A role of RnlA in the RNase LS activity from Escherichia coli

Escherichia coli ribonuclease LS is a potential antagonist of bacteriophage T4. When the T4 dmd gene is defective, RNase LS cleaves T4 mRNAs and antagonizes T4 reproduction. Our previous work demonstrated that E. coli rnlA is essential for RNase LS activity. Here we show that His-tagged RnlA cleaves T4 soc RNA at one of the sites also cleaved by RNase LS in a cell extract. The cleavage activities of His-tagged RnlA and the RNase LS activity in a cell extract were inhibited by Dmd encoded by T4 phage. Fractionation of the RNase LS activity in a cell extract showed that it sedimented through a sucrose density gradient as a 1000-kDa complex that included RnlA. Pull-down experiments revealed more than 10 proteins associated with His-tagged RnlA. Among these, triose phosphate isomerase exhibited a remarkable affinity to RnlA. These results suggest that RnlA plays a central role in RNase LS activity and that its activity is regulated by multiple components.     

A novel endoribonuclease, RNase LS, in Escherichia coli

The dmd gene of bacteriophage T4 is required for the stability of late-gene mRNAs. When this gene is mutated, late genes are globally silenced because of rapid degradation of their mRNAs. Our previous work suggested that a novel Escherichia coli endonuclease, RNase LS, is responsible for the rapid degradation of mRNAs. In this study, we demonstrated that rnlA (formerly yfjN) is essential for RNase LS activity both in vivo and in vitro. In addition, we investigated a role of RNase LS in the RNA metabolism of E. coli cells under vegetative growth conditions. A mutation in rnlA reduced the decay rate of many E. coli mRNAs, although there are differences in the mutational effects on the stabilization of different mRNAs. In addition, we found that a 307-nucleotide fragment with an internal sequence of 23S rRNA accumulated to a high level in rnlA mutant cells. These results strongly suggest that RNase LS plays a role in the RNA metabolism of E. coli as well as phage T4.     

RNase HI stimulates the activity of RnlA toxin in Escherichia coli

A type II toxin–antitoxin system in Escherichia coli, rnlA‐rnlB, functions as an anti‐phage mechanism. RnlA is a toxin with an endoribonuclease activity and the cognate RnlB inhibits RnlA toxicity in E. coli cells. After bacteriophage T4 infection, RnlA is activated by the disappearance of RnlB, resulting in the rapid degradation of T4 mRNAs and consequently no T4 propagation, when T4 dmd is defective: Dmd is an antitoxin against RnlA for promoting own propagation. Previous studies suggested that the activation of RnlA after T4 infection was regulated by multiple components. Here, we provide the evidence that RNase HI is an essential factor for activation of RnlA. The dmd mutant phage could grow on ΔrnhA (encoding RNase HI) cells, in which RnlA‐mediated mRNA cleavage activity was defective. RNase HI bound to RnlA in vivo and enhanced the RNA cleavage activity of RnlA in vitro. In addition, ectopic expression of RnlA in ΔrnlAB ΔrnhA cells has less effect on cell toxicity and RnlA‐mediated mRNA degradation than in ΔrnlAB cells. This is the first example of a direct factor for activation of a toxin.     

Opposite roles of the dmd gene in the control of RNase E and RNase LS activities

When the dmd gene of bacteriophage T4 is defective, expression of middle genes starts normally but drops abruptly. However, the residual expression of middle genes at late stages continues at a higher rate in cells infected with a dmd mutant than with the wild type. In order to understand the complex effects of the dmd gene, we followed changes in the quantity of mRNA from a middle gene, uvsY. The uvsY mRNA was degraded rapidly by RNase LS at middle stages but stabilized at late stages, suggesting that RNase LS targets middle-gene mRNAs only at middle stages. Furthermore, another RNase targeting middle mRNAs at late stages is also suggested to be inactivated when dmd is mutated. We found that RNase E was involved in the degradation of uvsY mRNA. Judging from the processing of gene-32 mRNA, RNase E activity declines after the beginning of the middle stage when dmd is defective.     

Structure–function studies of Escherichia coli RnlA reveal a novel toxin structure involved in bacteriophage resistance

Escherichia coli RnlA–RnlB is a newly identified toxin–antitoxin (TA) system that plays a role in bacteriophage resistance. RnlA functions as a toxin with mRNA endoribonuclease activity and the cognate antitoxin RnlB inhibits RnlA toxicity in E. coli cells. Interestingly, T4 phage encodes the antitoxin Dmd, which acts against RnlA to promote its own propagation, suggesting that RnlA‐Dmd represents a novel TA system. Here, we have determined the crystal structure of RnlA refined to 2.10 Å. RnlA is composed of three independent domains: NTD (N ‐t erminal d omain), NRD (N r epeated d omain) and DBD (D md‐b inding d omain), which is an organization not previously observed among known toxin structures. Small‐angle X‐ray scattering (SAXS) analysis revealed that RnlA forms a dimer in solution via interactions between the DBDs from both monomers. The in vitro and in vivo functional studies showed that among the three domains, only the DBD is responsible for recognition and inhibition by Dmd and subcellular location of RnlA. In particular, the helix located at the C‐terminus of DBD plays a vital role in binding Dmd. Our comprehensive studies reveal the key region responsible for RnlA toxicity and provide novel insights into its structure–function relationship.     

