MiCrObiology (1996), 142, 367-375 Printed in Great Britain Escherichia coli RNase II : characterization of the promoters involved in the transcription of rnb Rita Zilhao,’ Jacqueline Plumbridge,2 Eliane Hajnsdorf,2 Philippe RCgnier’ and Cecllia M. Arraiano’ Author for correspondence: Cecilia M. Arraiano. Tel: +351 1 442 6321. Fax: +351 1 442 8766. e-mail: cecilia@itqb.unl.pt 1 lnstituto de Tecnologia QuImica e Biolbgica, Universidade Nova de Lisboa, Apt 127, 2780 Oeiras, Portugal 2 lnstitut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France The mnb gene encodes ribonuclease II (RNase ll), one of the two major Escherichia coli exonucleases involved in mRNA degradation. In this paper, the mb transcript is characterized regarding its promoter and terminator regions. The combined results from S1 nuclease protectionanalysis, DNase I footprinting and gene fusions with lac2 have shown that mb is expressed from two promoters. S1 nuclease protection analysis and DNA footprinting have shown that mnb has two promoters, PI and P2. Transcriptional and translational lac2 reporter fusions, constructed to the mb gene, revealed that P2, the mb proximal promoter, is stronger than PI. However, P2 is not transcribed in vitro, suggesting that an additional factor is required in wivo. The 3’ end of the mb transcript mapped to a stem-loop structure immediately after the translated region. Keywords : Escberichia coli, mb, RNase 11, promoters, transcriptional control INTRODUCTION Ribonucleases are one of the main factors that determine the level of functional messengers in the cell (Deutscher, 1988). Accordingly, in the last years much research has focused on the identification, description and possible regulation of endo- and exonucleases (Belasco, 1993). RNase 11, a major processive exonuclease involved in 3’-5’ mRNA degradation in E. culi (Kaplan & Apirion, 1974; Donovan & Kushner, 1986) was first described in 1963 (Spahr & Schlessinger, 1963). Twenty years later the rnb gene was cloned (Donovan & Kushner, 1983), but the nucleotide sequence was only recently determined (Zilhiio et a/., 1993). Surprisingly, it has been suggested that RNase I1 may also have a stabilizing effect in the degradation of some messages (Hajnsdorf et al., 1994; Pepe et al., 1994). Strains carrying mutations like mb296, rnbS00, Arnb 150: : tet and Amb201: : tet are viable (Nikolaev et al., 1976; Donovan & Kushner, 1986; Piedade et al., 1995; Zilhiio et al., 1995a). However, double mutant strains (pnp7 rnbSOU), deficient in RNase I1 and polynu- cleotide phosphorylase (PNPase, encoded by pnp) are not viable, suggesting that these two enzymes are functionally equivalent (Donovan & Kushner, 1986). Non-viability of the double mutant is most likely due to the lack of exonucleolytic activity suggesting that there are probably no nucleases that can replace the role of both RNase I1 and PNPase. However, in vitro studies have shown that these two enzymes have a different capacity to overcome secondary structures in mRNA (Guarneros & Portier, 1990; McLaren et al., 1991). Other ribonucleases have been shown to be associated in complexes and to control their own expression (Miczak et al., 1991; Robert-Le Meur & Portier, 1992; Carpousis et al., 1994). RNase I1 is post-transcriptionally regulated by RNase I11 and RNase E (Zilhiio et al., 199513). The possibility that RNase I1 could also be regulated at the level of transcription encouraged us to investigate the expression of the mb gene. Based on a variety of in vitro and in vivu experimental approaches, we show that mb is transcribed from two promoters, P1 and P2, which have different expression levels. We suggest that in vim RNA polymerase needs an additional factor to initiate at P2. In this paper we also report that the mb transcript terminates in a rho- independent terminator as determined by S1 mapping and sequence analysis. METHODS The GenBanWEMBUDDBJ accession number for the corrected sequence of the 3’ end of mb is X67913. Strains, plasmids, phages and media. E. cob strains and plasmids used in this work are listed in Table 1. The cultures 0002-01 58 0 1996 SGM 367