View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

FEBS Letters 583 (2009) 2339–2342

journal homepage: www.FEBSLetters.org

RNase J is involved in the 50-end maturation of 16S rRNA and 23S rRNA in

Ramakanth Madhugiri, Elena Evguenieva-Hackenberg *

Institut für Mikrobiologie und Molekularbiologie, University of Giessen, Heinrich-Buff-Ring 26-32, 35392 Gießen, Germany

article info abstract

Article history: Sinorhizobium meliloti harbours genes encoding orthologs of ribonuclease (RNase) E and RNase J, Received 6 May 2009 the principle endoribonucleases in and subtilis, respectively. To analyse Revised 10 June 2009 the role of RNase J in S. meliloti, RNA from a mutant with miniTn5-insertion in the RNase J-encoding Accepted 15 June 2009 gene was compared to the wild-type and a difference in the length of the 5.8S-like ribosomal RNA Available online 21 June 2009 (rRNA) was observed. Complementation of the mutant, Northern blotting and primer extension 0 Edited by Ulrike Kutay revealed that RNase J is necessary for the 5 -end maturation of 16S rRNA and of the two 23S rRNA fragments, but not of 5S rRNA. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Ribonuclease RNase J 50-End 23S rRNA 16S rRNA Small RNA

1. Introduction sponding gene [9] and found out that it is involved in rRNA maturation. Endoribonucleases are important for maturation and degrada- In rRNA is transcribed in polycistronic form and is pro- tion of RNA. In Escherichia coli, ribonuclease (RNase) E plays a cen- cessed by RNases. The primary transcript is cleaved by RNase III, tral role in the maturation of 16S ribosomal RNA (rRNA), 5S rRNA, and the released 16S rRNA and 23S rRNA precursors undergo fur- tRNA, RNase P RNA as well as in mRNA decay [1–3]. In bacteria ther maturation [1]. In many 23S rRNA is lacking RNase E, such as Bacillus subtilis, the RNA processing and not continuous, but is fragmented near the 50-end resulting in a degradation mechanisms remained unclear for a long time. Re- 5.8S rRNA-like molecule and a large 2.6 kb rRNA; in some strains cently, it was shown that B. subtilis harbours two homologs of a no- the 2.6 kb rRNA is additionally fragmented [10–12]. Usually frag- vel type of endoribonuclease, RNase J1 and RNase J2 [4], which are mentation occurs at sites with intervening sequences, and is important for mRNA turnover and 50-end maturation of 16S rRNA dependent upon RNase III [11–13]. The resulting precursor rRNA [5–7]. The endonucleolytic properties of RNase J and RNase E are fragments then undergo further maturation by as yet uncharacter- similar, although they are not orthologs [4]. Surprisingly, it turned ized enzymes. The present work suggests that RNase J is responsi- out that RNase J has a 50–30 exoribonuclease activity [6]. In con- ble for the final maturation of the 50-termini of the two 23S rRNA trast, E. coli harbours only 30–50 exoribonucleases [8]. Thus, E. coli fragments and of 16S rRNA in S. meliloti (Fig. 1). and B. subtilis have different RNA processing mechanisms. How- ever, many bacteria including Sinorhizobium meliloti harbour genes encoding RNase E and RNase J. To analyse the role of RNase J in S. 2. Materials and methods meliloti, we used a strain with a miniTn5-insertion in the corre- 2.1. Bacterial strains

The wild-type S. meliloti Rm2011 [14], the mutant 3.10.CO3 Abbreviations: rRNA, ribosomal RNA; rDNA, rRNA gene; RNase, ribonuclease; nt, carrying miniTn5-insertion in SMc01929 [9], and the comple- ; rrn, ribosomal RNA operon mented mutant with pRK415::SMc01929 were grown in TY * Corresponding author. Fax: +49 641 9933549. E-mail address: [email protected] [15,16] at 32 °C with appropriate antibiotics (streptomycin, 1 1 1 (E. Evguenieva-Hackenberg). 250 lg ll , neomycin 120 lg ll and tetracycline 20 lg ll ).

