The overall goal of the project described in this proposal is to elucidate the mechanism of transcription termination in yeast mitochondria. To this end, one major aim of this project is to develop an in vitro transcription termination system for yeast mitochondria. For templates, plasmids will be constructed with regions containing putative termination regions fused to yeast mitochondrial promoters. Candidate termination regions will be selected on the basis of published in vivo evidence for transcription termination within polygenic transcriptional units. To identify transcription termination activity, mitochondrial preparations obtained with a minimum of fractionation will be used. In vitro transcripts will be analyzed by gel electrophoresis and S1 nuclease protection. Subsequently, the molecular basis of the termination process will be investigated.
One aim i s to characterize the DNA sequence or sequences which specify termination in the in vitro system. This will first be done by in vitro deletion mutagenesis to delineate required sequences. Oligonucleotide mutagenesis will also be used to alter specific critical bases. Another aim will be to determine if a factor in addition to the mitochondrial RNA polymerase is required for yeast mitochondrial termination; previous work has shown that a DNA binding factor does play a role in human mitochondrial termination. Preparations exhibiting transcription termination activity will be fractionated to separate or even purify putative factors involved in specifying termination. Termination factors which bind to DNA will be identified before and during fractionation by competitive DNA binding, gel retardation analysis, and DNA footprinting.
