Reverse Transcriptases in the Prokaryotes

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DESCRIPTION (provided by applicant): Contrary to the earlier belief, retroelements have also been found in bacteria Retrons are such elements as these existing in some wild strains of Escherichia coli (<10 percent of the population), Myxococcus xanthus (all natural isolates), and Vibrio cholereae (only in pathogenic strains). They encode reverse transcriptase (RT) evolutionarily related to retroviral RTs and other eukaryotic RTs. Bacterial RTs are responsible for the synthesis of a peculiar satellite single-stranded DNA called msDNA (multicopy single-stranded DNA), in which a single strand DNA (cDNA) is branched out from an internal rG residue of a highly structured RNA molecule forming a unique 2' ,5 '-phosphodiester linkage. The outstanding mysteries concerning msDNA synthesis are how individual bacterial RTs highly diverse each other are able to recognize their cognate RNA molecule, and how cDNA synthesis is primed from a specific internal G residue (branching G residue) in the single RNA molecule. We recently demonstrated that the 91-residue C-terminal domain of RT-Ec86 is responsible for the specific recognition of a stem-loop structure unique to the primer-template RNA molecule for RT-Ec86. Notably, this recognition stem-loop structure locates downstream of the branching G residue used for the cDNA priming reaction. On the basis of our recent results, we hypothesize that the seemingly primitive bacterial RTs retain the unique ability to utilize the C-terminal thumb domain region to specifically recognize their cognate stem-loop structure downstream of the branching G residue. This unique RNA-thumb domain interaction allows the 2'-OH group of the branching G residue to be correctly positioned at the active site of RTs to which the first nucleotide complimentary to the template RNA is added forming a 2', 5 '-phosphodiester linkage. In this proposal, we first attempt to construct molecular models of RT-Ec86 and its complexes with the primer-template RNA and the final msDNA product. On the basis of these models, we will take experimental approaches to determine the precise molecular mechanisms for the interaction between the thumb domain and the recognition stem-loop structure, for the cDNA priming reaction from the 2'-OH group of the branching G residue, and for the cDNA elongation reaction leading to the final msDNA product.