј leader and 3Ј trailer (Fig. 1A). Uniformly 32 P-labeled pre-tRNAs were synthesized in vitro with T7 RNA polymerase from the synthetic tDNA templates. T. maritima 3Ј tRNase cleaved pre-tRNA Arg (CCA), pre-tRNA Met (CCA), and pre-tRNA Phe (CCA) endonucleolytically (Fig. 1B). To determine the exact cleavage site, we performed the 3Ј tRNase cleavage reactions for unlabeled pre-tRNAs and subsequently 3Ј-end-labeled the products with [5Ј-32 P]pCp. Each 5Ј cleavage product was subjected to chemical RNA sequencing. Surprisingly, the T. maritima enzyme cleaved off 3Ј trailers precisely after the CCA residues in all three pre-tRNAs ( Fig. 1C).
Nucleotides at 75 and 76 Determine the Cleavage Site-To elucidate which nucleotides are the determinant for the cleavage after CCA, we tested for cleavage the three pre-tRNA Phe variants pre-tRNA Phe (UCA), pre-tRNA Phe (CUA), and pre-tRNA Phe (CCG), which contain UCA, CUA, and CCG, respectively, instead of CCA. These variants were also processed by T. maritima 3Ј tRNase with the exception of pre-tRNA Phe (CCG) ( Fig. 2A). Each cleavage site was determined as above. Interestingly, the cleavage site was shifted to 1-nt upstream in pre-tRNA Phe (CUA), whereas the cleavage site of pre-tRNA Phe -(UCA) was not changed.
Furthermore, three variants, pre-tRNA Arg (CCG), pre-tRNA Arg (GUG), and pre-tRNA Met (CCG), were examined. Pre-tRNA Arg (CCG) and pre-tRNA Arg (GUG) were cleaved after nt 75, whereas cleavage of pre-tRNA Met (CCG) was not detected (Fig. 2B). These results suggest that the CA residues at nt 75 and 76 are required for T. maritima 3Ј tRNase to cleave pre-tRNAs precisely after A at nt 76 and that otherwise the cleavage occurs after nt 75.
Cleavage of Pre-tRNA Met (UAG) after the Discriminator-We also tested for 3Ј tRNase cleavage another pre-tRNA Met (UAG) that is the only pre-tRNA without the encoded CCA. Intrigu- ingly, this exceptional pre-tRNA was cleaved after the discriminator (Fig. 3). This makes sense because pre-tRNAs without the CCA termini need to be cleaved after the discriminator at nt 73, to which tRNA nucleotidyltransferase adds the CCA residues, like eukaryotic pre-tRNAs. The reason why only pre-tRNA Met (UAG) among the other pre-tRNAs tested was cleaved after the discriminator may be because this pre-tRNA contains CCA residues at nt 71-73. T. maritima 3Ј tRNase may be able to recognize these CCA residues as the cleavage site determinant as discussed below.
A CCA Binding Domain in T. maritima 3Ј tRNase-The T. maritima enzyme shows its highest activity in 100 mM NaCl at pH 9 at 60°C in the presence of 10 mM MgCl 2 or 0.5 mM MnCl 2 (data not shown). The kinetic parameters K m and k cat for pre-tRNA Arg (CCA) and pre-tRNA Arg (GUG) were determined in the presence of MgCl 2 or MnCl 2 (Table III). The K m values for pre-tRNA Arg (CCA) in the presence of MgCl 2 and MnCl 2 were 0.3-and 0.4-fold, respectively, smaller than those for pre-tRNA Arg (GUG). This suggests that a specific domain for CCA binding exists in the enzyme. On the other hand, the k cat values for pre-tRNA Arg (CCA) were 0.4-and 0.6-fold, respectively, smaller than those for pre-tRNA Arg (GUG). This may reflect slower release of the CCA-containing product following the chemical cleavage step. As a result, the cleavage efficiency values k cat /K m for pre-tRNA Arg (CCA) were ϳ1.4-fold as large as those for pre-tRNA Arg (GUG) in both conditions. Two Amino Acid Residues Critical for the Cleavage Site Selection-Next we investigated which amino acid residues are responsible for making T. maritima 3Ј tRNase cleave after CCA. We assumed that such residues should be in well conserved regions and be different from residues in the other enzymes that cleave after the discriminator. We selected six residues to be examined; i.e. Val-30, Ser-31, Thr-33, Gly-49, Val-51, and Ala-55 (Fig. 4A). Expression plasmids for single or double amino acid-substituted variants, Tm(V30T), Tm(S31Q), Tm(T33Q), Tm(G49L), Tm(V51G), Tm(G49L/V51G), and Tm(A55L), were constructed by site-directed mutagenesis with PCR based on pQE/Tm(WT). The 3Ј tRNase variants were overexpressed in E. coli and purified as histidine-tagged proteins (Fig. 4B).
