A Novel Endonucleolytic Mechanism to Generate the CCA 3′ Termini of tRNA Molecules in Thermotoga maritima

The tRNA 3′-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′ and 3′ extra sequences. In the tRNAs that do not have the encoded CCA, the 3′ extra sequence after the discriminator nucleotide is usually cleaved off by the tRNA 3′ processing endoribonuclease (3′ tRNase, or RNase Z), and the 3′-terminal CCA residues are added thereto. Here we analyzed Thermotoga maritima 3′ 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-tRNAMet that is the only T. maritima pre-tRNA without the encoded CCA was cleaved after the discriminator. The kinetics data imply the existence of a CCA binding domain in T. maritima 3′ tRNase. We also identified two amino acid residues critical for the cleavage site selection and several residues essential for the catalysis. Analysis of cleavage sites by 3′ tRNases from another eubacteria Escherichia coli and two archaea Thermoplasma acidophilum and Pyrobaculum aerophilum corroborates the importance of the two amino acid residues for the cleavage site selection. The tRNA 3′-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′ and 3′ extra sequences. In the tRNAs that do not have the encoded CCA, the 3′ extra sequence after the discriminator nucleotide is usually cleaved off by the tRNA 3′ processing endoribonuclease (3′ tRNase, or RNase Z), and the 3′-terminal CCA residues are added thereto. Here we analyzed Thermotoga maritima 3′ 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-tRNAMet that is the only T. maritima pre-tRNA without the encoded CCA was cleaved after the discriminator. The kinetics data imply the existence of a CCA binding domain in T. maritima 3′ tRNase. We also identified two amino acid residues critical for the cleavage site selection and several residues essential for the catalysis. Analysis of cleavage sites by 3′ tRNases from another eubacteria Escherichia coli and two archaea Thermoplasma acidophilum and Pyrobaculum aerophilum corroborates the importance of the two amino acid residues for the cleavage site selection. Every single tRNA molecule ends with the sequence CCA (1Sprinzl M. Horn C. Brown M. Ioudovitch A. Steinberg S. Nucleic Acids Res. 1998; 26: 148-153Crossref PubMed Scopus (818) Google Scholar). This 3′-terminal sequence is essential for aminoacylation of the tRNAs (2Tamura K. Nameki N. Hasegawa T. Shimizu M. Himeno H. J. Biol. Chem. 1994; 269: 22173-22177Abstract Full Text PDF PubMed Google Scholar) and for translation on the ribosome (3Green R. Noller H.F. Annu. Rev. Biochem. 1997; 66: 679-716Crossref PubMed Scopus (425) Google Scholar) in all organisms. The tRNAs are transcribed as larger precursor molecules, which subsequently undergo various processing steps such as removal of 5′ and 3′ extra sequences to generate mature tRNAs (4Deutscher M.P. Nucleic Acids Res. Mol. Biol. 1990; 39: 209-240Crossref PubMed Scopus (105) Google Scholar). Because generally eukaryotic tRNA genes do not encode the CCA sequence, the eukaryotic tRNAs are supplemented with the CCA residues by tRNA nucleotidyltransferase (4Deutscher M.P. Nucleic Acids Res. Mol. Biol. 1990; 39: 209-240Crossref PubMed Scopus (105) Google Scholar, 5Schurer H. Schiffer S. Marchfelder A. Morl M. Biol. Chem. 2001; 382: 1147-1156Crossref PubMed Scopus (71) Google Scholar). It is believed that the discriminator nucleotide that protrudes from the aminoacyl stem, to which CCA is added, is generated by removing the 3′ extra sequence primarily with tRNA 3′ processing endoribonuclease (3′ tRNase, or RNase Z) 1The abbreviations used are: 3′ tRNase, tRNA 3′ processing endoribonuclease; nt, nucleotide(s); ATPγS, adenosine 5′-O-(3-thiotriphosphate). (6Castano J.G. Tobian J.A. Zasloff