Rescue of the RNA phage genome from RNase III cleavage.
The secondary structure of the RNA from the single-stranded RNA bacteriophages, like MS2 and Qb, has evolved to serve a variety of functions such as controlling gene expression, exposing binding sites for the replicase and capsid proteins, allowing strand separation and so forth. On the other hand, all of these foldings have to perform in bacterial cells in which various RNA splitting enzymes are present. We therefore examined whether phage RNA structure is under selective pressure by host RNases. Here we show this to be true for RNase III. A fully double-stranded hairpin of 17 bp, which is an RNase III target, was inserted into a non-coding region of the MS2 RNA genome. In an RNase III-host these phages survived but in wild-type bacteria they did not. Here the stem underwent Darwinian evolution to a structure that was no longer a substrate for RNase III. This was achieved in three different ways: (i) the perfect stem was maintained but shortened by removing all or most of the insert; (ii) the stem acquired suppressor mutations that replaced Watson-Crick base pairs by mismatches; (iii) the stem acquired small deletions or insertions that created bulges. These insertions consist of short stretches of non-templated A or U residues. Their origin is ascribed to polyadenylation at the site of the RNase III cut (in the + or - strand) either by Escherichia coli poly(A) polymerase or by idling MS2 replicase.
Klovins
J
J
Department of Biochemistry, Leiden Institute of Chemistry, Leiden University, 2300 RA Leiden, The Netherlands.
van Duin
J
J
Olsthoorn
R C
RC
D001483:Base Sequence; D019020:Directed Molecular Evolution; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000821:virology; D029968:Escherichia coli Proteins; D016679:Genome, Viral; D017909:Levivirus / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012330:RNA, Double-Stranded / Q000737:chemistry; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III; D014779:Virus Replication / Q000235:genetics
D029968:Escherichia coli Proteins; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D004722:Endoribonucleases; D043244:Ribonuclease III; C468366:ribonuclease III, E coli