Structure and Biosynthesis of Unbranched Multicopy Single-stranded DNA by Reverse Transcriptase in a Clinical Escherichia Coli Isolate

It has been shown that retrons, retro-elements in bacteria, produce a reverse transcriptase (RT) and multicopy single-stranded DNA (msDNA) whose 5' end is covalently linked to RNA (msdRNA) by a 2'-5' phosphodiester bond. Here, I show that a retron in clinical Escherichia coli strain 161 produces an msDNA unlinked to RNA. The msDNA produced by this retron is a 79-nucleotide-long single-stranded DNA with monophosphate on its 5' terminus. When the retron in strain 161 is cloned into E. coli K-12, the majority of msDNA produced in the clone is the same as the msDNA in the clinical strain. However, in the K-12 clone, about 10% of the msDNA produced is present as a DNA covalently linked to RNA. The DNA part of this RNA-DNA compound is an 83 nucleotides long with the same sequence as the unbranched msDNA, except for the presence of four additional nucleotides at the 5' side. From the analysis of the RNA-DNA compound and the results of in vitro synthesis, I show that the primary product of reverse transcription in this retron is an 83-nucleotide-long DNA covalently linked to RNA. This RNA-DNA compound is further processed to the final product, the 79-nucleotide-long msDNA with a terminal 5' monophosphate, by an endonucleolytic cleavage between the fourth and fifth positions of the DNA component of the RNA-DNA compound. The minimum region required for the production of such msDNA free of RNA contains only genes known to be required for the synthesis of branched msDNA-RNA compound in other retrons (msd, msr and ret). This suggests that either the RT has an endonuclease activity or that the msDNA-RNA compound is autocatalytically processed.