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• W2163048049 abstract "tRNA 3′ processing is one of the essential steps during tRNA maturation. The tRNA 3′-processing endonuclease tRNase Z was only recently isolated, and its functional domains have not been identified so far. We performed an extensive mutational study to identify amino acids and regions involved in dimerization, tRNA binding, and catalytic activity. 29 deletion and point variants of the tRNase Z enzyme were generated. According to the results obtained, variants can be sorted into five different classes. The first class still had wild type activity in all three respects. Members of the second and third class still formed dimers and bound tRNAs but had reduced catalytic activity (class two) or no catalytic activity (class three). The fourth class still formed dimers but did not bind the tRNA and did not process precursors. Since this class still formed dimers, it seems that the amino acids mutated in these variants are important for RNA binding. The fifth class did not have any activity anymore. Several conserved amino acids could be mutated without or with little loss of activity. tRNA 3′ processing is one of the essential steps during tRNA maturation. The tRNA 3′-processing endonuclease tRNase Z was only recently isolated, and its functional domains have not been identified so far. We performed an extensive mutational study to identify amino acids and regions involved in dimerization, tRNA binding, and catalytic activity. 29 deletion and point variants of the tRNase Z enzyme were generated. According to the results obtained, variants can be sorted into five different classes. The first class still had wild type activity in all three respects. Members of the second and third class still formed dimers and bound tRNAs but had reduced catalytic activity (class two) or no catalytic activity (class three). The fourth class still formed dimers but did not bind the tRNA and did not process precursors. Since this class still formed dimers, it seems that the amino acids mutated in these variants are important for RNA binding. The fifth class did not have any activity anymore. Several conserved amino acids could be mutated without or with little loss of activity. tRNA molecules are essential for protein synthesis, providing the amino acids during translation. They are not directly transcribed as functional molecules but as precursor RNAs, which require several processing steps to generate the functional tRNA molecule. Two of these processing steps are the removal of the additional 5′ and 3′ sequences of the tRNA. Although the removal of the additional 5′ sequence (the 5′ leader) is well understood (1Frank D.N. Pace N.R. Annu. Rev. Biochem. 1998; 67: 153-180Crossref PubMed Scopus (394) Google Scholar), maturation of the tRNA 3′ end is not as well studied, although a correctly generated tRNA 3′ end is essential for the addition of the CCA triplet and thus for aminoacylation (2Mörl M. Marchfelder A. EMBO Rep. 2001; 2: 17-20Crossref PubMed Scopus (81) Google Scholar). It has been shown that in Escherichia coli, tRNA 3′ maturation is a multistep process involving endo- as well as exonucleases, the final steps being performed by an exonuclease (3Deutscher M.P. Söll D. RajBhandary U. tRNA: Structure, Biosynthesis, and Function. ASM Press, Washington, D. C.1995: 51-65Google Scholar). In contrast, Bacillus subtilis employs an endonuclease, called tRNase 6The abbreviation used is: tRNasetRNA 3′ endonuclease. 6The abbreviation used is: tRNasetRNA 3′ endonuclease. Z (EC 3.1.26.11), which cleaves CCA-less tRNA precursors directly 3′ to the discriminator (4Pellegrini O. Nezzar J. Marchfelder A. Putzer H. Condon C. EMBO J. 2003; 22: 4534-4543Crossref PubMed Scopus (114) Google Scholar). Precursors, which do contain the CCA, are not processed by tRNase Z. Archaea and eukaryotes also use tRNase Z enzymes to process the tRNA 3′ trailer in a single-step mechanism (5Schiffer S. Rösch S. Marchfelder A. EMBO J. 2002; 21: 2769-2777Crossref PubMed Scopus (150) Google Scholar, 6Schierling K. Rösch S. Rupprecht R. Schiffer S. Marchfelder A. J. Mol. Biol. 2002; 316: 895-902Crossref PubMed Scopus (51) Google Scholar, 7Dubrovsky E.B. Dubrovskaya V.A. Levinger L. Schiffer S. Marchfelder