Phage-induced change in the stability of mRNAs

AbstractThe stability of mRNA in Escherichia coli cells changed after phage T4 infection. Stable E. coli mRNAs such as lpp and ompA were drastically destabilized immediately after infection. In contrast, T4 phage soc mRNA that had been unstable before infection became stabilized after infection. The host RNases E and G both contributed to the destabilization of these mRNAs. Accordingly, these RNases may alter their target RNAs before and after infection. An RNA chaperon, Hfq, and polyadenylation at 3′ ends of mRNA are known key factors for destabilization of ompA and lpp mRNAs in uninfected cells. However, they had no effect on the destabilization of E. coli mRNAs after infection. On the other hand, T4 infection in the presence of rifampicin or infection of a deletion mutant, Δtk2, did not destabilize the host mRNAs. These results strongly suggest that a phage-encoded factor is responsible for the destabilization of host mRNAs. Destabilization of host mRNAs was also observed after infection by phages T2 and T7

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

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

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

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