M. J. Biol. Chem. 1985; 260: 9002-9008Abstract Full Text PDF PubMed Google Scholar, 7Oommen A. Li X.Q. Gegenheimer P. Mol. Cell. Biol. 1992; 12: 865-875Crossref PubMed Scopus (33) Google Scholar, 8Nashimoto M. Nucleic Acids Res. 1997; 25: 1148-1155Crossref PubMed Scopus (73) Google Scholar, 9Levinger L. Bourne R. Kolla S. Cylin E. Russell K. Wang X. Mohan A. J. Biol. Chem. 1998; 273: 1015-1025Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 10Nashimoto M. Tamura M. Kaspar R.L. J. Mol. Biol. 1999; 287: 727-740Crossref PubMed Scopus (26) Google Scholar, 11Mohan A. Whyte S. Wang X. Nashimoto M. Levinger L. RNA (N. Y.). 1999; 5: 245-256Crossref PubMed Scopus (54) Google Scholar, 12Schiffer S. Rosch S. Marchfelder A. EMBO J. 2002; 21: 2769-2777Crossref PubMed Scopus (152) Google Scholar, 13Takaku H. Minagawa A. Takagi M. Nashimoto M. Nucleic Acids Res. 2003; 31: 2272-2278Crossref PubMed Scopus (152) Google Scholar, 14Schiffer S. Rosch S. Marchfelder A. Biol. Chem. 2003; 384: 333-342Crossref PubMed Scopus (23) Google Scholar) and possibly in some circumstances with some unidentified exoribonucleases (15Papadimitriou A. Gross H.J. Eur. J. Biochem. 1996; 242: 747-759Crossref PubMed Scopus (24) Google Scholar, 16Yoo C.J. Wolin S.L. Cell. 1997; 89: 393-402Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). 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 3′ trailers to generate the CCA termini (17Li Z. Deutscher M.P. Cell. 1996; 86: 503-512Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 18Deutscher M.P. Li Z. Prog. Nucleic Acids Res. Mol. Biol. 2001; 66: 67-105Crossref PubMed Scopus (120) Google Scholar). 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% CCA-coding 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.Table IGenes for tRNAs, 3′ tRNase, and exoribonucleasesSpeciesCCA/totalaThe number of CCA-containing tRNA genes versus the number of the total tRNA genes.EndoBNIIPNPPHDTBorrelia burgdorferi0/32+−+−−−−Chlamydophila pneumoniae0/38+−++−−−Chlamydophila trachomatis1/45+−++−−−Treponema pallidum10/31+−++−−−Buchnera sp.15/45+−+−++−Mycobacterium leprae15/45+−+−++−Staphylococcus aureus N31537/61+++−−−−Bacillus subtilis63/86+++++−−Aquifex aeolicus32/43+−+++−−Bacillus halodurans63/78+++−+−+Deinococcus radiodurans48/49+−+++−−Haemophilus influenzae55/56−++++++Thermotoga maritima45/46+−++−−−Campylobacter jejuni42/42−+++−−−Caulobacter crescentus51/51+−+++++Escherichia coli86/86+++++++Helicobacter pylori 2669536/36−+++−−−Mycoplasma genitalium35/35−−−+−−−Mycoplasma penetrans36/36−−−−−−−Patteurella multocida51/51+++++++Rickettsia prowazekii32/32−++−++−Pseudomonas aeruginosa62/62+++−+++Ureaplasma urealyticum29/29−−−+−−−Vibrio cholerae98/98+++++++Xylella fastidiosa49/49−++++++Archaeoglobus fulgidus0/46+−−+−−−Halobacterium sp.0/45+−−−+−−Thermoplasma volcanium0/45+−−−+−−Sulfolobus solfataricus1/45+−−−+−−Sulfolobus tokodaii1/45+−−−+−−Thermoplasma acidophilum1/45+−−−+−−Methanobacterium thermoautotrophicum1/39+−−−+−−Pyrobaculum aerophilum19/46+−−−+−−Methanococcus jannaschii25/36+−−−−−−Pyrococcus abyssi44/46+−−−+−−Pyrococcus furiosus46/46+−−−+−−Pyrococcus horikoshii46/46+−−−+−−Aeropyrum pernix46/46+−−−+−−a The number of CCA-containing tRNA genes versus the number of the total tRNA genes. Open table in a new tab In the course of compilation of tRNA, 3′ tRNase, and exoribonuclease genes from available prokaryote genome data, however, we became aware that the Thermotoga maritima genome (19Nelson K.E. Clayton R.A. Gill S.R. Gwinn M.L. Dodson R.J. Haft D.H. Hickey E.K. Peterson J.D. Nelson W.C. Ketchum K.A. McDonald L. Utterback T.R. Malek J.A. Linher K.D. Garrett M.M. Stewart A.M. Cotton M.D. Pratt M.S. Phillips C.A. Richardson D. Heidelberg J. Sutton G.G. Fleischmann R.D. Eisen J.A. White O. Salzberg S.L. Smith H.O. Venter J.C. Fraser C.M. Nature. 