tRNA 3′ End Maturation in Archaea has Eukaryotic Features: the RNase Z from Haloferax volcanii

doi:10.1006/jmbi.2001.5395 available online at http://www.idealibrary.com on

J. Mol. Biol. (2002) 316, 895±902

tRNA 30 End Maturation in Archaea has Eukaryotic Features: the RNase Z from Haloferax volcanii Karina Schierling, Sylvia RoÈsch, Renate Rupprecht, Steffen Schiffer and Anita Marchfelder* Molekulare Botanik, UniversitaÈt Ulm, 89069 Ulm, Germany

Here, we report the ®rst characterization and partial puri®cation of an archaeal tRNA 30 processing activity, the RNase Z from Haloferax volcanii. The activity identi®ed here is an endonuclease, which cleaves tRNA precursors 30 to the discriminator. Thus tRNA 30 processing in archaea resembles the eukaryotic 30 processing pathway. The archaeal RNase Z has a KCl optimum at 5 mM, which is in contrast to the intracellular KCl concentration being as high as 4 M KCl. The archaeal RNase Z does process 50 extended and intron-containing pretRNAs but with a much lower ef®ciency than 50 matured, intronless pretRNAs. At least in vitro there is thus no de®ned order for 50 and 30 processing and splicing. A heterologous precursor tRNA is cleaved ef®ciently by the archaeal RNase Z. Experiments with precursors containing mutated tRNAs revealed that removal of the anticodon arm reduces cleavage ef®ciency only slightly, while removal of D and T arm reduces processing effciency drastically, even down to complete inhibition. Comparison with its nuclear and mitochondrial homologs revealed that the substrate speci®city of the archaeal RNase Z is narrower than that of the nuclear RNase Z but broader than that of the mitochondrial RNase Z. # 2002 Elsevier Science Ltd.

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*Corresponding author

Keywords: archaea; tRNA; 3 end maturation; endonuclease; Haloferax volcanii

Introduction Since tRNA molecules are transcribed as precursor RNAs containing 50 and 30 extensions, several processing steps are required to generate functional tRNA molecules. While the enzyme catalysing the maturation of the tRNA 50 end, RNase P, has been studied to some extent and is similar in all organisms,1,2 little is known about maturation of the tRNA 30 end. In bacteria, tRNA 30 processing has been studied mainly in Escherichia coli, where removal of the 30 trailer is a multistep process.3 It is initiated by an endonucleolytic cleavage several nucleotides downstream of the tRNA 30 end and the remaining nucleotides are subsequently removed by exonucleases. The ®nal 30 end processing step can take place in E. coli only after RNase P has removed the 50 leader. In contrast to the multistep reaction in bacteria, the prevailing pathway in eukaryotes is a single-step reaction, involving an endonuclease E-mail address of the corresponding author: [email protected] 0022-2836/02/040895±8 $35.00/0

that cuts 30 to the discriminator (the discriminator is located 50 to the CCA and serves as an identity element in many tRNAs).4 The 30 end processing reaction is not necessarily dependent on the RNase P reaction, since some of the eukaryotic 30 processing activities have been reported to process 50 extended precursors.5 ± 8 The same single-step reaction as in the nucleus takes place in organelles, but organelles seem to follow a de®ned order for removal of the extensions, with removal of the 50 leader having to precede removal of the 30 trailer.9 ± 12 While some data have been published concerning archaeal tRNA 50 end maturation,13 ± 15 hitherto nothing is known about tRNA 30 end processing in archaea. To date, archaea are established as the third domain of life. Con®rmed by the ever increasing amount of genome sequences from representatives of all domains, it is now clear that archaea have a lot in common with bacteria and with eukarya. In archaea, tRNA genes have been identi®ed as clustered genes, as individual transcriptional units, and as multigene transcriptional units.16 These tRNA genes are transcribed into precursors con# 2002 Elsevier Science Ltd.