0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.06.026 2340 R. Madhugiri, E. Evguenieva-Hackenberg / FEBS Letters 583 (2009) 2339–2342

III? E? J III? III? E? J ?III III? E? J ?III

IGS IVS IGS 16S 5.8S 2.6 kb 5S

23S rRNA (pre-rRNA)

Fig. 1. Scheme of an rRNA operon in S. meliloti with proposed and verified ribonuclease cleavage sites. The rRNAs are indicated by bars, tRNAs in the intergenic sequence between 16S rRNA and 23S rRNA as well as downstream of 5S rRNA [19] are indicated by clover leafs. Sequences which are removed during processing of the primary transcript (intergenic sequences, IGS; intervening sequence, IVS; sequences at the 50- and 30-ends) are indicated by solid lines. RNase III is proposed to cleave out the pre-16S rRNA and the pre-23S rRNA, and, in addition, the IVS in helix 9 of 23S rRNA (indicated by arrows marked with III?) [12,13]. RNase E is proposed to cleave downstream of the RNase III cleavage sites at the 50-ends of the precursor rRNAs (indicated by arrows marked with E?) [23]. RNase J is shown to be responsible for the final maturation of the 50- ends of 16S rRNA and of the two 23S rRNA fragments (this work). Processed sequences and structural RNAs are not drawn to scale. pRK415::SMc01929 was transferred from E. coli S17-1 [17] into S. the region, in which 5S rRNA is expected (see below). In this region, meliloti 3.10.CO3 by diparental conjugation. two closely migrating bands were detectable in the wild-type probe (Fig. 2A). The identity of these bands was not clear: previ- 2.2. DNA manipulations ously they were described as 5S rRNA and 5.8S-like rRNA in Alpha- [11], but recently they were labelled as the 4.5S Manipulation of DNA was performed by standard methods [18]. RNA and 5S rRNA of S. meliloti 2011 [21]. To clarify their identity, Restriction enzymes and primers based on the S. meliloti genome we performed Northern blotting. The results show that the faster [19] were from Fermentas and Eurofins MWG Operon, respectively. migrating band of the prominent doublet is the 5S rRNA and the For PCR, the BIO-X-ACTTM DNA polymerase (Bioline) and total DNA slower migrating band is the 5.8S-like rRNA. The 4.5S RNA cannot were used; the amplificates were cloned into pDrive (Qiagen). A be assigned to a strong band comparable to the rRNA bands in the 3.6 kb ribosomal RNA operon (rrn) region was amplified with EtBr-stained gel (Fig. 2A). Smel-81721f 50-CTCGTCGATTC-AAAGTCAAC-30 and Smel-85308r 50-GTTTTCGCGGAG-AACCAGC-30. Sequencing was performed with 3.2. RNase J is necessary for the maturation of 23S rRNA fragments alpha-[32P]-dCTP and the T7 Sequencing Kit (USB). The solutions for 8% sequencing polyacrylamide gels were from AppliChem. We observed that the 5.8S-like rRNA is larger in the mutant RNase J of S. meliloti was identified by blastp (NCBI) using the B. than in the wild-type (Fig. 2B, lanes 1 and 2), suggesting that RNase subtilis RNase J2 as a query. The gene was amplified with Rnj-XbaI- J is necessary for the maturation of this rRNA. The suggestion was se 50-TCTAGAATGAAAAAAGCAAA-GGCGCATCAC-30 and RnjStrep- Kpn-as 50-GGTACCTCACTTCTCGAACTGCGGG-TGCGACCAGGAGGC GACCTTCGTGATAAAAACCGTCAC-30. The PCR product was re- cloned from pDrive into pRK415 [15] using XbaI and KpnI. 30µg 5 µg 2.6 kb rRNA 16S rRNA 5.8S 5S 2.3. RNA methods + + Probes 5S 5.8S 5S 4.5S 4.5S specific for

