| Popis: |
RNase E plays a central role in processing virtually all classes of cellular RNA in many bacterial species. A characteristic feature of RNase E and its paralogue RNase G, as well as several other unrelated ribonucleases, is their preference for 5'-monophosphorylated substrates. The basis for this property has been explored in vitro. At limiting substrate, cleavage of the rpsT mRNA by RNase E (residues 1-529) is inefficient, requiring excess enzyme. The rpsT mRNA is cleaved sequentially in a 5' to 3' direction, with the initial cleavage(s) at positions 116/117 or 190/191 being largely driven by direct entry, independent of the 5'-terminus or the 5'-sensor domain of RNase E. Generation of the 147 nt 3'-limit product requires sequential cleavages that generate 5'-monophosphorylated termini on intermediates, and the 5'-sensor domain of RNase E. These requirements can be bypassed with limiting enzyme by deleting a stem-loop structure adjacent to the site of the major, most distal cleavage. Alternatively, this specific cleavage can be activated substantially by a 5'-phosphorylated oligonucleotide annealed 5' to the cleavage site. This finding suggests that monophosphorylated small RNAs may destabilize their mRNA targets by recruiting the 5-sensor domain of RNase E 'in trans'. |
Plný text