The sequence of sites recognised by a member of the RNase E/G family can control the maximal rate of cleavage, while a 5'-monophosphorylated end appears to function cooperatively in mediating RNA binding.
Members of the RNase E/G family are multimeric, 5'-end-sensing, single-strand-specific endoribonucleases that are found in chloroplasts as well as bacteria, and have central roles in RNA processing and degradation. A well-studied member of this family is Escherichia coli RNase G. Recently, we have shown that the interaction of this enzyme with a 5'-monophosphorylated end can enhance substrate binding in vitro and the decay of mRNA in vivo. We show here that a single-stranded site despite not being sufficient for rapid cleavage makes a substantial contribution to the binding of RNase G. Moreover, we find that the sequence of a site bound by RNase G can moderate the maximal rate by at least an order of magnitude. This supports a model for the RNase E/G family in which a single-stranded segment(s) can cooperate in the binding of enzyme that subsequently cleaves preferentially at another site. We also provide evidence that in order to promote cleavage a 5'-monophosphorylated end needs to be linked physically to a single-stranded site, indicating that it functions cooperatively. Our results are discussed in terms of recent X-ray crystal structures and models for the initiation of bacterial mRNA degradation.