S. meliloti cells (OD600 0.5) were centrifuged at 6000g for 10 min at 4 °C, frozen in liquid nitrogen, disrupted with glass beads (Sigma) and Ribolyser (Hybaid), and total RNA was isolated using TRIzol (Invitrogen). RNA (3 lg) was separated on 10 % polyacryl- pre-5.8S rRNA amide–urea gels and stained by EtBr or transferred onto a nylon 5.8S rRNA 5.8 rRNA 5S rRNA membrane (Pall) for 4 h at 200 mA using a semidry blotter 5S rRNA 4.5S RNA (Peqlab). For detection of 5.8S-like rRNA, 5S rRNA and the signal recognition particle RNA (4.5S RNA) by Northern blotting, following 0 0 tRNA 5 -labelled primers were used: UP130 [10], 5 -GTTCGGAATGG- GAACGG-GTGCAG-30 and 50-GGCTGCTTCCTTCCGGACCTGAC-30. 1 2 3 4 5 6 7 The 4.5S RNA gene was identified by blastn (NCBI) using the corre- sponding E. coli gene as a query. 1 2 3 Primer extension was performed at 55 °C with 2 lg total RNA, pre-5.8S rRNA 20–30 units SuperscriptIII (Invitrogen) and the following 50-la- EtBr 5.8S rRNA belled primers: Smel-16SrRNA-PE (50-GCACTT-GCATGTGTTA 5S rRNA AGCCTGCC-30), Smel-5.8SrRNA-PE (50-CATGGATCAAAGCTCAT-TC 0 0 Northern blot, GCACG-3 ), Smel-2.6 kb-rRNA-PE (5 -CTTGCGGA-GTCTCGGT pre-5.8S rRNA 0 0 5.8S-probe TGATGTC-3 ) and Smel-5SrRNA-PE (5 -CTGGCAGCGA-CCTACTC 5.8S rRNA TCCC-30). Signals were detected using a molecular imager and the Northern blot, Quantity One software (BioRad). RNA secondary structures were 5S rRNA 5S-probe predicted with the program mfold [20]. Fig. 2. A precursor of the 5.8S rRNA accumulates in the RNase J mutant. (A) Detection of 5S rRNA, 5.8S-like rRNA and 4.5S RNA in total RNA from S. meliloti 3. Results wild-type. Panels 1 and 2, EtBr-stained RNA; panels 3–7, Northern blot hybridiza- tion; the loaded amount and the probes used, respectively, are given above the 3.1. Two closely migrating small RNAs are 5S rRNA and 5.8S-like rRNA panels. Prominent EtBr-stained bands are marked on the left side; molecules detected by hybridization are marked on the right side. (B) RNase J-dependent maturation of 5.8S-like rRNA. 1, Wild-type; 2, mutant; 3, complemented mutant. When RNA patterns of the wild-type and the mutant were com- The detected molecules are marked on the left side. The detection method is given pared after EtBr staining, a prominent difference was observed in at the right side. R. Madhugiri, E. Evguenieva-Hackenberg / FEBS Letters 583 (2009) 2339–2342 2341 confirmed by the successful complementation of the mutant (lane 50-ends. In the complemented mutant, 50-end maturation of the 3). We assumed that RNase J is involved in 50-end processing of 2.6 kb rRNA fragment was restored (lanes 1 and 3). rRNA and decided to analyse the 50-ends of all rRNA in S. meliloti. 3.3. RNase J is needed for the maturation of 16S rRNA but not of 5S The primer extension of the 5.8S-like rRNA revealed that the re- rRNA moval of 17 (nts) upstream of the mature 50-end is RNase J-dependent. In Fig. 3A, the mature 50-end in the wild-type The primer extension of 16S rRNA shows that the final trim- is marked with M, and the precursor end accumulating in the ming of the 50-end depends on RNase J (Fig. 3C). The experimen- RNase J-mutant is marked with P. The precursor is detectable in tally determined 50-end in the wild-type is in agreement with the wild-type, too (lanes 1 and 2). In the complemented mutant, the annotated 50-end. The major 50-end in the mutant is located maturation of the 5.8S-like rRNA was completely restored – the de- three nts upstream. Corresponding precursors are detectable in tected 50-ends are identical with those of the wild-type (lanes 1 the wild-type, too (lanes 1 and 2). The 50-ends detected in the com- and 3). The 50-end of 5.8S rRNA determined here is located six plemented mutant are identical with those in the wild-type (lanes nts downstream of the annotated 50-end of 23S rRNA and agrees 1 and 3). Thus, during 16S rRNA maturation, the last three nts at with the experimentally determined termini of the 5.8S-like rRNA the 50-end are removed by RNase J in S. meliloti. A minor signal in R. palustris and B. japonicum [11]. was detected at the position corresponding to very last nt (marked The primer extension of the 2.6 kb fragment of 23S rRNA re- with a thin arrow in the wild-type and complemented mutant). Fi- vealed that RNase J is needed for the removal of 40 nts upstream nally, primer extension of 5S rRNA demonstrated that 50-end mat- of the two positions representing the mature termini in the wild- uration of this rRNA is not RNase J-dependent (not shown). type (Fig. 3B; mature ends are marked with M). The positions of the 2.6 kb rRNA 50-ends agree with the 50-ends detected in other 4. Discussion Alphaproteobacteria [11,12]. At the position representing the ma- jor 50-end in the mutant (marked with P2), a precursor 50-end is Maturation of rRNA has been most intensely investigated in detectable in the wild-type, too (lanes 2 and 3). Two other precur- E. coli and B. subtilis, but there are still open questions [1].In sor 50-ends, which gave signals of similar intensity in the wild-type E. coli, following cleavage of the primary transcript by RNase III, and in the mutant, are detectable 9 and 10 nt upstream (marked the orthologous endoribonucleases RNase E and RNase G generate with P1). Further RNase J-dependent precursors ends (marked with the 50-end of 16S rRNA by sequential cleavages [22]. RNase E is also P3) were detected in the wild-type 5–6 nts upstream of the mature necessary for 50-end maturation of 5S rRNA. The final 30-ends of

