Control of Transcription Termination by an RNA Factor in Bacteriophage P4 Immunity: Identification of the Target Sites (original) (raw)
Related papers
Bacteriophage P4 immunity controlled by small RNAs via transcription termination
Molecular Microbiology, 1992
Satellite bacteriophage P4 immunity is encoded within a short DNA region 357 bp long containing the promoter PLE and 275 bp downstream. PLE is active both in the early post-infection phase, when genes necessary for P4 lytic cycle are transcribed from this promoter, and in the lysogenic condition, when expression of the above genes is prevented by prophage immunity. In order to understand how P4 immunity is elicited, we have characterized the transcription pattern during the establishment and the maintenance of the satellite phage P4 lysogenic condition. We found that prophage transcription starting at PLE ends prematurely and the transcripts do not extend beyond 300-400 nucleotides downstream of P|.E-Thus P4 immunity acts by causing premature transcription termination rather than by repressing transcription initiation. The P4 immunity region is transcribed in the prophage, but it does not seem to be translated; this region contains two elements {seqA and seqB) of a palindromic sequence. In addition to transcripts about 300 nucleotides long, P4 prophage produces a family of shorter transcripts, about 80 nucleotides long, containing seqA or seqB. Evidence is presented suggesting that SeqB RNA is the frans-acting immunity factor, and that interaction of SeqB RNA with the complementary nascent RNA containing seqA may be involved in bringing about premature transcription termination.
1999
In phage P4, transcription of the left operon may occur from both the constitutive P LE promoter and the regulated P LL promoter, about 400 nucleotides upstream of P LE . A strong Rho-dependent termination site, t imm , is located downstream of both promoters. When P4 immunity is expressed, transcription starting at P LE is efficiently terminated at t imm , whereas transcription from P LL is immunity insensitive and reads through t imm . We report the identification of two nested genes, kil and eta, located in the P4 left operon. The P4 kil gene, which encodes a 65-amino-acid polypeptide, is the first translated gene downstream of the P LE promoter, and its expression is controlled by P4 immunity. Overexpression of kil causes cell killing. This gene is the terminal part of a longer open reading frame, eta, which begins upstream of P LE . The eta gene is expressed when transcription starts from the P LL promoter. Three likely start codons predict a size between 197 and 199 amino acids for the Eta gene product. Both kil and eta overlap the t imm site. By cloning kil upstream of a tRNA reporter gene, we demonstrated that translation of the kil region prevents premature transcription termination at t imm . This suggests that P4 immunity might negatively control kil translation, thus enabling transcription termination at t imm . Transcription starting from P LL proceeds through t imm . Mutations that create nonsense codons in eta caused premature termination of transcription starting from P LL . Suppression of the nonsense mutation restored transcription readthrough at t imm . Thus, termination of transcription from P LL is prevented by translation of eta.
Immunity Determinant of Phage-plasmid P4 is a Short Processed RNA
Journal of Molecular Biology, 1995
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 Università 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.
Bacteriophage P4 sut1: a mutation suppressing transcription termination
Journal of General Virology, 2007
In the Escherichia coli satellite phage P4, transcription starting from P LE is prevalently controlled via premature termination at several termination sites. We identified a spontaneous mutation, P4 sut1 (suppression of termination), in the natural stop codon of P4 orf151 that, by elongating translation, suppresses transcription termination at the downstream t 151 site. Both the translational and the transcriptional profile of P4 sut1 differed from those of P4 wild-type. First of all, P4 sut1 did not express Orf151, but a higher molecular mass protein, compatible with the 303 codon open reading frame generated by the fusion of orf151, cnr and the intervening 138 nt. Moreover, after infection of E. coli, the mutant expressed a very low amount of the 1.3 and 1.7 kb transcripts originating at P LE and P LL promoters, respectively, and terminating at the intracistronic t 151 site, whereas correspondingly higher amounts of the 4.1 and 4.5 kb RNAs arising from the same promoters and covering the entire operon were detected. Thus the sut1 mutation converts a natural stop codon into a sense codon, suppresses a natural intracistronic termination site and leads to overexpression of the downstream cnr and a genes. This correlates with the inability of P4 sut1 to propagate in the plasmid state. By cloning different P4 DNA fragments, we mapped the t 151 transcription termination site within the 7633-7361 region between orf151 and gene cnr. A potential stem-loop structure, resembling the structure of a Rho-independent termination site, was predicted by mfold sequence analysis at 7414-7385.
