Adjacent single-stranded regions mediate processing of tRNA precursors by RNase E direct entry

The RNase E family is renowned for being central to the processing and decay of all types of RNA in many species of bacteria, as well as providing the first examples of endonucleases that can recognize 5′-monophosphorylated ends thereby increasing the efficiency of cleavage. However, there is increasing evidence that some transcripts can be cleaved efficiently by Escherichia coli RNase E via direct entry, i.e. in the absence of the recognition of a 5′-monophosphorylated end. Here, we provide biochemical evidence that direct entry is central to the processing of transfer RNA (tRNA) in E. coli, one of the core functions of RNase E, and show that it is mediated by specific unpaired regions that are adjacent, but not contiguous to segments cleaved by RNase E. In addition, we find that direct entry at a site on the 5′ side of a tRNA precursor triggers a series of 5′-monophosphate-dependent cleavages. Consistent with a major role for direct entry in tRNA processing, we provide additional evidence that a 5′-monophosphate is not required to activate the catalysis step in cleavage. Other examples of tRNA precursors processed via direct entry are also provided. Thus, it appears increasingly that direct entry by RNase E has a major role in bacterial RNA metabolism.