5‘ U A U G A C G H9 U 5‘ CG G A G C G A U P A A U A C G T 1 2 3 C A C G T 1 2 3 A G C A G C G C A U C A U C G A G U G G G P1 G C A U G C U A U G U U G A C G G U A U U A C C A C C C A G G G C C C G C G G C C G A GU G A G C U A C U G U A AGAA C C U A A P2 G C A G C A M U U A G U U A U G U A G 3‘ U G A A U C G P1, A U G A A RNase III ? A U 3‘ 5‘ A U G A U G G C A C G T 1 2 3 G C 5‘ G U A A A P3 U A A U C G P2, U AA H10 C A G C A C A RNase G U P A U E ? A U G A U U U H8 G UGU C G A A A A U U G U G A M A M C A AG G G A U A A A P3, G U A U U RNase J A C G C C G U A G 3‘ G C C G C A U G C A U U G M, RNase J U 5‘-U A A A 3‘ GCGAACGCAGGGAAC -3‘

Fig. 3. Determination of 50-ends of rRNA by primer extension. A, C, G, T each refer to the corresponding nucleotide of the rRNA gene (rDNA) sense strand as determined by sequencing. A part of the rRNA sequence is indicated on the right side of each panel. Positions of detected 50-ends are shown by arrows. P, precursor end; M, mature end. (A) Primer extension of the 5.8S rRNA. 1, Wild-type; 2, mutant; 3, complemented mutant. (B) Primer extension of the 2.6 kb rRNA. 1, Complemented mutant; 2, mutant; 3, wild- type. Other descriptions are as in (A). (C) Primer extension of 16S rRNA. Descriptions are as in (A). (D) Schematic representation of the helices 8–10 (H8–H10) of the 23S rRNA precursor in S. meliloti. The arrows mark the positions of 50-ends detected in (B). The proposed processing enzymes are given. 2342 R. Madhugiri, E. Evguenieva-Hackenberg / FEBS Letters 583 (2009) 2339–2342