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tRNA 3 0 Processing

taining 50 and 30 extensions and, in some cases, introns, all of these additional sequences have to be removed to yield functional tRNA molecules. Haloferax volcanii used in this study is a halophilic archaeon, which lives in an environment with high concentrations of salt. Halophilic archaea adapted to this environment by raising their intracellular concentration up to 4 M KCl, a salt concentration at which many conventional proteins are known to denature and precipitate17,18 and which converts some DNA sequences from the B form to the Z form.19 Here, we report the ®rst characterization and puri®cation of an archaeal tRNA 30 processing activity, the RNase Z from H. volcanii.

Results In Haloferax volcanii an endonuclease generates the mature tRNA 30 end To investigate tRNA 30 processing in H. volcanii, we isolated a soluble protein fraction (S100) from H. volcanii cells. In addition, we cloned tRNA genes for tRNATrp, tRNAMet and tRNAAla from H. volcanii to use them as templates for generating homologous tRNA precursors. Incubation of pretRNAAla with the protein extract yielded two processing products, which correspond in length to the 30 trailer and the tRNA (Figure 1). The second product, with the size of the 30 trailer, suggests that the activity involved in archaeal tRNA 30 processing is an endonuclease, which cleaves the precursor close to or directly at the tRNA 30 end. We termed this enzyme archaeal RNase Z. Partial purification of the archaeal RNase Z To characterize the activity further, we initiated puri®cation of the archaeal RNase Z. The S100 fraction isolated from H. volcanii cells was fractionated using PEG-precipitation and the 30 processing activity was found to precipitate with 3-10 % PEG. The RNase Z active PEG fraction was further fractionated using an ion-exchange column twice (Source Q), from which the enzyme eluted both times with 0.4 M KCl. The ®nal puri®cation step was a heparin column (RNase Z eluted with 0.1 M KCl). Comparatively few proteins remained in the RNase Z active heparin fraction, suggesting that only a few additional puri®cation steps are required to isolate the RNase Z from H. volcanii. Salt, pH and temperature requirements of RNase Z Since H. volcanii is living in an environment with high salt concentrations (2-4 M NaCl) and has similar salt concentrations inside the cell (2-4 M KCl), we were interested to determine the optimal processing conditions for this enzyme (Table 1). Interestingly, the optimal KCl concentration is only 5 mM KCl, much lower than the about 2-4 M intra-

Figure 1. The archaeal tRNA 30 processing activity is an endonuclease. PretRNAAla from H. volcanii was incubated with a protein extract from Haloferax volcanii. Lane k, incubation of precursor without proteins. Lane p, in vitro processing of pretRNAAla with the 3 %-10 % PEG fraction from H. volcanii. A DNA size marker is indicated at the right (sizes are given in nucleotides). Precursor and products are shown schematically at the left. The precursor is cleaved ef®ciently, yielding the tRNA and the 30 trailer. Thus, the tRNA 30 processing activity is an endonuclease that we termed RNase Z.

cellular KCl concentration in H. volcanii cells. We even detected reduction of activity at salt concentrations higher than 200 mM KCl. The optimal incubation temperature for the RNase Z is 40  C, which is in the range of the growth temperature of that organism. The halophile RNase Z has a slightly acid pH optimum, 5.5. Low concentrations of MgCl2 are optimal for 30 processing activity, which is similar to the eukaryotic endonucleolytic tRNA 30 processing activities studied so far (Table 1).5,10,20,21 Characterization of processing products After tRNA 30 end processing, the 30 -terminal CCA sequence has to be added to yield a func-

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tRNA 3 0 Processing Table 1. Comparison of optimal reaction conditions for RNase Z enzymes Parameter 

Temperature ( C) pH MgCl2 concentration (mM) KCl concentration (mM)