Journal of Molecular Biology, 2002
In the immune state bacteriophage P4 prevents expression of the replication functions by premature termination of transcription. A small RNA, the CI RNA, is the trans acting factor that regulates P4 immunity, by pairing to complementary target sequences and causing premature transcription termination. The CI RNA is matured by RNAse P and PNPase from the leader region of the same operon it regulates. In this work we better characterize this molecule. CI RNA copy number was determined to be around 500 molecules per lysogenic cell. By S 1 mapping we de®ned the 3 H -end at 8423(AE1); thus CI RNA is 79(AE1) nt long. The minimum region for correct processing requires two bases upstream of the CI RNA 5 H -end and the CCA sequence at the 3 H -end. Computer analysis by FOLD RNA of CI RNA sequence predicts a cloverleaf-like structure formed by a double-stranded stalk, a minor and a major stem loop, and a singlestranded bulge. We analysed several cI mutations, which fall either in the single or double-stranded CI RNA regions. Base substitutions in the main loop and in the single-stranded bulge apparently did not change CI RNA structure, but affected its activity by altering the complementarity with the target sequences, whereas a mutation in the secondary stem had a disruptive effect on CI RNA secondary structure. The effects of this latter mutation were suppressed by a base substitution that restored the complementarity with the corresponding base in the stem. Base substitutions in the main stem caused only local alterations in the secondary structure of CI. However, when the substitutions concerned either G8501 or its complementary base at the bottom of the stem, CI RNA was not correctly processed.
Characterization of the small antisense CI RNA that regulates bacteriophage P4 immunity1
Journal of Molecular Biology, 2002
In the immune state bacteriophage P4 prevents expression of the replication functions by premature termination of transcription. A small RNA, the CI RNA, is the trans acting factor that regulates P4 immunity, by pairing to complementary target sequences and causing premature transcription termination. The CI RNA is matured by RNAse P and PNPase from the leader region of the same operon it regulates. In this work we better characterize this molecule. CI RNA copy number was determined to be around 500 molecules per lysogenic cell. By S 1 mapping we de®ned the 3 H -end at 8423(AE1); thus CI RNA is 79(AE1) nt long. The minimum region for correct processing requires two bases upstream of the CI RNA 5 H -end and the CCA sequence at the 3 H -end. Computer analysis by FOLD RNA of CI RNA sequence predicts a cloverleaf-like structure formed by a double-stranded stalk, a minor and a major stem loop, and a singlestranded bulge. We analysed several cI mutations, which fall either in the single or double-stranded CI RNA regions. Base substitutions in the main loop and in the single-stranded bulge apparently did not change CI RNA structure, but affected its activity by altering the complementarity with the target sequences, whereas a mutation in the secondary stem had a disruptive effect on CI RNA secondary structure. The effects of this latter mutation were suppressed by a base substitution that restored the complementarity with the corresponding base in the stem. Base substitutions in the main stem caused only local alterations in the secondary structure of CI. However, when the substitutions concerned either G8501 or its complementary base at the bottom of the stem, CI RNA was not correctly processed.
Journal of Bacteriology, 1996
Bacteriophage P4's superinfection immunity mechanism is unique among those of other known bacteriophages in several respects: (i) the P4 immunity factor is not a protein but a short, stable RNA (CI RNA); (ii) in the prophage the expression of the replication operon is prevented by premature transcription termination rather than by repression of transcription initiation; (iii) transcription termination is controlled via RNA-RNA interactions between the CI RNA and two complementary target sequences on the nascent transcript; and (iv) the CI RNA is produced by processing of the same transcript it controls. It was thought that several host-encoded factors may participate in the molecular events required for P4 immunity expression, i.e., RNA processing, RNA-RNA interactions, and transcription termination. To identify such factors we searched for Escherichia coli mutations that affect P4 lysogenization. One such mutation, bfl-1, severely reduced P4's lysogenization frequency and delayed both the disappearance of the long transcripts that cover the entire replication operon and the appearance of the CI RNA. By physical mapping and genetic analysis we show that bfl-1 is allelic to pnp, which codes for polynucleotide phosphorylase, a 3-to-5 exonucleolytic enzyme. A previously isolated pnp null mutant (pnp-7) exhibited a phenotype similar to that of bfl-1. These results indicate that the polynucleotide phosphorylase of E. coli is involved with the maturation pathway of bacteriophage P4's RNA immunity factor.
Nonessential region of bacteriophage P4: DNA sequence, transcription, gene products, and functions
We sequenced the leftmost 2,640 base pairs of bacteriophage P4 DNA, thus completing the sequence of the 11,627-base-pair P4 genome. The newly sequenced region encodes three nonessential genes, which are called gop, 0, and clI (in order, from left to right). The gop gene product kills Escherichwa coli when the 0 protein is absent; the gop and ,3 genes are transcribed rightward from the same promoter. The cIl gene is transcribed leftward to a rho-independent terminator. Mutation of this terminator creates a temperature-sensitive phenotype, presumably owing to a defect in expression of the , gene.