These recombinant enzymes were tested for cleavage of pre-tRNA Arg (CCA) and pre-tRNA Arg (GUG), which were 5Ј-end-labeled with fluorescein. The reaction products were analyzed on a sequencing gel with an alkaline ladder to determine the The discriminators at nt 73 are denoted by semicircles. B, the 3Ј processing assays of the uniformly 32 P-labeled pre-tRNAs (0.1 pmol). The cleavage assays were performed with (ϩ) or without (Ϫ) the recombinant histidine-tagged T. maritima 3Ј tRNase (1 pmol). After a 10-min reaction, the products were separated on a denaturing gel. Bars with the letter S and arrowheads with the letter P denote pre-tRNA substrates and cleavage products, respectively. C, cleavage site determination by direct sequencing of a 5Ј-product. The cold pre-tRNAs were assayed and 3Ј-end-labeled with [5Ј-32 P]pCp. The cleavage site was determined by chemical RNA sequencing of each 5Ј-product. The 3Ј-terminal sequence of the 5Ј-product is shown. Each upper panel indicates the 5Ј cleavage product of uniformly 32 P-labeled each pre-tRNA under the same conditions as in B, but on a high resolving gel. cleavage sites. We found that the wild-type enzyme can cleave pre-tRNA Arg (CCA) at the additional minor site between nt 75 and 76 (Fig. 5A). The ratio of the minor to major products increased with the reaction time (data not shown), suggesting that this additional product is generated by the second cut of the original product and is an in vitro artifact.
All the above variants cleaved both pre-tRNAs, although the cleavage efficiency varied (Fig. 5, A and B). Among these variants, only Tm(S31Q) clearly changed the cleavage sites. In pre-tRNA Arg (CCA), the major cleavage site was shifted to 2-nt downstream, and some portion of the molecules were cleaved after the discriminator. In pre-tRNA Arg (GUG), about a half of the molecules were cleaved 1-nt downstream. With respect to Tm(T33Q), the cleavage of pre-tRNA Arg (CCA) after nt 75 was more dominant than that after nt 76. In addition, the cleavage was also detected after nt 74. Pre-tRNA Arg (GUG) was cleaved slightly after nt 74 as well as after nt 75. These results suggest that Ser-31 is a critical residue for selecting the cleavage site and that Thr-33 is also involved in the site selection.
These results also predict that, if 3Ј tRNases contain glutamines at the positions corresponding to residues 31 and 33, the enzymes would cleave pre-tRNAs after the discriminator. Indeed, ELAC1-type short 3Ј tRNases so far characterized have the corresponding two glutamines with the exception of the Arabidopsis thaliana enzyme, which contains a histidine and an alanine instead of glutamines (Fig. 4A). This exceptional  Fig. 1A. The discriminator at nt 73 is denoted by a semicircle. In the middle panels: the T. maritima pre-tRNA variants pre-tRNA Phe (UCA), pre-tRNA Phe (CUA), and pre-tRNA Phe (CCG) (0.1 pmol each) were incubated for 10 min with (ϩ) or without (Ϫ) the recombinant T. maritima 3Ј tRNase (1 pmol). Bars with the letter S and arrowheads with the letter P denote pre-tRNA substrates and cleavage products, respectively. The lower panels: the cleavage site was determined by chemical RNA sequencing of [5Ј-32 P]pCp-labeled each 5Ј-product. The 3Јterminal sequence of the 5Ј-product is shown. B, the T. maritima pre-tRNA variants pre-tRNA Arg (CCG), pre-tRNA Arg -(GUG), and pre-tRNA Met (CCG) were tested as above. case may be due to the lack of eight residues before the histidine motif.
Because three amino acids are missing in between residues 43 and 44 of T. maritima 3Ј tRNase compared with the other enzymes that cleave after the discriminator, we also thought that this difference could be partly responsible for the differential cleavage site selection. We tested the variant Tm(43LTR44), which contains three additional residues, LTR, in between Tyr-43 and Val-44, but no cleavage was observed (Fig. 5, A and B). Additionally, a variant, Tm(V38A), that contains a single amino acid substitution in a non-conserved region, was tested as a control. As expected, it cleaved both pre-tRNAs Arg , but cleavage sites were not shifted (Fig. 5, A  and B).