A. Nucleic Acids Res. 2004; 32: 255-262Crossref PubMed Scopus (103) Google Scholar, 8Takaku H. Minagawa A. Takagi M. Nashimoto M. Nucleic Acids Res. 2003; 31: 2272-2278Crossref PubMed Scopus (149) Google Scholar). tRNA 3′ endonuclease. tRNA 3′ endonuclease. The first tRNase Z, TRZ1, was isolated from Arabidopsis thaliana (5Schiffer S. Rösch S. Marchfelder A. EMBO J. 2002; 21: 2769-2777Crossref PubMed Scopus (150) Google Scholar). Data base analyses showed that TRZ1 homologues are present in organisms from all three kingdoms, bacteria, archaea, and eukarya (Fig. 1). The tRNase Z family of proteins (also called Elac1/Elac2) can be divided into two subgroups: the short tRNase Z proteins (being 250–350 amino acids long), tRNase ZS enzymes, and the long tRNase Z proteins (with 700–950 amino acids), the tRNase ZL enzymes. Although the tRNase ZS proteins are present in all kingdoms, the tRNase ZL enzymes can only be found in eukarya. Both subgroups are part of the same protein family since the C-terminal part of the tRNase ZL proteins has high sequence similarity to the tRNase ZS enzymes. TRZ1 belongs to the family of metal-dependent β-lactamases (9Tavtigian S.V. Simard J. Teng D.H. Abtin V. Baumgard M. Beck A. Camp N.J. Carillo A.R. Chen Y. Dayananth P. Desrochers M. Dumont M. Farnham J.M. Frank D. Frye C. Ghaffari S. Gupte J.S. Hu R. Iliev D. Janecki T. Kort E.N. Laity K.E. Leavitt A. Leblanc G. McArthur-Morrison J. Pederson A. Penn B. Peterson K.T. Reid J.E. Richards S. Schroeder M. Smith R. Snyder S.C. Swedlund B. Swensen J. Thomas A. Tranchant M. Woodland A.M. Labrie F. Skolnick M.H. Neuhausen S. Rommens J. Cannon-Albright L.A. Nat. Genet. 2001; 27: 172-180Crossref PubMed Scopus (478) Google Scholar), a group of metalloproteins that perform a variety of diverse functions (10Vogel A. Schilling O. Meyer-Klaucke W. Biochemistry. 2004; 43: 10379-10386Crossref PubMed Scopus (29) Google Scholar, 11Aravind L. In Silico Biol. 1999; 1: 69-91PubMed Google Scholar, 12Daiyasu H. Osaka K. Ishino Y. Toh H. FEBS Lett. 2001; 503: 1-6Crossref PubMed Scopus (272) Google Scholar). This metalloprotein family was classified into 16 subgroups (12Daiyasu H. Osaka K. Ishino Y. Toh H. FEBS Lett. 2001; 503: 1-6Crossref PubMed Scopus (272) Google Scholar), and the tRNase Z enzymes are part of the Elac1/Elac2 subgroup. Other subgroups include the 3′ mRNA cleavage and adenylation specificity factors (13Minvielle-Sebastia L. Keller W. Curr. Opin. Cell Biol. 1999; 11: 352-357Crossref PubMed Scopus (149) Google Scholar), SNM1 (also named PSO2), and Artemis (14Wolter R. Siede W. Brendel M. Mol. Gen. Genet. 1996; 250: 162-168PubMed Google Scholar, 15Pannicke U. Ma Y. Hopfner K.P. Niewolik D. Lieber M.R. Schwarz K. EMBO J. 2004; 23: 1987-1997Crossref PubMed Scopus (112) Google Scholar), proteins that are involved in DNA repair (16Callebaut I. Moshous D. Mornon J.P. de Villartay J.P. Nucleic Acids Res. 2002; 30: 3592-3601Crossref PubMed Google Scholar). Another subgroup consists of cAMP phosphodiesterase enzymes (17Francis S.H. Turko I.V. Corbin J.D. Prog. Nucleic Acid Res. Mol. Biol. 2001; 65: 1-52Crossref PubMed Google Scholar), which catalyze the hydrolysis of cAMP to the corresponding nucleoside 5′ monophosphate. The class II cAMP phosphodiesterase enzymes have been shown to bind two Zn2+ ions (17Francis S.H. Turko I.V. Corbin J.D. Prog. Nucleic Acid Res. Mol. Biol. 2001; 65: 1-52Crossref PubMed Google Scholar). In general, metallo-β-lactamases bind one or two metal ions, preferably zinc, iron, or manganese (10Vogel A. Schilling O. Meyer-Klaucke W. Biochemistry. 2004; 43: 10379-10386Crossref PubMed Scopus (29) Google Scholar). The E. coli tRNase Z enzyme has been shown to bind two zinc ions (18Vogel A. Schilling O. Niecke M. Bettmer J. Meyer-Klaucke W. J. Biol. Chem. 2002; 277: 29078-29085Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 19Schilling O. Vogel A. Kostelecky B. Natal da Luz H. Spemann D. Späth B. Marchfelder A. Tröger W. Meyer-Klaucke W. Biochem. J. 2005; 385: 145-153Crossref PubMed Scopus (31) Google Scholar). The recently published crystal structure of the tRNase Z enzymes from B. subtilis (20de la Sierra-Gallay I.L. Pellegrini O. Condon C. Nature. 