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 5′-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 5′ end. To investigate the mechanism by which 5′-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 5′ monophosphorylation accelerates the reaction not by improving substrate binding, but rather by enhancing the catalytic potency of these ribonucleases. Furthermore, the presence of a 5′ monophosphate 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 5′ phosphorylation state requires the formation of protein multimers. Among the molecular mechanisms that could account for these properties are those in which 5′-end binding by one enzyme subunit induces a protein structural change that accelerates RNA cleavage by another subunit

Bacteriophage T4 polynucleotide kinase triggers degradation of mRNAs

The bacteriophage T4-encoded RegB endoribonuclease is produced during the early stage of phage development and targets mostly (but not exclusively) the Shine–Dalgarno sequences of early genes. In this work, we show that the degradation of RegB-cleaved mRNAs depends on a functional T4 polynucleotide kinase/phosphatase (PNK). The 5′-OH produced by RegB cleavage is phosphorylated by the kinase activity of PNK. This modification allows host RNases G and E, with activity that is strongly stimulated by 5′-monophosphate termini, to attack mRNAs from the 5′-end, causing their destabilization. The PNK-dependent pathway of degradation becomes effective 5 min postinfection, consistent with our finding that several minutes are required for PNK to accumulate after infection. Our work emphasizes the importance of the nature of the 5′ terminus for mRNA stability and depicts a pathway of mRNA degradation with 5′- to 3′-polarity in cells devoid of 5′–3′ exonucleases. It also ascribes a role for T4 PNK during normal phage development