RNA cleavage linked with ribosomal action

Ribonuclease LS in Escherichia coli is a potential antagonist of bacteriophage T4. When T4 dmd is mutated, this RNase efficiently cleaves T4 mRNAs and leads to the silencing of late genes, thus blocking T4 growth. We previously found that, when two consecutive ochre codons were placed in the open reading frame of T4 soc, RNase LS cleaved soc mRNA at a specific site downstream of the ochre codons. Here, we demonstrate that RNase LS cleaves soc RNA at the same site even when only a single ochre codon is present or is replaced with either an amber or an opal codon. On the other hand, disruption of the Shine-Dalgarno sequence, a ribosome-binding site required for the initiation of translation, eliminates the cleavage. These results strongly suggest that RNase LS cleaves in a manner dependent on translation termination. Consistent with this suggestion, the cleavage dependency on an amber codon was considerably reduced in the presence of amber-codon-suppressing tRNA. Instead, two other cleavages that depend on translation of the region containing the target sites occurred farther downstream. Additional analysis suggests that an interaction of the ribosome with a stop codon might affect the site of cleavage by RNase LS in an mRNA molecule. This effect of the ribosome could reflect remodeling of the high-order structure of the mRNA molecule.     

Dmd of bacteriophage T4 functions as an antitoxin against Escherichia coli LsoA and RnlA toxins

Enterohaemorrhagic Escherichia coli O157:H7 harbours a cryptic plasmid, pOSAK1, that carries only three ORFs: mobA (involved in plasmid mobilization), ORF1 and ORF2. Predicted proteins encoded by these two ORFs were found to share a weak homology with RnlA and RnlB, respectively, a toxin–antitoxin system encoded on the E. coli K-12 chromosome. Here, we report that lsoA (ORF1) encodes a toxin and lsoB (ORF2) an antitoxin. In spite of the homologies, RnlB and LsoB functioned as antitoxins against only their cognate toxins and not interchangeably with each other. Interestingly, T4 phage Dmd suppressed the toxicities of both RnlA and LsoA by direct interaction, the first example of a phage with an antitoxin against multiple toxins.     

Escherichia coli endoribonucleases involved in cleavage of bacteriophage T4 mRNAs

The dmd mutant of bacteriophage T4 has a defect in growth because of rapid degradation of late-gene mRNAs, presumably caused by mutant-specific cleavages of RNA. Some such cleavages can occur in an allele-specific manner, depending on the translatability of RNA or the presence of a termination codon. Other cleavages are independent of translation. In the present study, by introducing plasmids carrying various soc alleles, we could detect cleavages of soc RNA in uninfected cells identical to those found in dmd mutant-infected cells. We isolated five Escherichia coli mutant strains in which the dmd mutant was able to grow. One of these strains completely suppressed the dmd mutant-specific cleavages of soc RNA. The loci of the E. coli mutations and the effects of mutations in known RNase-encoding genes suggested that an RNA cleavage activity causing the dmd mutant-specific mRNA degradation is attributable to a novel RNase. In addition, we present evidence that 5'-truncated soc RNA, a stable form in T4-infected cells regardless of the presence of a dmd mutation, is generated by RNase E.     

Alternative dimerization is required for activity and inhibition of the HEPN ribonuclease RnlA

The rnlAB toxin-antitoxin operon from Escherichia coli functions as an anti-phage defense system. RnlA was identified as a member of the HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding domain) superfamily of ribonucleases. The activity of the toxin RnlA requires tight regulation by the antitoxin RnlB, the mechanism of which remains unknown. Here we show that RnlA exists in an equilibrium between two different homodimer states: an inactive resting state and an active canonical HEPN dimer. Mutants interfering with the transition between states show that canonical HEPN dimerization via the highly conserved RX_4-6H motif is required for activity. The antitoxin RnlB binds the canonical HEPN dimer conformation, inhibiting RnlA by blocking access to its active site. Single-alanine substitutions mutants of the highly conserved R255, E258, R318 and H323 show that these residues are involved in catalysis and substrate binding and locate the catalytic site near the dimer interface of the canonical HEPN dimer rather than in a groove located between the HEPN domain and the preceding TBP-like domain. Overall, these findings elucidate the structural basis of the activity and inhibition of RnlA and highlight the crucial role of conformational heterogeneity in protein function.     