2005; 433: 657-661Crossref PubMed Scopus (128) Google Scholar) and Thermotoga maritima (21Ishii R. Minagawa A. Takaku H. Takagi M. Nashimoto M. Yokoyama S. J. Biol. Chem. 2005; Google Scholar) confirms that the tRNase Z enzymes belong to the family of metal-dependent β-lactamases since they contain the metallo-β-lactamase fold. Structural data also show that the enzyme is a homodimer with the monomers arranged head to head. The two monomers jointly form the active site cleft, which can readily accommodate single-stranded RNA. The exosite (an element outside the active site that participates in substrate binding) protrudes from the main protein body pointing toward the solvent. We are currently analyzing the functional modules of the eukaryotic tRNase Z enzyme, TRZ1, from A. thaliana. It is a short tRNase Z enzyme of 280 amino acids containing two potential leucine zippers and a histidine motif that is part of a metallo-β-lactamase motif containing 3 highly conserved histidine residues. To identify amino acids involved in dimerization, tRNA binding, and catalysis, 24 point mutations were made. In addition, several deletion variants were generated that have one of the potential motifs removed or carry a deletion from the C terminus. Our results show that several conserved amino acids can be mutated without or with little loss of tRNA processing activity. We identify 4 conserved amino acids to be required for dimerization and 1 amino acid and three regions important for tRNA binding. In addition, 6 amino acids were shown to be required for catalytic activity. Mutagenesis Strategy—The TRZ1 cDNA was excised from pET32a-nuz (5Schiffer S. Rösch S. Marchfelder A. EMBO J. 2002; 21: 2769-2777Crossref PubMed Scopus (150) Google Scholar) (using NcoI and XhoI) and cloned into pBlue KSII (digested with NcoI and XhoI) yielding pBlue-nuz. Inverse PCR was employed using pBlue-nuz as template to generate the TRZ1 variants. For deletion variants, the primers (primer sequences are available upon request) spared the region to be deleted. For point variants, one of the primers carried the mutation. PCR products were ligated to yield the pBlue-mutant clones. Depending on the nature of the amino acids to be mutated, the resulting amino acids were one of the hydrophobic amino acids, glycine, alanine, leucine, valine, isoleucine, and serine. The pBlue-mutant clones were digested with NcoI and XhoI to release the cDNA and the mutated cDNA was subcloned into pET32a and pET29a (Novagen), respectively (both digested with NcoI and XhoI), yielding the pET32a-mutant clones and pET29a-mutant clones, respectively. A list of the variants obtained is shown in TABLE ONE. All constructs were sequenced to confirm the mutations.TABLE ONEtRNase Z variants An overview over the mutations made is shown. The Mutation column indicates where the mutation was made; (c) identifies amino acids conserved in at least four of the five tRNase Z sequences aligned in Figure 1. The xlink column results from the cross-link experiments; +, protein forms dimers; o, weak dimerization observed; -, no dimerization observed. The EMSA column results from the electrophoretic mobility shift assays; +, protein binds to tRNA; o, weak binding observed; -, no binding observed. The ivp column results from in vitro processing experiments. Wild type activity was set to 100%, all TRZ1 variants were compared to the wild type activity. Processing activity is given in % activity compared to wild type activity. The Class column shows the classification of the mutant. Class 1 has wild type activity (80–100 % compared to wild type); class two has reduced catalytic activity (20–80 %); class three forms dimers, binds tRNA but has no processing activity (below 20 %); class four forms dimers but does not bind tRNA and has no catalytic activity; and class five does not form dimers, does not bind tRNA and has no activity. wt, wild type protein TRZ1.MutationxlinkEMSAivpClass–++100–Deletion 51–60o––4Deletion 149–164o––4Deletion 200–212o––4Deletion 270–280Multimers––5C25G++332C40G++991F51L (c)++951H54L (c)+o–3H56L (c)oo–3D58A (c)++–3H59L (c)Multimers––5G62V (c)++262P64A (c)++981P83L (c)Multimers––5H133L (c)+o–3Y140L (c)o+302P178A (c)++742G184V (c)o––4D185G (c)++73T186I (c)Multimers––5K203I (c)Multimers––5L205I (c)o+562E208A (c)++552T210I (c)o+851H226L (c)++–3H248L (c)Multimers––5R252G (c)++262Deletion of Arg-252++–3Y253S (c)++232 Open table in a new tab Overexpression of Recombinant Proteins—Expression of TRZ1 and TRZ1 variants was done as described previously (5Schiffer S. Rösch S. Marchfelder A. EMBO J. 2002; 21: 2769-2777Crossref PubMed Scopus (150) Google Scholar) with the following modification: the strain BL21(DE3)pLys was used for expression (Supplemental Fig. 1). Class 3 variants were separated from GroEL (22Houry W.A. Frishman D. Eckerskorn C. Lottspeich F. Hartl F.U. Nature. 