H. volcanii

Potato mt

Rat mt

Wheat

X. laevis

40 5.5 5 5

30 8.0 5 30

37 7.2 0-2 10-25

35 8.4 0 0

37 6.5-8.5 1-5 100

Optimal reaction conditions of the RNase Z enzymes from Haloferax volcanii (this work), potato mitochondria,5 rat mitochondria,11 wheat5 and Xenopus laevis20 are listed. All RNase Z enzymes require similar temperature and salt conditions.

tional tRNA molecule. The terminal tRNA nucleotidyl transferase that is resposible for that reaction requires a 30 hydroxyl group at the tRNA 30 end. To determine whether the Haloferax RNase Z generates a 30 hydroxyl group at the tRNA 30 end, we analysed the end groups of both processing products. An in vitro processing assay with unlabelled pretRNA was performed, the RNA was precipitated and incubated with [a-32P]pCp and RNA ligase (data not shown). Since the RNA ligase also requires a 30 hydroxyl group to add the [a-32P]pCp, only RNA molecules with 30 hydroxyl groups are labelled in this reaction. The tRNA was labelled ef®ciently in this ligation experiment, showing that RNase Z indeed leaves a hydroxyl group at the tRNA 30 end. To con®rm the presence of a 50 phosphoryl group at the 30 trailer, pretRNAAla was transcribed with [a-32P]UTP to label the 50 nucleo(30 trailer sequence tide of the 30 trailer 0 50 UGGUAAGCAGUA3 ), yielding also a molecule labelled throughout. After incubation with RNase Z, the 30 trailer was eluted and subjected to digest by RNases A and T1 to generate 30 -monophosphate nucleosides (Np). The digestion mix was subsequently separated on 2D TLC, and ®ve spots were detected by autoradiography (data not shown). Four spots corresponded to Ap, Up, Gp and Cp, while the ®fth was, according to its position on the 2D TLC, pUp. Thus, since the ®rst nucleotide of the 30 trailer is a U, the phosphoryl group remains at the 30 trailer resulting in a single pUp nucleotide upon digestion with RNases. Determination of cleavage site Initial experiments had shown that an endonuclease is responsible for tRNA 30 end maturation in H. volcanii. To investigate whether the endonuclease cleaves the precursor directly 30 to the discriminator or some nucleotides downstream, we performed a primer extension experiment (Figure 2). Primer extension analysis of the 30 trailer yielded a cDNA, which corresponds to a cleavage event directly 30 to the discriminator. Substrate specificity of the archaeal RNase Z The archaeal RNase Z processes also 5 0 extended precursor tRNA molecules Most of the tRNA 30 processing enzymes analysed so far have been shown to require a mature

tRNA 50 end. Thus, it is only after removal of the 50 leader by RNase P that RNase Z is able to act on the pretRNA. We incubated a 50 extended pretRNAAla with the Haloferax RNase Z to study the in¯uence of the 50 extension on the archaeal RNase Z (Figure 3). The precursor molecule is converted into two processing products, corresponding in size to the 30 trailer and the tRNA plus 50 leader. Although cleavage ef®ciency is not as high as with the 50 matured precursor (Figure 1), the 50 extended pretRNA is still accepted as substrate by the Haloferax RNase Z. Processing of intron containing pretRNAs To analyse whether intron-containing tRNAs are substrates for the archaeal RNase Z, pretRNAMet containing a 75 nt long intron was incubated with the RNase Z active protein fraction (Figure 4). Both products of the tRNA 30 processing reaction are detectable, although the processing ef®ciency seems to be lower than with the intronless precursor pretRNAAla (Figure 1). Heterologous precursor molecules are substrates for the archaeal 30 processing enzyme Whether heterologus pretRNAs can be substrates for the archaeal RNase Z was tested in additional experiments. PretRNATyr from plant mitochondria was incubated with an RNase Z active fraction (data not shown). The plant tRNA precursor is cleaved ef®ciently yielding two products, the mature tRNA and the 30 trailer. Processing of tRNA variants The general tRNA structure seems to be important for recognition by the Haloferax RNase Z, since homologous and heterologous tRNA precursors are accepted as substrates. To get an idea of which parts of the tRNA are essential for an RNase Z substrate, we constructed a couple of tRNA variants missing one or more tRNA arms (Figure 5).22 The proposed secondary structures were con®rmed using structure probing experiments.22 These tRNA variants were incubated as pretRNAs (containing a 51 nt long 30 trailer) with the Haloferax RNase Z. In vitro processing experiments showed that the removal of the anticodon arm (variants T3 and T6) resulted in a reduction of cleavage ef®ciency to 73 % and 42 %, respectively, while the