A promiscuous antitoxin of bacteriophage T4 ensures successful viral replication

Bacteria are constantly threatened by predation from bacteriophage parasites and, in response, have evolved an array of resistance mechanisms. These resistance mechanisms then place greater selection pressure on the infecting bacteriophages, which develop counter-strategies in a perpetual 'arms race' between virus and host. Toxin-antitoxin (TA) loci are widespread in bacteria and can confer multiple benefits, including resistance to bacteriophages. The study by Otsuka and Yonesaki, published in this issue of Molecular Microbiology, describes a new plasmid-encoded TA system, lsoAB, which confers resistance to a dmd(-) mutant of bacteriophage T4 through the activity of the LsoA toxin. Infections with wild-type T4, however, are unaffected as the Dmd protein acts as an alternative antitoxin to LsoA, thus preventing its anti-bacteriophage activity. Dmd has also been shown to negate the activity of a related toxin, RnlA. This is a striking result indicating that Dmd can act as a promiscuous antitoxin, binding and inhibiting multiple toxin partners, when antitoxin activity is generally considered to be limited to a single cognate toxin. This study is an exciting addition to both the bacteriophage resistance and TA fields, and suggests a greater role for TA system-based resistance and counter-resistance in the world's oldest predator-prey relationship.     

Bacteriophage T4 encodes an RNase H which removes RNA primers made by the T4 DNA replication system in vitro.

RNase H activity increases markedly after bacteriophage T4 infection of Escherichia coli MIC2003, an RNase H-deficient host. We have extensively purified the RNase H from these T4-infected cells and have shown that the RNase H activity copurifies with a 5' to 3' DNA exonuclease activity. The N-terminal sequence of a 35-kDa protein copurifying with the RNase H activity matches the terminus of the predicted product of an open reading frame (designated ORF A or 33.2) upstream of T4 gene 33, identified previously by Hahn and co-workers (Hahn, S., Kruse, U., and Rüger, W. (1986) Nucleic Acids Res. 14, 9311-9327). Plasmids containing ORF A under the control of the T7 promoter express RNase H and 5' to 3' DNA exonuclease activities as well as a protein that comigrates on sodium dodecyl sulfate-polyacrylamide gels with the 35-kDa protein present in the RNase H purified from T4-infected cells. T4 RNase H removes the pentamer RNA primers from DNA chains initiated by the T4 primase-helicase (gene products 61 and 41). Addition of T4 RNase H and T4 DNA ligase leads to extensive joining of discontinuous lagging strand fragments in the T4 DNA replication system in vitro.     

Either bacteriophage T4 RNase H or Escherichia coli DNA polymerase I is essential for phage replication.

Bacteriophage T4 rnh encodes an RNase H that removes ribopentamer primers from nascent DNA chains during synthesis by the T4 multienzyme replication system in vitro (H. C. Hollingsworth and N. G. Nossal, J. Biol. Chem. 266:1888-1897, 1991). This paper demonstrates that either T4 RNase HI or Escherichia coli DNA polymerase I (Pol I) is essential for phage replication. Wild-type T4 phage production was not diminished by the polA12 mutation, which disrupts coordination between the polymerase and the 5'-to-3' nuclease activities of E. coli DNA Pol I, or by an interruption in the gene for E. coli RNase HI. Deleting the C-terminal amino acids 118 to 305 from T4 RNase H reduced phage production to 47% of that of wild-type T4 on a wild-type E. coli host, 10% on an isogenic host defective in RNase H, and less than 0.1% on a polA12 host. The T4 rnh(delta118-305) mutant synthesized DNA at about half the rate of wild-type T4 in the polA12 host. More than 50% of pulse-labelled mutant DNA was in short chains characteristic of Okazaki fragments. Phage production was restored in the nonpermissive host by providing the T4 rnh gene on a plasmid. Thus, T4 RNase H was sufficient to sustain the high rate of T4 DNA synthesis, but E. coli RNase HI and the 5'-to-3' exonuclease of Pol I could substitute to some extent for the T4 enzyme. However, replication was less accurate in the absence of the T4 RNase H, as judged by the increased frequency of acriflavine-resistant mutations after infection of a wild-type host with the T4 rnh (delta118-305) mutant.     

The C-terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo

RNase E is an essential Escherichia coli endonuclease, which controls both 5S rRNA maturation and bulk mRNA decay. While the C-terminal half of this 1061-residue protein associates with polynucleotide phosphorylase (PNPase) and several other enzymes into a 'degradosome', only the N-terminal half, which carries the catalytic activity, is required for growth. We characterize here a mutation (rne131 ) that yields a metabolically stable polypeptide lacking the last 477 residues of RNAse E. This mutation resembles the N-terminal conditional mutation rne1 in stabilizing mRNAs, both in bulk and individually, but differs from it in leaving rRNA processing and cell growth unaffected. Another mutation (rne105 ) removing the last 469 residues behaves similarly. Thus, the C-terminal half of RNase E is instrumental in degrading mRNAs, but dispensable for processing rRNA. A plausible interpretation is that the former activity requires that RNase E associates with other degradosome proteins; however, PNPase is not essential, as RNase E remains fully active towards mRNAs in rne+pnp mutants. All mRNAs are not stabilized equally by the rne131 mutation: the greater their susceptibility to RNase E, the larger the stabilization. Artificial mRNAs generated by E. coli expression systems based on T7 RNA polymerase can be genuinely unstable, and we show that the mutation can improve the yield of such systems without compromising cell growth.