Mechanism of inhibition of Rho-dependent transcription termination by bacteriophage P4 protein Psu.

In the Escherichia coli satellite phage P4, transcription starting from PLE 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 t151 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 PLE and PLL promoters, respectively, and terminating at the intracistronic t151 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 α genes. This correlates with the inability of P4 sut1 to propagate in the plasmid state. By cloning different P4 DNA fragments, we mapped the t151 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.

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Translation of Two Nested Genes in Bacteriophage P4 Controls Immunity-Specific Transcription Termination

Journal of Bacteriology ◽

10.1128/jb.181.17.5225-5233.1999 ◽

1999 ◽

Vol 181 (17) ◽

pp. 5225-5233 ◽

Cited By ~ 8

Author(s):

Francesca Forti ◽

Simona Polo ◽

Kirk B. Lane ◽

Erich W. Six ◽

Gianpiero Sironi ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Nonsense Mutation ◽

Transcription Termination ◽

Open Reading Frame ◽

Terminal Part ◽

Reading Frame ◽

Bacteriophage P4 ◽

Nested Genes ◽

Nonsense Codons

ABSTRACT In phage P4, transcription of the left operon may occur from both the constitutive PLE promoter and the regulated PLL promoter, about 400 nucleotides upstream of PLE. A strong Rho-dependent termination site,timm , is located downstream of both promoters. When P4 immunity is expressed, transcription starting at PLE is efficiently terminated attimm , whereas transcription from PLL is immunity insensitive and reads throughtimm . 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 PLE 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 PLE. Theeta gene is expressed when transcription starts from the PLL promoter. Three likely start codons predict a size between 197 and 199 amino acids for the Eta gene product. Bothkil and eta overlap thetimm site. By cloning kil upstream of a tRNA reporter gene, we demonstrated that translation of thekil region prevents premature transcription termination attimm . This suggests that P4 immunity might negatively control kil translation, thus enabling transcription termination at timm . Transcription starting from PLL proceeds throughtimm . Mutations that create nonsense codons ineta caused premature termination of transcription starting from PLL. Suppression of the nonsense mutation restored transcription readthrough at timm . Thus, termination of transcription from PLL is prevented by translation of eta.

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