898

Figure 2. The archaeal RNase Z cleaves next to the discriminator. To determine the exact cleavage site, a primer extension experiment was carried out. Sequencing reactions (lanes A,C,G,T) and primer extension (lane pex) were started from primer Ala7. The coding strand sequence is shown at the right. The cleavage site is indicated with an arrow. Reverse transcription of the 30 trailer with primer Ala7 yields a cDNA that corresponds to cleavage 30 to the discriminator. Thus, the archaeal RNase Z cleaves the precursor directly 30 to the discriminator.

deletion of the D arm (T4 and T5) had more drastic effects, yielding only 3 % and 5 % cleavage ef®ciencies, respectively. If anticodon, D and variable arms (T6a) are removed, cleavage ef®ciency is similarly very low, 3 %. The T arm seems to be essential for processing, since variants T1 and T2, missing this particular arm, are not processed at all (Figure 5).

Discussion Processing by the archaeal RNase Z does not seem to require nucleotide modi®cation, since all substrates used were generated with phage T7

tRNA 3 0 Processing

Figure 3. A 50 extended pretRNA is processed by the archaeal RNase Z. PretRNAAla containing a 50 leader was incubated with the archaeal RNase Z. Lane m, DNA size marker (given in nucleotides). Lane k, control reaction without proteins. Lane p, in vitro processing of the 50 extended pretRNAAla. Two processing products are generated, corresponding in size to the 30 trailer and to tRNA plus 50 leader. A 50 extended pretRNA is thus a substrate for the archaeal RNase Z, although with a much lower ef®ciency than the 50 matured pretRNA (Figure 1).

polymerase. To date, no data are available about the length of the native 50 and 30 extensions in archaeal tRNA precursors. Experiments presented here show that even archaeal tRNA precursors containing long 30 trailers can be processed ef®ciently by the endonuclease characterized here. The endonucleolytic pathway seems to be the prevailing way to generate mature tRNA 30 ends In organelles, the tRNA 30 end is generated by an endonucleolytic cut next to the discriminator.9 ± 12,23 The same is true for the majority of eukaryotic tRNA processing systems studied so far.7,20,24 ± 29 Only in E. coli are the ®nal maturation steps in the tRNA 30 processing pathway performed by exonucleases.3 In H. volcanii, and probably also in other archaea, tRNA 30 trailers are removed by an endonuclease that cleaves directly at the tRNA 30 end. No additional enzymes are required to yield the mature tRNA 30 end. In this respect, archaeal tRNA 30 processing resembles

899

tRNA 3 0 Processing

The halophilic RNase Z is inactivated by high salt concentrations

Figure 4. Intron-containing pretRNAMet is processed by the archaeal RNase Z. PretRNAMet containing a 75 nt intron was incubated with the archaeal RNase Z. Lane p, in vitro processing of pretRNAMet. Lane k, incubation without proteins. Lane m, DNA size standard (sizes are given in nucleotides). Precursor and products are shown schematically at the left. The intron-containing precursor is cleaved by the archaeal RNase Z to yield the 30 trailer and the tRNA with intron. The archaeal RNase Z is able to process this precursor, albeit with a lower ef®ciency than an intronless precursor (Figure 1).

more the eukaryotic than the bacterial processing pathway. Biochemical characterization of the archaeal RNase Z The optimal reaction conditions for the archaeal RNase Z determined in this work are similar to those observed for eukaryotic tRNA 30 processing activities (Table 1). They all require low Mg2‡ concentrations for activity and are active over broad temperature ranges.5,10,20,21 The optimal pH observed for the halophilic RNase Z is more acidic than for other RNase Z enzymes. This might be due to the fact that halophilic enzymes often contain an unusually high percentage of acidic amino acid residues.