1999; 399: 323-329Crossref PubMed Scopus (1212) Google Scholar) encodes orthologues to 3′ 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 3′ tRNase is responsible for removal of 3′ 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 3′ tRNases so far characterized are believed to cleave pre-tRNAs immediately after the discriminator (6Castano J.G. Tobian J.A. Zasloff M. J. Biol. Chem. 1985; 260: 9002-9008Abstract Full Text PDF PubMed Google Scholar, 7Oommen A. Li X.Q. Gegenheimer P. Mol. Cell. Biol. 1992; 12: 865-875Crossref PubMed Scopus (33) Google Scholar, 8Nashimoto M. Nucleic Acids Res. 1997; 25: 1148-1155Crossref PubMed Scopus (73) Google Scholar, 9Levinger L. Bourne R. Kolla S. Cylin E. Russell K. Wang X. Mohan A. J. Biol. Chem. 1998; 273: 1015-1025Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 10Nashimoto M. Tamura M. Kaspar R.L. J. Mol. Biol. 1999; 287: 727-740Crossref PubMed Scopus (26) Google Scholar, 11Mohan A. Whyte S. Wang X. Nashimoto M. Levinger L. RNA (N. Y.). 1999; 5: 245-256Crossref PubMed Scopus (54) Google Scholar, 12Schiffer S. Rosch S. Marchfelder A. EMBO J. 2002; 21: 2769-2777Crossref PubMed Scopus (152) Google Scholar, 13Takaku H. Minagawa A. Takagi M. Nashimoto M. Nucleic Acids Res. 2003; 31: 2272-2278Crossref PubMed Scopus (152) Google Scholar, 14Schiffer S. Rosch S. Marchfelder A. Biol. Chem. 2003; 384: 333-342Crossref PubMed Scopus (23) Google Scholar). 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 3′ tRNase for enzymatic properties using various pre-tRNAs. Here we show that T. maritima 3′ 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 3′ tRNase to select the cleavage site must have co-evolved with the gain or loss of the CCA sequence in tRNA genes. Construction of Expression Plasmids for 3′ tRNases from T. maritima, Thermoplasma acidophilum, and Pyrobaculum aerophilum—Twelve DNA fragments were chemically synthesized to produce the double-stranded DNA encoding T. maritima 3′ tRNase, in which the codons are optimized for translation in E. coli. The full-length DNA for 3′ tRNase (data not shown) was created by PCR with the primer pair 5′Tm/3′Tm (Table II) using Pyrobest DNA polymerase (Takara Shuzo) and cloned between the SphI and sites of the The was to produce 3′ tRNase containing used for and to Open table in a new tab The full-length 3′ tRNase of E. coli T. acidophilum and P. aerophilum were from their The primer and (Table II) were used for the of E. T. acidophilum, and P. aerophilum was cloned between the and sites of We that the of and are the as the sequences and Construction of Expression Plasmids for T. maritima 3′ tRNase we its to in E. coli T. maritima 3′ tRNase for the enzyme containing a for the enzyme containing a for the enzyme containing a for the enzyme containing a for the enzyme containing a for the enzyme containing a for the enzyme containing a for the enzyme containing a for the enzyme containing a for the enzyme containing a for the enzyme containing a for the enzyme containing a for the enzyme containing for the enzyme containing and and for the enzyme containing between residues and were generated by by using PCR H. Minagawa A. Takagi M. Nashimoto M. Nucleic Acids Res. 2003; 31: 2272-2278Crossref PubMed Scopus (152) Google Scholar, PubMed Scopus Google Scholar, M. Nashimoto C. Tamura M. Kaspar R.L. K. J. Mol. Biol. 2001; PubMed Scopus Google Scholar) and by the DNA with DNA and DNA The primer used for the are in We that the sequences are by DNA Expression and of coli that a for 3′ tRNase was at in a containing the of the this the was by at for the were by were in a containing The were and at for The was with at for the were from the with of the containing