Primers for vertebrate mitochondrial leading-strand DNA replication are products 
of transcription synthesized by mitochondrial RNA polymerase. The precursor 
primer RNA exists as a persistent RNA-DNA hybrid, known as an R-loop, formed 
during transcription through the replication origin (Xu, B., and Clayton, D. A. 
(1996) EMBO J. 15, 3135-3143). In an effort to examine the precise structure of 
this primer RNA intermediate, we have used two methods to reconstitute model 
R-loops containing the mouse mitochondrial DNA origin sequence. First, we 
demonstrate that bacteriophage SP6 RNA polymerase can efficiently catalyze the 
formation of an R-loop at the mouse mtDNA origin sequence. Second, the R-loop 
can be assembled by annealing presynthesized RNA and supercoiled DNA template in 
the presence of formamide. R-loop formation by either method is dependent on 
specific template sequences. The reconstituted R-loop is exceptionally stable 
and exhibits an unexpected structure. Structural studies indicate that the RNA 
strand is organized within the RNA-DNA base-paired region, suggesting that the 
heteroduplex interaction occurs through a specific conformation. We propose that 
the organized structure of the R-loop is critical for primer RNA function in 
vivo with important implications for the RNA processing and DNA replication 
machinery.