An interesting observation is that the Haloferax RNase Z requires only low concentrations of K‡, as do the eukaryotic 30 processing enzymes. In fact, the enzyme is inhibited in vitro by K‡ concentrations higher than 200 mM. These results suggest that the enzyme itself is not able to tolerate the high-salt concentrations up to 2-4 M KCl prevalent in H. volcanii cells and may need special molecules for stabilization in vivo. A couple of other enzymes from halophiles have been reported to be inhibited by high salt concentrations.18 In general, enzymes from halophiles can be separated into two types; the ®rst group requires high salt concentrations in vitro, and the second prefers low salt concentrations in vitro and is sometimes even inhibited by high salt concentrations.18 The halophilic RNase Z identi®ed here shows characteristics of the latter group. The nature of the end-groups of the processing products shows that this enzyme, like previously identi®ed RNase Z enzymes, is a phosphodiesterase, which cleaves the phosphodiester bond such that 50 phosphoryl and 30 hydroxyl groups are left on the processing products. Since the archaeal RNase Z cleaves immediately 30 to the discriminator and leaves a hydroxyl group at the tRNA 30 end, the terminal tRNA nucleotidyl transferase can directly add the CCA triplet after RNase Z cleavage. Order of processing events According to our in vitro experiments, a 50 matured precursor is not required for processing by the archaeal RNase Z. Thus, at least in vitro, it is not necessary that RNase P cleavage precedes 30 processing. In contrast, analyses in vivo showed that 50 processing occurs before 30 processing.30 However, the differences in the order of processing steps might be due to the fact that Palmer et al. analysed processing of a yeast tRNA precursor in Haloferax cells.30 Analyses of the plant nuclear RNase Z showed that the ef®ciency of processing of 50 extended pretRNAs varied from pretRNA to pretRNA.5 Other reports found that in some eukaryotes, 50 end processing precedes 30 end processing,20,27 while in other eukaryotes the order is reversed.7 These results suggest that no de®ned order of processing is required to generate mature tRNA ends in all organisms. Since intron-containing precursors can be substrates for the archaeal RNase Z, albeit with a lower processing ef®ciency, it seems that, at least in vitro, 30 end processing can occur before intron splicing. Generally, it has been reported that the order of processing depends on the precursor concentration. When pretRNAs are present in physiological concentrations, splicing occurs before tRNA end maturation, but at high precursor concentrations end maturation occurs before splicing.31

900

tRNA 3 0 Processing

Figure 5. Comparison of substrate cleavage ef®ciencies by the archaeal, the mitochondrial and the nuclear RNase Z. Substrates containing a 51 nt long trailer were incubated with archaeal, mitochondrial and nuclear RNase Z (this work and see Schiffer et al.22). Reactions were repeated three times and cleavage ef®ciencies were determined and averaged. Wild-type cleavage ef®ciency was set to 100 %. Wild-type and variant substrates are shown schematically along the x-axis. Relative cleavage ef®ciency is shown at the y-axis (in %). The archaeal RNase Z processes the wildtype substrate most ef®ciently. While variants T3 and T6 are cleaved with 73 % and 42 % ef®ciency, respectively, variants T4, T5 and T6a are processed less ef®ciently (3 %, 5 % and 3 %, respectively). Variants T1 and T2 are not cleaved at all. The nuclear RNase Z has, compared to the archaeal and mitochondrial RNase Z, the broadest substrate spectrum, cleaving all variants except those with missing T arm, T1 and T2.