Essential Residues for the Catalysis-We also examined how substitutions of conserved amino acids affect the cleavage by 3Ј tRNase. Tm(H48A), Tm(H50A), and Tm(H53A), in which the histidines in the histidine motif were substituted with alanines, were found not to cleave both pre-tRNAs Arg at all (Fig. 5,  A and B). Likewise, Tm(D25A) and Tm(D52A) did not process both substrates. In contrast, cleavages by Tm(G27A) were observed at the original sites like the wild-type enzyme. These results suggest that the three histidines and one aspartate in the histidine motif and the aspartate at residue 25 are essential components for the catalysis. Furthermore, it should be noted that the percent cleavage by Tm(G49L) and Tm(G49L/ V51G) decreased significantly (Fig. 5), suggesting that the glycine at residue 49 in the histidine motif is important for the T. maritima 3Ј tRNase activity.
Cleavage Site Selection by Other Prokaryotic 3Ј tRNases-To corroborate the above notion on the cleavage site selection, we investigated properties of 3Ј tRNases from another eubacteria E. coli and two archaea T. acidophilum and P. aerophilum (Fig.  4A). These enzymes were purified as histidine-tagged proteins (Fig. 6A), and assayed for in vitro tRNA 3Ј processing of T. maritima pre-tRNA Arg (CCA), pre-tRNA Arg (GUG), and human pre-tRNA Arg (GUG).
The E. coli enzyme cleaved human pre-tRNA Arg (GUG) primarily after the discriminator (Fig. 6D). This is consistent with the notion that the two glutamines are critical determinants for the cleavage after the discriminator (Fig. 4A). The T. maritima pre-tRNA Arg (CCA) and pre-tRNA Arg (GUG) were not substrates for this enzyme (Fig. 6, B and C). The T. acidophilum enzyme, which has two glutamines as the site selection residues, cleaved human pre-tRNA Arg (GUG) after the discriminator and after G at nt 74 (Fig. 6D). Although T. maritima pre-  tRNA Arg (CCA) was not a substrate, T. maritima pre-tRNA Arg -(GUG) was cleaved after G at nt 74 (Fig. 6, B and C). 3Ј tRNase from P. aerophilum, which contains an arginine at 33 (in the numbering system of T. maritima 3Ј tRNase) instead of the second glutamine (Fig. 4A), processed only T. maritima pre-tRNA Arg (CCA), and the cleavage site was after C at nt 75 (Fig.  6B). On the whole, these results agree with the notion as to the cleavage site selection.

DISCUSSION
The Role of 3Ј tRNase in T. maritima Cells-To the best of our knowledge, T. maritima 3Ј tRNase is the only endoribonuclease so far identified that can cleave pre-tRNAs after CCA. We showed that this enzyme can cleave three T. maritima pre-tRNAs, pre-tRNA Arg (CCA), pre-tRNA Met (CCA), and pre-tRNA Phe (CCA), after the CCA residues. This implies that the enzyme can also cleave the other 42 CCA-containing pre-tRNAs after CCA. The exceptional pre-tRNA Met (UAG) was shown to be processed at the discriminator site, and the resulting pre-tRNA would be a good substrate for the CCA-adding enzyme. Thus, in theory, the CCA termini of all 46 T. maritima tRNAs should be able to be generated by these two enzymes. This consideration suggests that T. maritima cells probably utilize this novel mechanism to generate the CCA 3Ј-termini of tRNA molecules. RNase II and polynucleotide phosphorylase may be involved in shortening of long 3Ј trailers in the same fashion as E. coli (23).
T. maritima 3Ј tRNase processed pre-tRNAs both with and without 7-nt 5Ј leaders (Figs. 1 and 6D) like the mammalian enzyme (24). Although we have currently no information about how long the cellular 5Ј leaders are, if they are less than 7 nt, T. maritima 3Ј tRNase would cleave off 3Ј trailers regardless of 5Ј processing by RNase P (25).