23S rRNA and 5S rRNA are created by the exoribonuclease RNase T. References However, it is still not clear which enzymes generate the 50-ends of 23S and 5S rRNA, and the 30-end of 16S rRNA in E. coli [1].InB. sub- [1] Deutscher, M.P. (2009) Maturation and degradation of ribosomal RNA in 0 bacteria. Prog. Mol. Biol. Transl. Sci. 85, 369–391. tilis, RNase J generates the 5 -end of 16S rRNA [5,6], but other un- [2] Carpousis, A.J., Luisi, B.F. and McDowall, K.J. (2009) Endonucleolytic initiation known RNases are also involved in 16S rRNA processing. The of mRNA decay in Escherichia coli. Prog. Mol. Biol. Transl. Sci. 85, 91–135. termini of 23S and 5S rRNA are generated by the RNases Mini-III [3] Hartmann, R.K., Gößringer, M., Späth, B., Fischer, S. and Marchfelder, A. (2009) and M5, respectively [1]. The making of tRNAs and more – RNase P and tRNase Z. Prog. Mol. Biol. Transl. Sci. 85, 319–368. Based on this knowledge, we assumed that RNase III cleaves the [4] Even, S., Pellegrini, O., Zig, L., Labas, V., Vinh, J., Bréchemmier-Baey, D. and primary rrn transcript in S. meliloti (Fig. 1). The 23S rRNA precursor Putzer, H. (2005) Ribonucleases J1 and J2: two novel endoribonucleases in B. molecule is probably cleaved by RNase III, too, because the position subtilis with functional homology to E. coli RNase E. Nucleic Acids Res. 33, 2141–2152. of the P1 precursor ends detected in Fig. 3B perfectly matches pre- [5] Britton, R.A., Wen, T., Schaefer, L., Pellegrini, O., Uicker, W.C., Mathy, N., Tobin, viously mapped RNase III cleavage sites in helix 9 of other Alpha- C., Daou, R., Szyk, J. and Condon, C. (2007) Maturation of the 50 end of Bacillus proteobacteria [11–13] (Fig. 3D). Since our results show that subtilis 16S rRNA by the essential ribonuclease YkqC/RNase J1. Mol. Microbiol. 0 63, 127–138. RNase J is necessary for generation of the 5 -termini of 16S rRNA [6] Mathy, N., Bénard, L., Pellegrini, O., Daou, R., Wen, T. and Condon, C. (2007) 50- and of the 23S rRNA fragments, we suggest that RNase J acts in a to-30 exoribonuclease activity in bacteria: role of RNase J1 in rRNA maturation processing pathway downstream of RNase III and of another endo- and 50 stability of mRNA. Cell 129, 681–692. [7] Mäder, U., Zig, L., Kretschmer, J., Homuth, G. and Putzer, H. (2008) MRNA ribonuclease, which creates the precursor ends that accumulate in processing by RNases J1 and J2 affects Bacillus subtilis gene expression on a the RNase J-mutant. This enzyme is probably RNase E, because S. global scale. Mol. Microbiol. 70, 183–196. meliloti harbours no RNase G, and RNase E was found to cleave [8] Andrade, J.M., Pobre, V., Silva, I.J., Domingues, S. and Arraiano, C.M. (2009) The role of 30–50 exoribonucleases in RNA degradation. Prog. Mol. Biol. Transl. Sci. in vitro transcripts of R. leguminosarum and R. capsulatus at the po- 85, 187–229. sition corresponding to the P2 precursor end found in vivo in [9] Pobigaylo, N., Wetter, D., Szymczak, S., Schiller, U., Kurtz, S., Meyer, F., Alphaproteobacteria (Fig. 3B and D; [12,23]). It cannot be excluded, Nattkemper, T.W. and Becker, A. (2006) Construction of a large signature- however, that RNase J and RNase E, have overlapping functions and tagged mini-Tn5 transposon library and its application to mutagenesis of Sinorhizobium meliloti. Appl. Environ. Microbiol. 72, 4329–4337. cleave endonucleolytically at the same positions in S. meliloti, since [10] Selenska-Pobell, S. and Evguenieva-Hackenberg, E. (1995) Fragmentations of RNase J of B. subtilis and RNase E of E. coli have similar cleavage the large-subunit rRNA in the family . J. Bacteriol. 177, 6993– specificity [4] The further 50-end maturation is RNase J-dependent, 6998. [11] Zahn, K., Inui, M. and Yukawa, H. (2000) Divergent mechanisms of 50 23S rRNA and the precursors are probably directly processed by the enzyme. IVS processing in the alpha-proteobacteria. Nucleic Acids Res. 28, 4623–4633. Fig. 3D summarizes our model for generation of the 50-end of the [12] Evguenieva-Hackenberg, E. (2005) Bacterial ribosomal RNA in pieces. Mol. 2.6 kb rRNA fragment. In Fig. 1, the proposed and verified ribonuc- Microbiol. 