Substrate specificity of the archaeal RNase Z Heterologous pretRNAs are cleaved by the archaeal RNase Z, showing that the overall tRNA structure is suf®cient for cleavage. Incubation of tRNA variants with the archaeal RNase Z revealed that the anticodon arm is not necessarily required for cleavage. In contrast, the T arm is essential for processing and variants lacking the D arm are very poor substrates. The archaeal RNase Z thus requires parts of the ancient tRNA (acceptor stem and T arm) and parts of the modern tRNA (D arm) for ef®cient cleavage. The same tRNA variants have been incubated with the mitochondrial and nuclear RNase Z (Figure 5).22 While the nuclear RNase Z has a broad substrate spectrum, processing variants without the D arm rather ef®ciently, the mitochondrial RNase Z does not tolerate the removal of the D arm at all. Thus, with respect to substrate speci®city, the archaeal RNase Z is to be placed between the nuclear and the mitochondrial enzyme, having a narrower substrate spectrum than the nuclear RNase Z but a wider one than the mitochondrial RNase Z. A common ancestor for the archaeal and the eukaryotic enzyme? Taken together, the 30 processing pathway in archaea is similar to the same process in eukarya,

but distinctly different from 30 maturation in E. coli. tRNA 30 processing in archaea is thus another process resembling its eukaryotic counterpart and further analyses will show whether the eukaryotic enzymes are derived from an archaeal ancestor.

Materials and Methods Strains and culture conditions H. volcanii strain DS2 (DSM 3757, NCPIB 2012, ATCC 29605) was grown aerobically at 37  C in modi®ed growth medium (18 % SW) (Halohandbook: http:// www.microbiol.unimelb.edu.au/micro/staff/mds/HaloHandbook/index.html). Isolation of tRNA genes from H. volcanii The genes for tRNATrp, tRNAMet and tRNAAla were ampli®ed from H. volcanii DNA by PCR (primer sequences and PCR programs are available upon request). The resulting PCR products contain the tRNA gene, additional 50 and 30 sequences and, in the case of tRNATrp and tRNAMet, introns. PCR products were subcloned into pBluescript SK II to yield clones pblue-Trp, pblue-Met and pblue-Ala. Substrate preparation Templates for 50 matured pretRNATrp, pretRNAMet, pretRNAAla and 50 extended pretRNAAla from H. volcanii were synthesized from clones pblue-Trp, pblue-Met and

901

tRNA 3 0 Processing pblue-Ala using PCR. The resulting templates pHvTrp, pHvMet, pHvAla and pHvAla ‡ 5 contain the phage T7 promotor, the respective tRNA gene (tRNATrp, 74 nt with a 105 nt intron; tRNAMet, 74 nt with a 75 nt intron; and tRNAAla, 79 nt) and a 30 trailer (length of 30 trailer for: pHvTrp, 70 nt; pHvMet, 105 nt; and pHvAla, 101 nt). Template pHvAla ‡ 5 contains, in addition, a 79 nt long 50 leader. In vitro transcription and puri®cation of transcripts were performed as described.32 Purification of RNase Z from H. volcanii H. volcanii (16 l) were grown to an A600 of 0.8. Cells were collected by centrifugation at 7500 g for 15 minutes at 4  C. After washing the cell pellet with buffer C (50 mM Tris-HCl (pH 7.5), 2 M KCl, 5 mM MgCl2), cells were disrupted by soni®cation and a high-speed supernatant (S100) was obtained by ultracentrifugation at 100,000 g for 60 minutes at 4  C.