of the steps were at pre-tRNAs were synthesized in vitro with RNA polymerase (Takara Shuzo) from the containing its The were in the presence or absence of the by the (Takara and the transcribed pre-tRNAs were The pre-tRNAs were subsequently with to the after the removal of the of the with (Takara the were with using polynucleotide (Takara a single was the The pre-tRNAs with were In tRNA 3′ 3′ processing for or pre-tRNA were with 3′ tRNases of various in a containing and in at The assays for 3′ tRNases from other species T. maritima were at of the on a the was using at or analyzed with a RNA pre-tRNA was with T. maritima 3′ tRNase the at for with and with The in were with (Takara Shuzo) and at for The 5′ cleavage was and its 3′-terminal sequence was by the RNA S. A. PubMed Scopus Google Scholar). The was using at T. maritima 3′ tRNase after of we chemically synthesized DNA fragments to produce the double-stranded DNA encoding T. maritima 3′ tRNase number in which the codons are optimized for translation in E. coli. The full-length DNA for 3′ tRNase (data not shown) was created by PCR using DNA polymerase and cloned the The which is to produce 3′ tRNase containing was E. coli The enzyme was in the and with We the 3′ tRNase for in vitro processing of and pre-tRNAs from T. maritima, which both 5′ and 3′ pre-tRNAs were synthesized in vitro with RNA polymerase from the T. maritima 3′ tRNase cleaved and To the cleavage we the 3′ tRNase cleavage for pre-tRNAs and subsequently the with 5′ cleavage was to RNA the T. maritima enzyme cleaved off 3′ trailers precisely after the CCA residues in all pre-tRNAs at 75 and 76 the which nucleotides are the for the cleavage after CCA, we for cleavage the variants and which and respectively, of variants were also by T. maritima 3′ tRNase with the exception of cleavage site was as the cleavage site was to 1-nt in the cleavage site of was not and were and were cleaved after cleavage of was not suggest that the CA residues at 75 and 76 are required for T. maritima 3′ tRNase to cleave pre-tRNAs precisely after A at 76 and that the cleavage occurs after of after the also for 3′ tRNase cleavage another that is the only pre-tRNA without the encoded Intriguingly, this pre-tRNA was cleaved after the discriminator This because pre-tRNAs without the CCA termini to be cleaved after the discriminator at to which tRNA nucleotidyltransferase the CCA residues, eukaryotic pre-tRNAs. The reason why only the other pre-tRNAs was cleaved after the discriminator be because this pre-tRNA CCA residues at T. maritima 3′ tRNase be to CCA residues as the cleavage site as A CCA in T. maritima 3′ T. maritima enzyme its activity in at at in the presence of or (data not The and for and were in the presence of or (Table The for in the presence of and were and respectively, for This that a domain for CCA binding in the the other the for were and respectively, for This of the CCA-containing the cleavage a the cleavage for were as as for in both for pre-tRNA cleavage by T. maritima 3′ Open table in a new tab for the we which amino acid residues are responsible for T. maritima 3′ tRNase cleave after We assumed that such residues should be in and be from residues in the other that cleave after the discriminator. We six residues to be and Expression for single or amino and were by with PCR on The 3′ tRNase variants were in E. coli and as were for cleavage of and which were with The were analyzed on a with to the cleavage We found that the enzyme cleave at the additional site between 75 and 76 The of the to with the (data not that this additional is generated by the of the and is in vitro the above variants cleaved both although the cleavage A and only the cleavage In the cleavage site was to 2-nt and some of the molecules were cleaved after the discriminator. In a of the molecules were cleaved 1-nt downstream. to the cleavage of after 75 was that after In the cleavage was also after was cleaved after as as after suggest that is a critical for the cleavage site and that is also involved in the site selection. also if 3′ tRNases at the to residues and the would cleave pre-tRNAs after the discriminator. 