J. Mol. Biol. (1995) 249, 869–878 Immunity Determinant of Phage-plasmid P4 is a Short Processed RNA Francesca Forti, Pierangela Sabbattini, Gianpiero Sironi Sandro Zangrossi, Gianni Deho ` and Daniela Ghisotti* In the phage-plasmid P4, both lysogenic and lytic functions are coded by the Dipartimento di Gentica e di Biologia dei Microrganismi same operon. Early after infection the whole operon is transcribed from the constitutive promoter P LE . In the lysogenic condition transcription from P LE Universita ` di Milano terminates prematurely and only the immunity functions, which are Via Celoria 26, 20133 Milano proximal to the promoter, are thus expressed. Fragments of the P4 immunity Italy region were cloned in an expression vector. A DNA fragment as short as 91 bp was sufficient, when transcribed, to express P4 immunity and to complement P4 immunity deficient mutants. This fragment, like prophage P4, produced a 69 nt long RNA (CI RNA). A shorter P4 fragment neither expressed immunity nor synthesized the CI RNA. Thus the CI RNA is the P4 trans -acting immunity factor. The 5' end of the CI RNA, mapped by primer extension, does not correspond to the transcription initiation point, thus suggesting that the CI RNA is produced by processing of the primary transcript. In an RNase P mutant host the processing of the 5' end and the production of a functional CI RNA were impaired. The requirement of RNase P for the correct processing of CI appears to be related to the predicted secondary structure of the precursor CI RNA. A region (seqB) within the CI RNA shows complementarity with two cis -acting sequences (seqA and seqC) required for P4 immunity, suggesting that transcription termination may be caused by pairing of the CI RNA with the complementary target sequences on the nascent transcript. Keywords: prophage immunity; RNA processing; RNA–RNA interactions; RNase P; transcription termination *Corresponding author Introduction In most known temperate bacteriophages such as , Mu, P2, and P1, immunity is elicited by a repressor protein that prevents transcription initiation at key promoters for lytic gene expression (Gussin et al ., 1983; Howe, 1987; Bertani & Six, 1988; Yarmolinsky & Sternberg, 1988). The immunity system of P4 differs from this model in that expression of the genes involved in the lytic cycle is not prevented by repression of transcription initiation, but by premature termination of transcription from the constitutive promoter P LE (Deho ` et al ., 1988, 1992). This control is exerted also in trans on a superinfecting phage, thus eliciting superinfection immunity. The satellite phage P4 enjoys multiple develop- mental pathways. Following infection of its host, Escherichia coli , P4 enters a short period of uncommitted replication followed by the choice between lytic/plasmid propagation or lysogenic maintenance. Lytic growth is dependent on the presence in the bacterial cell of a helper phage, such as P2, that provides P4 with the morphogenetic and lytic functions. In the absence of the helper, P4 replicates as a multicopy plasmid. In the lytic/plas- mid condition, autonomous replication of P4 is active, and expression of the P4 replication genes is needed. In contrast, lysogenization requires the integration of the phage genome into the bacterial chromosome and the establishment of prophage immunity, which prevents expression of P4 replica- tion genes (for reviews, see Bertani & Six, 1988; Christie & Calendar, 1990; Lindqvist et al ., 1993). Central to the choice between the lysogenic versus Present address: Pierangela Sabbattini, Medical Research Council, Clinical Sciences Center, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 0NN, UK. Abbreviations used: IPTG, isopropyl--D- thiogalactopyranoside; p.f.u., plaque forming units. 0022–2836/95/250869–10 $08.00/0  1995 Academic Press Limited