1999; 402: 147-154Crossref PubMed Scopus (434) Google Scholar), which copurifies during S tag purification, using a MiniQ column, to confirm that loss of processing activity was not due to the presence of GroEL. The column was equilibrated using buffer A (40 mm Tris-HCl, pH 8), a step gradient was applied using buffer B (buffer A with 1 m KCl), and tRNase Z was eluted with 120 mm KCl. The tRNase Z fraction was concentrated and dialyzed against buffer A. All recombinant proteins (wild type and variants) were expressed at the same (low) amounts (100–120 μg of protein/l E. coli culture). Cross-linking Assay—For cross-linking assays with glutaraldehyde, 1 μg of protein was incubated with glutaraldehyde (final concentration 0.05%) in double distilled water in 10 μl for 30 min at room temperature. After the addition of 1 μl of 1 m lysine, the sample was loaded onto a SDS-PAGE. Proteins were transferred to a nitrocellulose membrane (Hybond C, Amersham Biosciences). TRZ1 monomers and homodimers were detected using a primary rabbit antibody against TRZ1. Electrophoretic Mobility Shift Assay—tRNA from wheat (tRNA isolated from wheat, Type V, Sigma) was 3′-end-labeled with [32P]pCp. 1 fmol of labeled tRNA was heated for 5 min at 80 °C in binding buffer (10 mm Tris-Cl, pH 8.0, 5 mm MgCl2, 10 mm KCl, 10 μg/ml bovine serum albumin, and 5% glycerol) and allowed to cool down to room temperature. Recombinant TRZ1 (100 ng), phenylmethylsulfonyl fluoride, and dithiothreitol were added (to a final concentration of 0.5 and 1 mm, respectively), and the reaction was incubated for 20 min at room temperature. Finally, 2 μl of 100% glycerol was added, and the reaction was loaded onto 8% native PAGE, which was run 60 min at 4 °C with 10 Vcm-1 in TG buffer (10 mm Tris, pH 8, 58 mm glycine). Gels were analyzed by autoradiography. Substrate Preparation—Precursor tRNATyr from Oenothera berteriana was transcribed from template pTyrII as described (23Kunzmann A. Brennicke A. Marchfelder A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 108-113Crossref PubMed Scopus (70) Google Scholar). In Vitro Processing Assays—All processing assays were carried out with 100 ng of recombinant protein in a reaction volume of 100 μl in nuz-IVP buffer (40 mm Tris, pH 8.4, 2 mm MgCl2, 2 mm KCl, and 2 mm dithiothreitol) at 37 °C for 30 min. To investigate the effect of imidazole upon the reaction, imidazole (1 and 10 mm final concentration) was added to the reaction mixture. Processing reactions were terminated by phenol and chloroform extractions. Nucleic acids were precipitated, and reaction products were analyzed on 8% polyacrylamide gels. Gels were analyzed by autoradiography. Metal Dependence of the in Vitro Processing Reaction—To analyze which metal ions are required for the processing reaction, reactions were preincubated for 5 min at 37 °C with 1 and 10 mm 1,10-phenantroline or for 1 h at 4°C with 10 mm EDTA. Chelators and chelatormetal complexes were subsequently removed by dialysis. In vitro processing reactions were carried out in metal-IVP buffer (40 mm Tris, pH 8.5, 2 mm dithiothreitol), and different metal ions (0.2 mm Mn2+, Fe2+, or Zn2+ according to Ref. 18Vogel A. Schilling O. Niecke M. Bettmer J. Meyer-Klaucke W. J. Biol. Chem. 2002; 277: 29078-29085Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar; 2 mm Mg2+ according to Ref. 24Mayer M. Schiffer S. Marchfelder A. Biochemistry. 