In vivo oligo(A) insertions in phage MS2: role of Escherichia coli poly(A) polymerase.
Previously we introduced an RNase III site into the genome of RNA phage MS2 by extending a hairpin with a perfect 18 bp long stem. One way in which the phage escaped from being killed by RNase III cleavage was to incorporate uncoded A residues on either side of the stem. This oligo(A) stretch interrupts the perfect stem that forms the RNase III site and thus confers resistance. In this paper we have analyzed the origin of these uncoded adenosines. The data strongly suggest that they are added by the host enzyme poly(A) polymerase. Apparently the 3'-OH created by RNase III cleavage becomes a substrate for poly(A) polymerase. Subsequently, MS2 replicase makes one contiguous copy from the two parts of the genome RNA. The evolutionary conversion from RNase III sensitivity to resistance provides a large spectrum of solutions that could be an important tool to understand what essentially constitutes an RNase III site in vivo.
van Meerten
D
D
Leiden Institute of Chemistry, Department of Biochemistry, Leiden University, Gorlaeus Laboratories, PO Box 9502, 2300 RA Leiden, The Netherlands,
Zelwer
M
M
Régnier
P
P
Duin
J
J
D000227:Adenine Nucleotides; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000821:virology; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000502:physiology; D017909:Levivirus / Q000235:genetics*; D016254:Mutagenesis, Insertional; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides; D011062:Polynucleotide Adenylyltransferase / Q000235:genetics / Q000502:physiology*; D012367:RNA, Viral / Q000737:chemistry
D000227:Adenine Nucleotides; D029968:Escherichia coli Proteins; D009843:Oligoribonucleotides; D012367:RNA, Viral; C023505:2',5'-oligoadenylate; D011062:Polynucleotide Adenylyltransferase; C063643:pcnB protein, E coli; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V