The involvement of free radical-induced oxidative damage to mitochondrial (mt) proteinsand DNA has been suggested as a cause of the 'vicious cycle' that results in mt dysfunction inage-related pathologies and neurodegenerative diseases. However, it has recently been shownthat the levels of oxidative damage in mt RNA may be greater than in mt DNA, and that theaccumulation of defective transcripts may be a major cause of mt dysfunction. To maintain the quality of transcripts mitochondria employ an extensive system ofposttranscriptional RNA surveillance and must balance the rates of RNA synthesis anddegradation in order to prevent swamping of this system. However, the molecular mechanismsthat might adjust the level of transcription under various conditions are not known. One approach to this question is to identify proteins that are associated with the mttranscription complex under various conditions, and to examine their effects on mt RNAPactivity in a purified in vitro system. In preliminary experiments, proteins that are associatedwith the yeast mt RNAP were successfully identified by affinity pulldown of TAP-tagged mtRNAP from isolated organelles. One of the most abundant proteins identified was Mss116p,which had previously been implicated in coordinating rates of RNA degradation and synthesisby genetic studies. Strikingly, we found that Mss116p enhances the stability of transcriptioncomplexes in vitro, and reduces the concentration of nucleoside triphosphate that is required toextend RNA from a paused complex. These effects are similar to those of elongation factors formultisubunit RNAPs and suggest that Mss116p might act to decrease the abundance ofaberrant transcripts by allowing mt RNAP to complete RNA synthesis more efficiently, especiallywhen the abundance of NTP substrates is limited, as would occur under oxidative stressconditions or oxygen/energy deprivation. Our preliminary results in the yeast system demonstrate the utility of this approach inidentifying protein factors that may modulate mt transcription in mammalian. In the proposedstudies we intend to apply this approach to identify protein factors that interact with the mtRNAP in neural tissue under normal and stress conditions using cultured rat primary corticalastrocytes (RPCA) as a model system, and to define their genome-wide localization andmolecular mechanism of action by in vitro methods. Characterization of mt transcriptionalplasticity in neural tissue may allow the delivery of new targets of potential therapies todecrease the rate of age-associated neural cell death.
These studies will provide insight as to how mitochondria respond to oxidative damage and toaging; and will allow identification of cis- and trans-acting factors that regulate the activity of mtgenome and may serve as targets of longevity-improving interventions and therapies.