2000; 39: 2096-2105Crossref PubMed Scopus (43) Google Scholar) were added to analyze their effect on processing. No difference was observed whether the protein was preincubated with metal ions for 3 h at 24 °Cor whether the reaction was started (by the addition of the tRNA precursor) immediately after the addition of the metal ions. All buffers were treated with Chelex 100 (Bio-Rad) to remove metal ions. Determination of Cleavage Efficiency—To determine the cleavage efficiencies, in vitro processing products of internally labeled precursors were separated by PAGE gels, which were subsequently dried. Gels were analyzed using a Fuji BAS 1000 instrument (FujiFilm), and processing products were quantified using the software MacBAS (FujiFilm). All experiments were carried out in triplicates, and the resulting data were averaged. The cleavage efficiency of the wild type precursors was set to 100%. Gel filtration analyses suggested that TRZ1 might be active as homodimer. Thus we performed cross-linking assays with the recombinant TRZ1 and all variants to identify potential multimers. To investigate how tightly TRZ1 binds to the substrate and/or to the product, we performed electrophoretic mobility shift assays with TRZ1 and all variants. As shown previously, the recombinant tRNase Z TRZ1 from A. thaliana cleaves tRNA precursors efficiently in vitro (5Schiffer S. Rösch S. Marchfelder A. EMBO J. 2002; 21: 2769-2777Crossref PubMed Scopus (150) Google Scholar). Thus as a test for catalytic activity, all variants were incubated with precursor tRNAs. Characteristics of the Wild Type Protein TRZ1—Cross-linking experiments with TRZ1 showed that the enzyme is indeed a homodimer (Fig. 2). Electrophoretic mobility shift assay analyses revealed that TRZ1 binds tightly to tRNA molecules but only weakly to the synthetic precursor tRNA (Fig. 3 and data not shown). The fact that TRZ1 did not bind well to the precursor tRNA under the conditions employed could be due to the fact that the precursor RNA used was made in vitro, and thus tRNA nucleotides were not modified and the precursor molecules might not fold correctly. Thus the following RNA binding studies were made with wheat tRNA. Incubation with precursor tRNATyr confirmed the catalytic activity of TRZ1 (Fig. 4).FIGURE 3tRNase Z binds to tRNAs. TRZ1 and variants were incubated with wheat tRNA to analyze whether the proteins bind to tRNA. The reaction was loaded onto a non-denaturing polyacrylamide gel, which was subsequently analyzed by autoradiography. The autoradiograph clearly shows that all four proteins bind to the tRNA (although H54L with lower efficiency). Lane wt, wild type protein TRZ1 incubated with tRNA; lanes C25G, C40G, and H54L, incubation of variants with tRNA; lane c, control without the addition of proteins. The tRNA and the tRNA/protein-complex are shown at the right schematically.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4tRNase Z processes precursor tRNAs. To investigate whether the mutations made interfere with the catalytic activity, proteins were incubated with precursor tRNA molecules. Lane m, DNA size marker; lane c, control reaction without proteins; lane wt (wild type protein TRZ1) and lanes C25G, C40G, and H54L, incubation with the respective proteins. TRZ1 (100% activity, see also TABLE ONE) and C40G (99% when compared with TRZ1) cleave the precursor tRNA efficiently, C25G shows only weak activity (33% when compared with TRZ1), and H54L does not cleave the precursor. DNA size markers are shown in nucleotides at the left. Precursor and products are shown schematically at the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Rationale for Mutant Selection—To identify important regions and amino acids of TRZ1 for dimerization, RNA binding, and catalysis, we initiated an extensive mutational study of TRZ1. Alignment of tRNase Z protein sequences from different organisms revealed conserved regions and amino acids that we chose for mutations (Fig. 1, TABLE ONE). Motif search software predicted three regions to be a histidine motif and two potential leucine zippers, respectively. Therefore we made three internal deletions: one, del51–60, which spans the histidine motif, and the other two, del149–164 and del200–212, which span the two potential leucine zippers (starting and ending with the 1st and 3rd leucine). In addition, we made a deletion from the C terminus (del270–280) to define a shorter tRNase Z enzyme. Point variants were generated of amino acids conserved between tRNase Z proteins (Fig. 1). Of particular interest were the amino acids from the histidine motif, which are conserved between the metal-dependent β-lactamases (His-54, His-56, Asp-58, and His-59) and the two histidines (His-133 and His-226) that might be involved in metal binding (11Aravind L. In Silico Biol. 1999; 1: 69-91PubMed Google Scholar). In addition to mutation of conserved amino acids, we also mutated 3 amino acids that are specific for TRZ1. Cys-25 and Cys-40 can only be found in the TRZ1 sequence and could be involved in dimer formation. Lys-203 was also unique for the TRZ1 sequence and was at a position where all other tRNase Z proteins have a conserved aspartic acid. All TRZ1 variants were expressed at the same levels as wild type TRZ1 in soluble form and purified with S protein-agarose. The E. coli protein GroEL (22Houry W.A. Frishman D. Eckerskorn C. Lottspeich F. Hartl F.U. Nature. 1999; 402: 147-154Crossref PubMed Scopus (434) Google Scholar) copurified with all recombinant proteins, but the wild type protein is not influenced by the presence of GroEL in any of the three activities tested (dimerization, tRNA binding, and processing). Thus only class 3 variants were separated from GroEL using anion exchange chromatography yielding pure proteins to confirm that the loss of processing activity in these variants was not due to the presence of GroEL. Indeed, activity tests with variants with and without GroEL gave the same results. Only in a few cases (Pro-83, Thr-186, del270–280) was it not possible to separate GroEL from the TRZ variants. Interestingly, these proteins were also not able to form dimers. GroEL does not interfere with dimerization since the other proteins dimerize also in the presence of GroEL. All variants were analyzed in respect to their ability to form dimers, to bind to tRNA, and to catalyze tRNA processing. During this study, structures of two bacterial tRNase Z enzymes have been solved. To discuss the results of this mutational study in a structural context, all mutations were mapped onto the B. subtilis structure based on the tRNase Z alignment (Fig. 5). Metal Ions Are Required for tRNA Processing Activity—It was shown previously that metal ions are required for tRNA processing activity of TRZ1 since preincubation with EDTA resulted in loss of activity (24Mayer M. Schiffer S. Marchfelder A. Biochemistry. 2000; 39: 2096-2105Crossref PubMed Scopus (43) Google Scholar). Interestingly, the metal required seems to be already bound by the enzyme since the addition of metal ions to the recombinant enzyme after expression in E. coli is not necessary. To elucidate which metal is required for activity, we preincubated the reaction with chelators 1,10-phenantroline and EDTA (Fig. 6). Preincubation with 10 mm 1,10-phenantroline and EDTA inhibited the reaction. Of several metal ions tested, only the addition of Mn2+ and Mg2+ rescued the activity, showing that these metal ions are required for the activity. Removal of Several Internal Amino Acids Leads to Inactivation of the Protein—Deletions of the histidine motif (del51–60) and the potential leucine zipper motifs (del149–164 and del200–212) (TABLE ONE and Supplemental Figs. 2–4) resulted in almost complete inactivation of the processing activity. Cross-linking experiments showed that these deletion variants barely form dimers anymore (TABLE ONE and Supplemental Fig. 2), that they were not able to bind to tRNAs anymore, and that they could not process precursor tRNAs. The C Terminus Is Essential for tRNA Binding and Processing Activity—Removal of 11 amino acids at the C terminus resulted in variant del270–280, which does not form dimers but does form multimers. In addition, this variant does not bind to tRNAs and does not have catalytic activity (TABLE ONE, Supplemental Figs. 2–4). Therefore the 11 amino acids from the C terminus are essential for tRNase Z activity. Amino Acids That Are Not Essential for Activity (Class One)—Variants C40G (Figs. 2, 3, 4), F51L, P64A, and T210I (Supplemental Figs. 2–4) showed the same activity or nearly the same activity (80–100%) as the wild type protein, suggesting that amino acids Cys-40, Phe-51, Pro-64, and Thr-210 are not essential for dimerization, tRNA binding, and catalysis. Amino acid Cys-40 was not conserved throughout the tRNase Z proteins and present only in TRZ1 from Arabidopsis; therefore it was not too surprising that mutation of that amino acid does not interfere