Structural and mechanistic basis of anti-termination of Rho-dependent transcription termination by bacteriophage P4 capsid protein Psu

ABSTRACTRho is a hexameric molecular motor that functions as a conserved transcription terminator in the majority of bacterial species and is a potential drug target. Psu is a bacteriophage P4 capsid protein that inhibitsEscherichia coliRho by obstructing its ATPase and translocase activities. In this study, we explored the anti-Rho activity of Psu for Rho proteins from different pathogens. Sequence alignment and homology modeling of Rho proteins from pathogenic bacteria revealed the conserved nature of the Psu-interacting regions in all these proteins. We chose Rho proteins from various pathogens, includingMycobacterium smegmatis,Mycobacterium bovis,Mycobacterium tuberculosis,Xanthomonas campestris,Xanthomonas oryzae,Corynebacterium glutamicum,Vibrio cholerae,Salmonella enterica, andPseudomonas syringae. The purified recombinant Rho proteins of these organisms showed variable rates of ATP hydrolysis on poly(rC) as the substrate and were capable of releasing RNA from theE. colitranscription elongation complexes. Psu was capable of inhibiting these two functions of all these Rho proteins.In vivopulldown assays revealed direct binding of Psu with many of these Rho proteins.In vivoexpression ofpsuinduced killing ofM. smegmatis,M. bovis,X. campestris, andE. coliexpressingS. entericaRho indicating Psu-induced inhibition of Rho proteins of these strains under physiological conditions. We propose that the “universal” inhibitory function of the Psu protein against the Rho proteins from both Gram-negative and Gram-positive bacteria could be useful for designing peptides with antimicrobial functions and that these peptides could contribute to synergistic antibiotic treatment of the pathogens by compromising the Rho functions.IMPORTANCEBacteriophage-derived protein factors modulating different bacterial processes could be converted into unique antimicrobial agents. Bacteriophage P4 capsid protein Psu is an inhibitor of theE. colitranscription terminator Rho. Here we show that apart from antagonizingE. coliRho, Psu is able to inhibit Rho proteins from various phylogenetically unrelated Gram-negative and Gram-positive pathogens. Upon binding to these Rho proteins, Psu inhibited them by affecting their ATPase and RNA release functions. The expression of Psuin vivokills various pathogens, such asMycobacteriumandXanthomonasspecies. Hence, Psu could be useful for identifying peptide sequences with anti-Rho activities and might constitute part of synergistic antibiotic treatment against pathogens.

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Mechanism of Inhibition of Rho-dependent Transcription Termination by Bacteriophage P4 Protein Psu

Journal of Biological Chemistry ◽

10.1074/jbc.m603982200 ◽

2006 ◽

Vol 281 (36) ◽

pp. 26491-26500 ◽

Cited By ~ 32

Author(s):

Bibhusita Pani ◽

Sharmistha Banerjee ◽

Jisha Chalissery ◽

Muralimohan Abishek ◽

Ramya Malarini Loganathan ◽

...

Keyword(s):

Transcription Termination ◽

Mechanism Of Inhibition ◽

Bacteriophage P4

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The Psu protein of bacteriophage P4 is an antitermination factor for rho-dependent transcription termination.

Journal of Bacteriology ◽

10.1128/jb.173.21.6722-6731.1991 ◽

1991 ◽

Vol 173 (21) ◽

pp. 6722-6731 ◽

Cited By ~ 25

Author(s):

N A Linderoth ◽

R L Calendar

Keyword(s):

Transcription Termination ◽

Bacteriophage P4

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Bacteriophage P4 sut1: a mutation suppressing transcription termination

Journal of General Virology ◽

10.1099/vir.0.82605-0 ◽

2007 ◽

Vol 88 (3) ◽

pp. 1041-1047

Author(s):

Susanna Terzano ◽

Ilaria Oliva ◽

Francesca Forti ◽

Claudia Sala ◽

Francesca Magnoni ◽

...

Keyword(s):

Stop Codon ◽

Transcription Termination ◽

Spontaneous Mutation ◽

Wild Type ◽

Stem Loop ◽

Reading Frame ◽

E Coli ◽

Stem Loop Structure ◽

Bacteriophage P4 ◽

Sense Codon

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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