used: Mes for pH 4.5 to pH 7.0, Tris-HCl for pH 7.5 to 9.0. Processing reactions were terminated by extractions with phenol and chloroform. Nucleic acids were precipitated, and reaction products were analysed on 8 % polyacrylamide gels. Products were quanti®ed by measuring signal intensities of an exposed X-ray ®lm with a Fuji BAS 1000 (FujiFilm) and the software MacBAs (FujiFilm). According to the optimal reaction conditions determined, all processing reactions were carried out with 5 mg of protein (3-10 % PEG fraction) in 100 ml of buffer ivp-a (40 mM Mes (pH 5.5), 5 mM MgCl2, 5 mM KCl) for 30 minutes at 40  C if not stated otherwise. Characterization of processing products To analyse the nature of the 50 and 30 terminal groups of the processing products, experiments were carried out as described.10

PEG precipitation

Determination of cleavage site

A 3-10 % PEG-fraction was prepared as follows: a 40 % (w/w) PEG stock solution (40 % PEG 6000 in buffer E (50 mM Tris-HCl (pH 7.8), 20 mM MgCl2)) was added slowly to the S100 fraction until the ®nal PEG concentration was 3 % (w/w). The solution was stirred for 60 minutes and the precipitate was pelleted for 30 minutes at 30,000 g. To the resulting supernatant, a 40 % PEG stock solution was added to a ®nal concentration of 10 %. After centrifugation, the resulting pellet was dissolved in 1 ml of buffer E, yielding the fraction 3 %-10 % PEG.

An in vitro processing reaction using unlabelled pretRNAAla was performed. To identify signals resulting from endogenous RNA molecules, a control reaction without addition of precursor was carried out. After extraction with phenol/cloroform, RNAs were precipitated and subsequently used in a reverse transcription reaction using primer Ala7. The same primer was used for the sequencing reaction.

Source 30Q column

Mitochondrial pretRNATyr from Oenothera berteriana and tRNA variants thereof were prepared as described.22

The 10 % PEG fraction was loaded onto a 6 ml Source 30Q column (AmershamPharmacia). The column was washed with buffer A (40 mM Tris-HCl (pH 7.0), 5 mM MgCl2, 5 % (v/v) glycerol) and bound proteins were eluted with a KCl step gradient (0.2, 0.4, 2.0 M KCl in buffer A). RNase Z eluted with 0.4 M KCl. This fraction was dialyzed against buffer A using Centriplus ®ltration units (Millipore) and again loaded onto the Source Q column. The same fractionation steps were applied and RNase Z eluted again with 0.4 M KCl. Heparin-Sepharose column After desalting, the 0.4 M Source Q fraction was further fractionated on a 5 ml heparin-Sepharose column (AmershamPharmacia). After equilibration with buffer B (50 mM Mes (pH 6.5), 5 mM MgCl2, 5 % glycerol) the dialyzed fraction was loaded and proteins were eluted with a KCl step gradient (0.1, 0.3, 0.5, 2.0 M KCl in buffer B). RNase Z eluted with 0.1 M KCl. Aliquots from the RNase Z active PEG, Source and heparin fractions were analysed by SDS/10 % PAGE as described.33 Optimization of processing assays The initial processing assay was performed with 5 mg of 10 % PEG pellet fraction in buffer E-DTT (buffer E with 2 mM DTT) in a volume of 100 ml at 37  C for 30 minutes. PretRNAAla was incubated in different buffers depending on the parameter examined. All experiments were repeated three times and resulting data were averaged. For pH determination, the following buffers were

Synthesis of mitochondrial pretRNATyr and tRNA variants

Acknowledgements We thank Elli Bruckbauer and Claudia Gautsch for expert technical assistance. Thanks are due to Annika Lieberoth and Silvia Kirchner for preparation of the 3 %10 % PEG fraction from Haloferax volcanii cells. The work presented here was funded by AnfangsfoÈrderung Ulm and the VolkswagenStiftung.

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Edited by W. Baumeister (Received 5 November 2001; received in revised form 13 December 2001; accepted 17 December 2001)