3′ tRNases so far characterized have the two with the exception of the which a and of This be to the of residues the Because amino are in between residues and of T. maritima 3′ tRNase with the other that cleave after the we also that this be responsible for the cleavage site selection. We the which additional residues, in between and but no cleavage was observed A and a that a single amino acid in a was as a it cleaved both but cleavage sites were not A and for the also of amino the cleavage by and in which the in the were with were found not to cleave both at all A and and not both In contrast, cleavages by were observed at the sites the suggest that the and one in the and the at are essential for the catalysis. it should be that the cleavage by and that the at in the is for the T. maritima 3′ tRNase by 3′ the above on the cleavage site we properties of 3′ tRNases from another eubacteria E. coli and two archaea T. acidophilum and P. aerophilum were as and for in vitro tRNA 3′ processing of T. maritima and The E. coli enzyme cleaved primarily after the discriminator This is with the that the two are critical for the cleavage after the discriminator The T. maritima and were not for this enzyme and The T. acidophilum which has two as the site selection residues, cleaved after the discriminator and after at T. maritima was not a T. maritima was cleaved after at and 3′ tRNase from P. which at the of T. maritima 3′ of the only T. maritima and the cleavage site was after at 75 the with the as to the cleavage site selection. The of 3′ tRNase in T. maritima the of T. maritima 3′ tRNase is the only endoribonuclease so far identified that cleave pre-tRNAs after We that this enzyme cleave T. maritima and after the CCA This that the enzyme also cleave the other CCA-containing pre-tRNAs after The was to be at the discriminator and the pre-tRNA would be a substrate for the in the CCA termini of all 46 T. maritima tRNAs should be to be generated by two This that T. maritima probably utilize this mechanism to generate the CCA of tRNA RNase II and polynucleotide phosphorylase be involved in of 3′ trailers in the as E. coli Z. Deutscher M.P. RNA (N. Y.). 2002; PubMed Scopus Google Scholar). T. maritima 3′ tRNase pre-tRNAs both with and without 5′ and the enzyme M. S. Tamura M. Kaspar R.L. Nucleic Acids Res. 1999; PubMed Scopus Google Scholar). we have no the 5′ if are nt, T. maritima 3′ tRNase would cleave off 3′ trailers of 5′ processing by RNase R. D. S. Nucleic Acids Res. 2001; PubMed Scopus Google Scholar). of reason why the containing of CCA at was cleaved after the discriminator by T. maritima 3′ tRNase be because the enzyme the CCA residues at as the cleavage site in some mechanism is that the enzyme would of the in which the CCA residues at from the and at to the discriminator The T. maritima enzyme would this of the containing a in the as CCA-containing pre-tRNAs and would cleave it after A at This to be by pre-tRNA variants containing that the of for 3′ tRNase cleavage and The of T. maritima 3′ tRNase with of that the a of the site A. S. E. M. C. O. EMBO J. PubMed Scopus Google Scholar). data suggest that the in T. maritima 3′ tRNase essential of the and that the and one a for the catalysis. 3′ tRNase the nucleotide from the at M. Nucleic Acids Res. 1997; 25: 1148-1155Crossref PubMed Scopus (73) Google Scholar, 11Mohan A. Whyte S. Wang X. Nashimoto M. Levinger L. RNA (N. Y.). 