Involvement of the Escherichia coli endoribonucleases G and E in the secondary processing of RegB-cleaved transcripts of bacteriophage T4

Abstract

Sequence-specific endoribonuclease RegB of bacteriophage T4 cleaves early phage mRNAs and facilitates the transition between early and subsequent phases of T4 gene expression. The great majority of RegB targets have been identified in the intergenic regions of T4 transcripts, frequently in the Shine-Dalgarno sequences. Here we show that localization of RegB targets is not restricted to intergenic regions of mRNA. We detected 30 intragenic RegB sites in T4 transcripts that are differently susceptible to cleavage. Four RegB-processed mRNAs were previously shown to undergo further processing at so-called "secondary sites". We have found three additional transcripts carrying clear targets for both RegB and another endoribonuclease. We show that secondary cuts within RegB-processed T4 mRNAs are generated mainly by Escherichia coli RNase G, but that in some cases RNase E can recognize the same targets. Using plasmid-phage systems we demonstrate that T4 infection favours cleavage by the host endoribonucleases at these sites.

Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation

“…Some efficient RegB cuts have also been detected at GGAG/U within coding sequences. RegB cleavages can be detected very soon after infection, earlier than 45 seconds at 30°C [5,9-14]. …”

Bacteriophage T4 polynucleotide kinase triggers degradation of mRNAs

“…In this case, RNase G contributes to only one and RNase E contributes to the other of the two secondary cleavages (Fig. S1B) (15). Both cleavages disappeared in the absence of PNK (Fig.…”

“…Phosphorylation of the 5′-OH terminus by the kinase activity of PNK permits attack by the host RNases G and E, two endoribonucleases that have strong preference for 5′-monophosphorylated RNA termini (4)(5)(6)22). RNases E and G cut first in the vicinity of the initial RegB cleavage site, generating the so-called secondary cleavages (10,14,15). Because the cleavage products of RNases E and G bear 5′-PO 4 groups, they are themselves substrates for additional cleavage by these endonucleases, causing a cascade of endonucleolytic cuts with 5′-to 3′-polarity that leads to mRNA destabilization.…”

“…Here, we showed that the PNK protein accumulates slowly after infection, suggesting that the delay between primary and secondary cuts reflects the time necessary for PNK to accumulate in sufficient quantities after infection. The work by Zajanckauskaite et al (15) proposed that a phage factor can modify RNases E and G, permitting them to attack the RegB-processed transcripts with a 5′-OH terminus.…”

“…One likely model is that RegB accelerates mRNA decay by increasing the number of entry sites for 3′-5′ exoribonucleases within long polycistronic mRNAs. Another mechanism was suggested by the finding that some early transcripts undergo cleavages by RNases G and E that depend on prior cleavage by RegB a few nucleotides upstream (10,14,15). These cuts, called secondary cuts, were detected in seven different early transcripts.…”