Members of the RNase E/G family are multimetric, 5'-end-sensing, single-strand-specific endoribonucleases leases 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 (C) 2009 Elsevier Inc All rights reserved