23 research outputs found
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
50
-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 50
-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 50 side of a tRNA precursor triggers a
series of 50
-monophosphate-dependent cleavages.
Consistent with a major role for direct entry
in tRNA processing, we provide additional evidence
that a 50
-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
Catalytic activation of multimeric RNase E and RNase G by 5â˛-monophosphorylated RNA
RNase E is an endonuclease that plays a central role in RNA processing and degradation in Escherichia coli. Like its E. coli homolog RNase G, RNase E shows a marked preference for cleaving RNAs that bear a monophosphate, rather than a triphosphate or hydroxyl, at the 5Ⲡend. To investigate the mechanism by which 5â˛-terminal phosphorylation can influence distant cleavage events, we have developed fluorogenic RNA substrates that allow the activity of RNase E and RNase G to be quantified much more accurately and easily than before. Kinetic analysis of the cleavage of these substrates by RNase E and RNase G has revealed that 5Ⲡmonophosphorylation accelerates the reaction not by improving substrate binding, but rather by enhancing the catalytic potency of these ribonucleases. Furthermore, the presence of a 5Ⲡmonophosphate can increase the specificity of cleavage site selection within an RNA. Although monomeric forms of RNase E and RNase G can cut RNA, the ability of these enzymes to discriminate between RNA substrates on the basis of their 5Ⲡphosphorylation state requires the formation of protein multimers. Among the molecular mechanisms that could account for these properties are those in which 5â˛-end binding by one enzyme subunit induces a protein structural change that accelerates RNA cleavage by another subunit