with activity. However, Phe-51, Pro-64, and Thr-210 are conserved in at least four of the five protein sequences aligned in Fig. 1; thus it is interesting that these amino acids do not seem to be essential for processing activity. Variants That Have Only Reduced Catalytic Activity (20–80%) (Class Two)—Variants C25G (Figs. 2, 3, 4), G62V, Y140L, P178A, L205I, E208A, R252G, and Y253S (Supplemental Figs. 2–4) are still able to form dimers and still bind to tRNAs. However, they cleave tRNA precursors with reduced activity (20-80% wild type activity, TABLE ONE). Since these variants all still formed dimers and bind to tRNAs, the mutation seemed to affect only the catalytic activity. Variants That Do Not Have Catalytic Activity (Class Three)—Variants H54L (Figs. 2, 3, 4), H56L, D58A, H133L, D185G, H226L, and delR252 (Supplemental Figs. 2–4) still form dimers and bind to tRNA but are not able to process precursor tRNAs. This suggests a direct involvement of amino acids His-54, His-56, Asp-58, His-133, Asp-185, and His-226 in catalysis. The mutation of amino acid R252G only reduced the catalytic activity (down to 26%, see above); deletion of this amino acid, however, resulted in total loss of activity. In the case of ribozyme-mutants, which were not catalytically active anymore, the addition of imidazole rescued the catalytic activity (25Perrotta A.T. Shih I. Been M.D. Science. 1999; 286: 123-126Crossref PubMed Scopus (253) Google Scholar). To test whether the addition of imidazole to the histidine mutants can rescue the catalytic activity, we added 1 and 10 mm imidazole to the processing reaction with mutants H54L, H56L, H133L, and H226L. The addition of imidazole did, however, not rescue the catalytic activity of these mutants (data not shown). Variants That Do Not Bind or Bind Only Weakly to tRNAs (Class Four)—Several variants (del51–60, del149–164, del200–212, and G184V) were still able to form dimers but did not bind to tRNAs anymore. They also did not process precursor tRNAs (Supplemental Figs. 2–4), suggesting that the mutated amino acid Gly-184 and that the deleted regions are important for RNA binding. Variants That Do Not Form Dimers (Class Five)—Variants H59L, P83L, T186I, K203I, H248L, and del270–280 did not form dimers anymore but seemed to aggregate since glutaraldehyde cross-linking results in bigger complexes, probably representing multimers. This suggested that the change of these amino acids somehow disturbs the whole protein structure, which leads to complete loss of activity. Supporting this hypothesis is the fact that these variants cannot be separated from the E. coli chaperonin GroEL. Neither anion exchange chromatography nor gel filtration analysis (even with the addition of ATP and Mg2+) succeeded in separating these TRZ1 variants from GroEL. Although tRNA 3′ end processing is vital for tRNA maturation and subsequent aminoacylation, little is known at present about the functional domains of the tRNA 3′-processing enzyme tRNase Z. We performed an extensive mutational study of the tRNase Z from A. thaliana to identify amino acids and regions important for dimerization, tRNA binding, and catalysis. During this study, the structures of the bacterial tRNase Z enzymes from B. subtilis and T. maritima were solved (20de la Sierra-Gallay I.L. Pellegrini O. Condon C. Nature. 2005; 433: 657-661Crossref PubMed Scopus (128) Google Scholar, 21Ishii R. Minagawa A. Takaku H. Takagi M. Nashimoto M. Yokoyama S. J. Biol. Chem. 2005; Google Scholar). Our results obtained from the mutational study of the eukaryotic short tRNase Z enzyme presented here were thus compared with the bacterial structure. Characteristics of the tRNase Z Enzyme From A. thaliana—The TRZ1 protein is a homodimer, as are the homologous proteins from E. coli (18Vogel A." @default.
• W2163048049 created "2016-06-24" @default.
• W2163048049 creator A5024161694 @default.
• W2163048049 creator A5026450279 @default.
• W2163048049 creator A5034833088 @default.
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• W2163048049 date "2005-10-01" @default.
• W2163048049 modified "2023-10-03" @default.
• W2163048049 title "Analysis of the Functional Modules of the tRNA 3′ Endonuclease (tRNase Z)" @default.
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