Control of transcription termination by an RNA factor in bacteriophage P4 immunity: identification of the target sites

Abstract

Prophage P4 immunity is elicited by a short, 69-nucleotide RNA (CI RNA) coded for within the untranslated leader region of the same operon it controls. CI RNA causes termination of transcription that starts at the promoter P LE and prevents the expression of the distal part of the operon that codes for P4 replication functions (␣ operon). In this work, we identify two sequences in the untranslated leader region of the ␣ operon, seqA and seqC, that are the targets of the P4 immunity factor. seqA and seqC exhibit complementarity to a sequence internal to the CI RNA (seqB). Mutations in either seqA or seqC that alter its complementarity to seqB abolished or reduced P4 lysogenization proficiency and delayed the shutoff of the long transcripts originating from P LE that cover the entire operon. Both seqA and seqC single mutants were still sensitive to P4 prophage immunity, whereas P4 seqA seqC double mutants showed a virulent phenotype. Thus, both functional sites are necessary to establish immunity upon infection, whereas a single site appears to be sufficient to prevent lytic gene expression when immunity is established. A mutation in seqB that restored complementarity to both seqA and seqC mutations also restored premature termination of P LE transcripts, thus suggesting an important role for RNA-RNA interactions between seqB and seqA or seqC in P4 immunity.In most known temperate bacteriophages, prophage immunity is determined by a repressor protein which prevents transcription initiation at promoters controlling the expression of replication functions and of other genes involved in the lytic cycle. Satellite bacteriophage P4 deviates from this model in two respects. (i) Prophage immunity does not prevent transcription initiation of the operon encoding the replication functions (␣ operon); rather, it promotes premature termination of transcription that initiates at P LE , the constitutive promoter of the ␣ operon (9, 10, 12). (ii) The immunity factor is not a protein but a small, 69-nucleotide (nt) RNA (CI RNA) exhibiting complementarity with the nontranslated leader region of the transcripts it regulates (10, 11a, 12).P4 depends on all the morphogenetic genes of a helper phage such as P2 to assemble the viral particle. Autonomous P4 replication requires the expression of a primase-helicase coded for by the P4 ␣ gene (14, 35) and leads either to the lytic cycle (when a helper phage genome is present in the host cell) or to a multicopy plasmid state (in the absence of the helper).As an episome, P4 may also integrate in the host chromosome and establish the lysogenic condition (for a review, see reference 21).Central to the choice and the maintenance of the lysogenic versus the lytic or plasmid condition is the regulation of P4 replication genes (Fig. 1). Soon after infection of a sensitive host, transcription of the ␣ operon starts at the constitutive promoter P LE . Transcription from P LE seems to be intrinsically termination prone, and transcripts of different lengths (0.3 to 0.5, 1.3, and 4.1 kb) are produced....

Complete Genomic Sequence of SfV, a Serotype-Converting Temperate Bacteriophage of Shigella flexneri

“…ysis of the HinfI/BamHI fragment indicated that it was predicted to contain the following P4 TT features (Allison and Verma, unpublished): the P LE 70 promoter (Ϫ35 sequence, TTGATT, nt 27568 to 27573; Ϫ10 sequence, TACACT, nt 27591 to 27596); cI RNA containing seqA, seqB, seqCЈ, and seqCЉ and folding in the conserved secondary RNA structure of the P4 cI RNA; and a nested ORF, orf-77, commencing downstream of the cI RNA (the ATG start codon is located at nt 27846) and reading in frame with orf-37. In the immune state of phage P4, the cI RNA molecule (69 nt), which is the product of processing of a transcript initiated from constitutive promoter P LE , mediates TT and superinfection immunity through RNA-RNA interactions with complementary sequences located up-and downstream in the nascent transcript, thus directly preventing the expression of downstream genes involved in the lytic cycle (14,42). The cI RNA has a complex predicted secondary structure and contains seqB, which is complementary to upstream seqA and downstream seqCЈ and seqCЉ (14,42).…”

“…In the immune state of phage P4, the cI RNA molecule (69 nt), which is the product of processing of a transcript initiated from constitutive promoter P LE , mediates TT and superinfection immunity through RNA-RNA interactions with complementary sequences located up-and downstream in the nascent transcript, thus directly preventing the expression of downstream genes involved in the lytic cycle (14,42). The cI RNA has a complex predicted secondary structure and contains seqB, which is complementary to upstream seqA and downstream seqCЈ and seqCЉ (14,42). P LE , seqA, seqB, and seqC are located within the eta gene.…”

Functional Analysis of the Protein Machinery Required for Transport of Lipopolysaccharide to the Outer Membrane of Escherichia coli

“…The waaL::cam locus was transferred into FL905, FL907, AM661, and AM679 by P1 transduction (34). Bacteria were grown in LD broth (33). When required, 0.2% L-arabinose (as an inducer of the araBp promoter), 0.2% glucose, 0.5 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) for overexpression of LptC with a C-terminal His 6 tag (LptC-H) or 1 mM IPTG, 100 g/ml ampicillin, 50 g/ml kanamycin, and 25 g/ml chloramphenicol were added.…”

Mycobacterium tuberculosis FurA Autoregulates Its Own Expression

“…E. coli XL1-Blue (5) was grown in LD medium (28) supplemented, when necessary, with kanamycin (50 g/ml). M. smegmatis mc 2 155 was grown in LD medium containing 0.2% (vol/vol) glycerol and 0.05% (vol/vol) Tween 80 and supplemented when necessary with kanamycin (20 g/ml).…”