The proposed research involves continued biochemical-genetic studies of mitochondrial [plasmids and group II introns, two types of retroelements that have been found in organelles and bacteria. These retroelements encode unique species of reverse transcriptases (RTs) and use novel replication and mobility mechanisms that may provide insight into the early evolution of retroelements and introns. The Mauriceville and Varkud mitochondrial retroplasmids (mRPs) found in certain strains of Neurospora are closely related, small circular DNAs that replicate via reverse transcription. During the current grant period, we found that the mRP- encoded RT functions analogously to viral RNA-dependent RNA polymerases in initiating (-) strand cDNA synthesis at a tRNA-like structure at the 3' end of the mRP transcript and has the unprecedented ability for a DNA polymerase to initiate DNA synthesis de novo (i.e., without a primer). These characteristics are expected for a primitive RT that evolved from an RNA-dependent RNA polymerase, and they support the hypothesis that the mRPs are primitive retroelements related to the early ancestors of retroviruses. In the proposed research, we would continue to study the mRPs, their RT and their mechanisms of replication and integration. Group II introns, the second subject of the proposed research, are catalytic RNAs believed to be related to the progenitors of nuclear pre-mRNA introns. The yeast mtDNA group II introns COX1-I1 and - I2 (aI1 and aI2) had been shown to encode RT-like proteins that function in splicing the intron in which they are encoded. In collaboration with Drs. Philip Perlman and Ronald Butow (u. Texas), we showed that the aI1 and aI2 proteins are also active, intron-specific RTs that function in intron mobility. In the proposed research, we would continue to study how the group II intron RTs function in both intron mobility and RNA splicing. The continued studies of mRPs and group II introns should provide novel insight into reverse transcription and replication mechanisms, structural and evolutionary relationships between different types of nucleic acid polymerases, and the evolution of retroelements and RNA viruses, groups that include HIV and other important human pathogens.
Specific aims are: (1) To continue biochemical analysis of the mRP RT, focusing on the de novo initiation reaction and the modes of template and primer recognition. (2) To study other aspects of the mRP replication pathway, including RT translation, (+) strand synthesis, DNA circularization and integration. (3) To develop methods for expression of the mRP RT for structure-function analysis. (4) To continue collaborative experiments with Drs. Philip Perlman and Ronald Butow to study the function of the yeast tDNA group II intron RTs in intron mobility and RNA splicing. (5) To develop methods for the expression of bacterial or other group II intron RTs in E. coli.
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