1999; 5: 245-256Crossref PubMed Scopus (54) Google Scholar). In the 3′ trailers and one or two additional 3′ be by this enzyme from the other that the enzyme has a binding domain for the CCA From the kinetics data (Table a CCA binding domain also to in the T. maritima 3′ tRNase cleaves pre-tRNAs after the discriminator or after the CCA residues be by the between the and the The amino acid residues at and the of T. maritima 3′ to be critical for this two residues are the site be in the of the binding and the cleavage after the discriminator. the residues are other amino such as and the site be the A binding and pre-tRNAs be cleaved after The of the and residues be critical for this 3′ tRNase, which has a and a at the cleaves pre-tRNAs after the and 3′ tRNase, which a and a at the also cleaves pre-tRNAs after the discriminator H. Minagawa A. Takagi M. Nashimoto M. Nucleic Acids Res. 2003; 31: 2272-2278Crossref PubMed Scopus (152) Google Scholar, J. D.H. M. A. P. M. M. D. C. S. R. D. T. K.E. A. J. A. Peterson S. M. Smith R. J. A. M. A.M. S. J. 2001; PubMed Scopus Google Scholar). with 3′ the and 3′ tRNases and residues, respectively, between the site selection residues and the and the enzyme also has several additional residues the site selection residues J. D.H. M. A. P. M. M. D. C. S. R. D. T. K.E. A. J. A. Peterson S. M. Smith R. J. A. M. A.M. S. J. 2001; PubMed Scopus Google Scholar). This be the reason why the selection for 3′ tRNases not to in 3′ of pre-tRNAs containing other CA at 75 and 76 occurs after 75 This be because do not the domain and the be or cleavage of the pre-tRNAs be to with the of by 3′ tRNases and in the 3′ tRNases in substrate and cleavage If we a to pre-tRNA substrates, this would a to of the roles of enzyme in the E. coli 3′ tRNase cleaved only after the discriminator The that this enzyme cleave T. maritima because removal of the CCA residues would be The roles of 3′ tRNase in E. coli are not because the exoribonucleases are for tRNA 3′ This have for a in of in the CCA-coding this enzyme be to other RNA substrates, pre-tRNAs that the CCA residues M.P. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). We not why T. maritima was not This is not to the presence of the 5′ because with a 5′ was as as without the (data not In to the T. maritima genome, the CCA sequences are not encoded in all tRNA genes in the T. acidophilum genome with one exception A. M.L. C. D. S. Nature. PubMed Scopus Google Scholar). The pre-tRNAs would probably be by 3′ tRNase, although the cleavage site vary from after to after the discriminator depending on pre-tRNA from in vitro Because T. maritima was not cleaved by T. acidophilum 3′ tRNase, the CCA-containing pre-tRNA would be by RNase PH, which is encoded in the genome as the only RNase the six E. coli the P. aerophilum genome H. S. A. 2002; PubMed Scopus Google Scholar) CCA-coding and tRNA P. aerophilum 3′ tRNase cleaved only the CCA-containing in vitro the pre-tRNAs with CCA would be by 3′ tRNase or RNase PH, which is encoded in the genome as the only RNase the six to the other RNase 3′ trailers to the or other unidentified be This with the the genome has CCA-coding tRNA and 3′ tRNase cleaves only pre-tRNAs O. J. Marchfelder A. H. C. EMBO J. 2003; PubMed Scopus Google Scholar). of the to the CCA 3′ above that the mechanism for 3′ tRNase to select 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 be used depending on the presence or absence of CCA in pre-tRNAs. the T. maritima enzyme with not with or with K. A.M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The presence of the two 3′ tRNase and enzyme involved in the 3′-terminal CCA that T. maritima has We M. for