How is msDNA normally produced?

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This page has a lot of very good history about msDNA. [ msDNA History and References]

Here is a 2005 paper on msDNA.

How is msDNA [multicopy single-stranded DNA] normally produced?

In some strains of E. coli, msDNA linked to RNA is produced by a reverse transcriptase (RT) encoded by a retron.[1] The retron is a genetic substructure containing the gene for reverse transcriptase (RT) as well as the msr-msd region (msr gene codes for msdRNA/ msd gene codes for msDNA) of the transcript, which serves as a template as well as a primer for the RT reaction. Bacterial reverse transcriptases (RTs) are distinctive amongst other RTs in terms of the priming reactions for cDNA (complementary DNA) synthesis. Because the single RNA molecule is used as both template and starting point for DNA replication, the RT forms an unusual 2′,5′-phosphodiester linkage between an internal G residue of the RNA molecule and the 5′ end of the cDNA to initiate cDNA . In this process the bacterial RTs synthesize msDNA. [2] Lampson confirmed that the msDNA contained the DNA-RNA duplex and that the RNA molecule existed as a template for AT in his study of a clinical strain of E. coli (Cl-1).This msDNA formed a secondary hairpin structure, and the RNA portion formed a stem-and loop structure.[3]

(1)Lima 1995 Isolation and Characterization of Host Mutants Defective in msDNA Synthesis: Role of Ribonuclease H in msDNA Synthesis

(2) Lima 1997 A Novel Retron That Produces RNA-less msDNA inEscherichia coliUsing Reverse Transcriptase,


--Alyn the violist 14:23, 25 April 2009 (EDT)

The retron needed to synthesize msDNA is integrated into the bacterial genome at a single locus. All E. coli RTs encoded by an open reading frame at this locus has transposon-like elements similar to HIV-1 RT (1). All three genes at the locus can be transcribed by one promoter for RNA polymerase, producing one long transcript. Alternatively, it is also possible to produce msDNA in vivo when the msr-msd region and RT gene are expressed under separate promoters. In all 12 msDNA-retrons found, the sequence immediately surrounding the branching internal G residue is highly conserved.

The secondary structures formed in the RNA immediately after the G residue are essential for the reverse transcriptase to initiate the 2’-OH priming reaction (1,2). Notably, the primary RNA transcript contains two kinds of inverted repeats, a1/a2 and b1/b2. a2 and a1 are directly upstream and downstream of the msr-msd primary transcript, and b1/b2 are located within the msd gene. This placement allows folding the transcript into the stable hairpin structure (1,2). Furthermore, it positions the branched G for RT priming. The formation of the secondary structure allows a priming reaction and initiation of reverse transcription. As elongation of the DNA strand continues along the RNA template, the RNA template is concomitantly removed within the growing DNA-RNA duplex.

It is possible to produce the msDNA-retron in a cell-free system composed of the purified RT and its cognate (1), suggesting no other factors may be needed for this process. The msDNA reproduced in E. coli is 65–163 bases of single-stranded DNA (3). Further study found that at least the first 36 bases of the msDNA may be critical for reproduction (4).

(1) Yamanaka K, Shimamoto T, Inouye S, Inouye M. Retrons. In: Mobile DNA II. Craig NL, Craigie R, Gellert M, Lambowitz AM, eds. (2002). Washington, DC: ASM Press. 784–95.

(2) Lampson, B.C, Inouye S, Inouye M. (1991). msDNA of bacteria. Prog. Nucleic Acid Res. Mol. Biol. 40: 1–24.

(3) Inouye M and Inouye S. (1992). Retrons and multi-copy single-stranded DNA. J. Bacteriology 174: 2419–24.

(4) Mao, J., Shimada, M., Inouye S., Inouye M. (1995). Gene regulation by antisense DNA produced in vivo. J. Biol. Chem. 270.

-- Olivia H. 15 May 2009 2:31 EDT