Possible Conformational Change of Pre-tRNA Met (UAG)-The reason why the pre-tRNA Met (UAG) containing UAG instead of CCA at nt 74 -76 was cleaved after the discriminator by T. maritima 3Ј tRNase (Fig. 3B) may be because the enzyme can recognize the CCA residues at nt 71-73 as the cleavage site determinant in some way. One possible mechanism is that the enzyme would recognize an alternative conformation of the pre-tRNA Met (UAG), in which the CCA residues at nt 71-73 protrude from the acceptor stem and G at nt 70 shifts to the discriminator position (Fig. 3A). The T. maritima enzyme would recognize this form of the pre-tRNA Met (UAG) containing a distorted T-stem-loop in the same fashion as normal CCAcontaining pre-tRNAs and would cleave it after A at nt 73. This supposition needs to be tested by examining pre-tRNA variants containing base substitutions that affect the stability of each conformer for 3Ј tRNase cleavage and structure probing.
The Interactions of T. maritima 3Ј tRNase with Pre-tRNAs-The crystal structure of Bacillus cereus ␤-lactamase suggested that the histidine motif forms a part of the active site (26). Consistently, our data suggest that the histidine motif in T. maritima 3Ј tRNase forms an essential part of the catalytic core and that especially the three histidines and one aspartate play a central role for the catalysis.
Pig 3Ј tRNase clearly discriminates the nucleotide C from the others at nt 74 (8,11). In addition, the very short 3Ј trailers 74 CC 75 , 74 CCA 76 , and 74 CCA 76 plus one or two additional 3Ј nt can be distinguished by this enzyme from the other trailers, suggesting that the pig enzyme has a binding domain for the CCA residues. From the present kinetics data (Table III), a CCA binding domain also appears to exist in the T. maritima enzyme.
Whether 3Ј tRNase cleaves pre-tRNAs after the discriminator or after the CCA residues may be determined by the relative position between the catalytic core and the CCA-binding domain. The amino acid residues at 31 and 33 (in the numbering system of T. maritima 3Ј tRNase) appear to be critical for this positioning. When these two residues are glutamines, the active site may be located in the vicinity of the first C binding site, and the cleavage may occur after the discriminator. When the residues are other amino acids such as serine and threonine than glutamine, the catalytic site may be positioned near the A binding site, and pre-tRNAs may be cleaved after CCA. The hydroxyl groups of the serine and threonine residues may be critical for this positioning.
Human ELAC2-type long 3Ј tRNase, which has a phenylalanine and a glutamine at the corresponding sites, cleaves pre-tRNAs after the discriminator, and Saccharomyces cerevisiae ELAC2-type long 3Ј tRNase, which contains a leucine and a threonine at the corresponding sites, also cleaves pre-tRNAs after the discriminator (13,27). Compared with ELAC1-type short 3Ј tRNases, the human and yeast long 3Ј tRNases contain 5 and 6 more residues, respectively, between the site selection residues and the histidine motif, and the yeast enzyme also has several additional residues before the site selection residues (27). This could be the reason why the selection rule for short 3Ј tRNases does not seem to hold in long 3Ј tRNases.
Cleavage of pre-tRNAs containing bases other than CA at nt 75 and 76 occurs after nt 75 (Fig. 2). This may be because these bases do not fit the CCA-binding domain well, and the catalytic core cannot be placed properly. Non-or inefficient cleavage of the CCG-containing pre-tRNAs (Fig. 2) may be attributed to somehow unfavorable interaction with the CCA-binding domain.
Discrimination of Pre-tRNA Species by Prokaryotic 3Ј tRNases and Their Roles in the Cells-The four prokaryotic 3Ј tRNases tested here differed in substrate specificities and cleavage sites. If we can find out a rule to discriminate pre-tRNA substrates, this would become a clue to elucidation of the physiological roles of each enzyme in the cells.
E. coli 3Ј tRNase cleaved only human pre-tRNA Arg (GUG) after the discriminator (Fig. 6). The property that this enzyme cannot cleave T. maritima pre-tRNA Arg (CCA) makes sense, because removal of the CCA residues would be wasteful. The physiological roles of 3Ј tRNase in E. coli cells are not clear, because the exoribonucleases are sufficient for tRNA 3Ј processing. This gene thus might have been preserved for a backup system in case of occasional mutagenesis in the CCA-coding regions. Alternatively, this enzyme may be utilized to process other RNA substrates, including T4 bacteriophage pre-tRNAs FIG. 5. Pre-tRNA cleavage assays using the recombinant T. maritima 3 tRNase variants. A, the 3Ј tRNase variants (1 pmol) were assayed for cleavage of T. maritima pre-tRNA Arg (CCA) (0.1 pmol), which was 5Ј-end-labeled with fluorescein. After a 15-min reaction, the products were separated on a denaturing gel. The cleavage sites were identified using alkaline ladders (L) of the fluoresceinlabeled pre-tRNA Arg (CCA) and the 5Ј cleavage products by the wild-type 3Ј tRNase as size standards. The 5Ј cleavage products are indicated by arrows with the cleavage site nucleotides. I, input RNA with no enzyme; W, wild type; 43-44, the variant Tm(43LTR44). The other variants are represented by the substituted amino acid positions. B, the same assays as above were performed with respect to the fluorescein-labeled T. maritima pre-tRNA Arg (GUG) (0.1 pmol). that lack the CCA residues (28). We could not explain why T. maritima pre-tRNA Arg (GUG) was not cleaved. This is not due to the presence of the 5Ј leader, because human pre-tRNA Arg (GUG) with a 7-nt 5Ј leader was processed as effi-ciently as human pre-tRNA Arg (GUG) without the leader (data not shown).
