The wobble nucleotide-excising anticodon nuclease RloC is governed by the zinc-hook and DNA-dependent ATPase of its Rad50-like region.

The conserved bacterial anticodon nuclease (ACNase) RloC and its phage-excluding homolog PrrC comprise respective ABC-adenosine triphosphatase (ATPase) and ACNase N- and C-domains but differ in three key attributes. First, prrC is always linked to an ACNase silencing, DNA restriction-modification (R-M) locus while rloC rarely features such linkage. Second, RloC excises its substrate's wobble nucleotide, a lesion expected to impede damage reversal by phage transfer RNA (tRNA) repair enzymes that counteract the nick inflicted by PrrC. Third, a distinct coiled-coil/zinc-hook (CC/ZH) insert likens RloC's N-region to the universal DNA damage checkpoint/repair protein Rad50. Previous work revealed that ZH mutations activate RloC's ACNase. Data shown here suggest that RloC has an internal ACNase silencing/activating switch comprising its ZH and DNA-break-responsive ATPase. The existence of this control may explain the lateral transfer of rloC without an external silencer and supports the proposed role of RloC as an antiviral contingency acting when DNA restriction is alleviated under genotoxic stress. We also discuss RloC's possible evolution from a PrrC-like ancestor.

RloC: a wobble nucleotide-excising and zinc-responsive bacterial tRNase.

SUMMARY: The conserved bacterial protein RloC, a distant homologue of the tRNA(Lys) anticodon nuclease (ACNase) PrrC, is shown here to act as a wobble nucleotide-excising and Zn(++)-responsive tRNase. The more familiar PrrC is silenced by a genetically linked type I DNA restriction-modification (R-M) enzyme, activated by a phage anti-DNA restriction factor and counteracted by phage tRNA repair enzymes. RloC shares PrrC's ABC ATPase motifs and catalytic ACNase triad but features a distinct zinc-hook/coiled-coil insert that renders its ATPase domain similar to Rad50 and related DNA repair proteins. Geobacillus kaustophilus RloC expressed in Escherichia coli exhibited ACNase activity that differed from PrrC's in substrate preference and ability to excise the wobble nucleotide. The latter specificity could impede reversal by phage tRNA repair enzymes and account perhaps for RloC's more frequent occurrence. Mutagenesis and functional assays confirmed RloC's catalytic triad assignment and implicated its zinc hook in regulating the ACNase function. Unlike PrrC, RloC is rarely linked to a type I R-M system but other genomic attributes suggest their possible interaction in trans. As DNA damage alleviates type I DNA restriction, we further propose that these related perturbations prompt RloC to disable translation and thus ward off phage escaping DNA restriction during the recovery from DNA damage.

[Gene silencing RNAi technology: possible application to therapy].

RNA interference (RNAi), i.e. gene silencing, or gene expression down-regulation is the process whereby a double-stranded RNA (dsRNA) induces the homology-dependent degradation of cognate messenger RNA (mRNA). When dsRNA is introduced into cells, an RNA-induced silencing complex (RISC) is assembled. RISC serves as cellular machinery that is responsible for the specific mRNA degradation. This process results in the subsequent reduction of the specific protein translated from appropriate mRNA. Short RNA duplexes (21 nucleotide), called small interfering RNA (siRNA), have become the major tool for induction of gene silencing. With the human genome mapped and sequenced, attempts are currently being made to manipulate the expression of genes involved in viral diseases, carcinogenesis and other disorders with the aim of developing novel therapies.