57, 318–325. [13] Evguenieva-Hackenberg, E. and Klug, G. (2000) RNase III processing of leolytic cleavages in the primary rrn transcript of S. meliloti are intervening sequences found in helix 9 of 23S rRNA in the alpha subclass of summarized. Proteobacteria. J. Bacteriol. 182, 4719–4729. At present, it is not known whether RNase J of S. meliloti has [14] Meade, H.M. and Signer, E.R. (1977) Genetic mapping of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA 74, 2076–2078. endo- and exoribonucleolytic activity. An increase in the minor sig- [15] Keen, N.T., Tamaki, S., Kobayashi, D. and Trollinger, D. (1988) Improved broad- nals one nt upstream of the mature 50-ends of 16S rRNA and of the host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70, 2.6 kb fragment (Fig. 3B and C) might indicate that the last nt is re- 191–197. 0 0 [16] Beringer, J.E. (1974) R factor transfer in Rhizobium leguminosarum. J. Gen. moved more slowly during a 5 -3 exonucleolytic trimming of the Microbiol. 84, 188–198. termini. Alternatively, these signals might also represent minor [17] Simon, R., Priefer, U. and Pühler, A. (1982) A broad host range mobilization endonucleolytic cleavage products. Thus, the precise mechanism system for in vivo genetic engineering: transposon mutagenesis in gram- for final 50-end maturation of rRNA by RNase J in S. meliloti remains negative bacteria. Biotechnology 1, 784–791. [18] Ausubel, F.M., Brent, R., Kingston, D.D., Moore, J., Seidman, G., Smith, J.A. and to be determined. Struhl, K. (1989) Current Protocols in Molecular Biology, vol. I, Greene Finally, it is important to notice that B. subtilis harbours two Publishing Associates and Wiley Interscience, New York, NY. RNase J homologs, one of which (RNase J1) is essential. S. meliloti [19] Galibert, F., Finan, T.M., Long, S.R., Puhler, A., Abola, P., Ampe, F., Barloy-Hubler, F., Barnett, M.J., Becker, A., Boistard, P., Bothe, G., Boutry, M., Bowser, L., has only enzyme of the RNase J-type, and the protein shows higher Buhrmester, J., Cadieu, E., Capela, D., Chain, P., Cowie, A., Davis, R.W., Dreano, similarity to RNase J2 then to RNase J1 of B. subtilis. Our data and the S., Federspiel, N.A., Fisher, R.F., Gloux, S., Godrie, T., Goffeau, A., Golding, B., fact that two RNase J-mutants are present in the mini-Tn5 library of Gouzy, J., Gurjal, M., Hernandez-Lucas, I., Hong, A., Huizar, L., Hyman, R.W., Jones, T., Kahn, D., Kahn, M.L., Kalman, S., Keating, D.H., Kiss, E., Komp, C., S. meliloti [9] show that RNase J is not essential in this organism. The Lelaure, V., Masuy, D., Palm, C., Peck, M.C., Pohl, T.M., Portetelle, D., Purnelle, B., two mutants have identical defects in rRNA maturation (results not Ramsperger, U., Surzycki, R., Thebault, P., Vandenbol, M., Vorholter, F.J., shown), and carry transposon insertions in the 37. (used here) and Weidner, S., Wells, D.H., Wong, K., Yeh, K.C. and Batut, J. (2001) The composite genome of the legume symbiont Sinorhizobium meliloti. Science 2001 (293), 60. codon of the gene, respectively [9]. The finding that rRNA is not 668–672. properly maturated in the RNase J-mutant of S. meliloti shows that [20] Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization although the cellular role of RNase J in B. subtilis and of RNase E in prediction. Nucleic Acids Res. 31, 3406–3415. [21] Valverde, C., Livny, J., Schlüter, J.P., Reinkensmeier, J., Becker, A. and Parisi, G. E. coli are similar, there is differentiation of function between the (2008) Prediction of Sinorhizobium meliloti sRNA genes and experimental two enzymes when they are present in the same cell. detection in strain 2011. BMC 9, 416. [22] Li, Z., Pandit, S. and Deutscher, M.P. (1999) RNase G (CafA protein) and RNase 0 Acknowledgements E. RNase G (CafA protein) and RNase E are both required for the 5 maturation of 16S ribosomal RNA. EMBO J. 18, 2878–2885. [23] Klein, F. and Evguenieva-Hackenberg, E. (2002) RNase E is involved in 50-end We thank Gabriele Klug (Universität Giessen) for generous sup- 23S rRNA processing in alpha-Proteobacteria. Biochem. Biophys. Res. port. We are grateful to Anke Becker (Universität Freiburg) for Commun. 299, 780–786. sending us the S. meliloti mutant strains. This work was supported by the Deutsche Forschungsgemeinschaft (Ev42/4-1).