In good contrast to the T. maritima genome, the CCA sequences are not encoded in all 45 tRNA genes in the T. aci-FIG. 6. Pre-tRNA 3 processing activities of 3 tRNases from E. coli, T. acidophilum, and P. aerophilum. A, protein profiles of the 3Ј tRNases. The histidine-tagged 3Ј tRNases from E. coli (Ec), T. acidophilum (Ta), and P. aerophilum (Pa) were produced in E. coli and purified with nickel agarose. The purified proteins (0.5-1 g) were analyzed on an SDS-10% polyacrylamide gel, and visualized by staining the gel with Coomassie Brilliant Blue R-250. M denotes size standards. B, the 3Ј processing assays for these enzymes (1 pmol) using the fluorescein-labeled T. maritima pre-tRNA Arg (CCA) (0.1 pmol). The reaction mixtures were incubated for 15 min, and the products were subsequently analyzed on a denaturing gel. The 5Ј cleavage products are indicated by arrows with the cleavage site nucleotides. The 5Ј cleavage products by the wild-type T. maritima 3Ј tRNase are used as size standards. I, input RNA; L, alkaline ladder. The major cleavage site by each enzyme is indicated on the pre-tRNA secondary structure. C, the same assays as above were performed with respect to the fluorescein-labeled T. maritima pre-tRNA Arg (GUG) (0.1 pmol). D, the same assays as above were performed with respect to human pre-tRNA Arg (GUG) (0.1 pmol). The 73-nt 5Ј cleavage product by pig 3Ј tRNase (P) was used as a size standard (8,21). L, the alkaline ladder of the fluorescein-labeled T. maritima pre-tRNA Arg (CCA).
dophilum genome with one exception (29). The cellular pre-tRNAs would probably be 3Ј-processed by 3Ј tRNase, although the cleavage site could vary from after nt 74 to after the discriminator depending on pre-tRNA species, judging from our in vitro data. Because T. maritima pre-tRNA Arg (CCA) was not cleaved by T. acidophilum 3Ј tRNase, the exceptional CCAcontaining pre-tRNA would be 3Ј-processed by RNase PH, which is encoded in the genome as the only RNase orthologue among the six E. coli enzymes.
Although the P. aerophilum genome (30) contains 19 CCAcoding and 27 non-CCA-coding tRNA genes, P. aerophilum 3Ј tRNase cleaved only the CCA-containing pre-tRNA Arg in vitro (Fig. 6). Thus, the 19 pre-tRNAs with CCA would be processed by either 3Ј tRNase or RNase PH, which is encoded in the genome as the only RNase orthologue among the six exoribonucleases. With respect to the other 27 pre-tRNAs, RNase PH could trim 3Ј trailers up to the discriminator, or other unidentified RNases could be involved. This case contrasts sharply with the Bacillus subtilis case, where the genome has ϳ73% CCA-coding tRNA genes, and B. subtilis 3Ј tRNase cleaves only CCA-less pre-tRNAs (31).
Evolution of the Mechanism to Generate the CCA 3Ј Termini-The above consideration implies that the mechanism for 3Ј tRNase to select substrates and cleavage sites must have co-evolved with the gain or loss of exoribonuclease genes and the CCA sequence encoded in tRNA genes and would currently be used properly depending on the presence or absence of CCA in pre-tRNAs. Curiously, the T. maritima CCA-adding enzyme groups with A-adding enzymes, not with CCA-adding enzymes or with CC-adding enzymes (32). The presence of the two unusual enzymes 3Ј tRNase and CCA-adding enzyme involved in the 3Ј-terminal CCA generation suggests that T. maritima has evolved very uniquely.