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.
Jourdan
Stefanie Simone
SS
Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK.
Kime
Louise
L
McDowall
Kenneth John
KJ
D001483:Base Sequence; D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D020871:RNA Stability; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*
D029968:Escherichia coli Proteins; D009711:Nucleotides; D012329:RNA, Bacterial; D012333:RNA, Messenger; D004722:Endoribonucleases; C081905:rng protein, E coli; C022537:ribonuclease E