Studies on mitochondrial tRNA from animal cells: II. Hybridization of aminoacyl-tRNA from rat liver mitochondria with heavy and light complementary strands of mitochondrial DNA

Abstract

The sequence homology of mitochondrial and cytoplasmic aminoacyl-tRNA's with mitochondrial DNA of rat liver has been studied by performing hybridization at low temperatures in the presence of 50% formamide. The tRNA was acylated with radioactive amino acids so that specific aminoacyl-tRNA-DNA hybridization could be followed in the presence of other species of RNA. With a constant amount of DNA and increasing RNA concentrations, a saturation plateau was reached with all tRNA's tested. Competition experiments showed that mitochondrial aminoacyl-tRNA competed far more efficiently with mitochondrial tyrosyl-, phenylalanyl-, seryl- or leucyl-tRNA for hybridization sites on mitochondrial DNA than did cytoplasmic aminoacyl-tRNA. No hybridization was observed with rat liver nuclear DNA or Escherichia coli DNA. Hybridization of mitochondrial aminoacyl-tRNA's was also performed with isolated complementary heavy (H) and light (L) strands of mitochondrial DNA. The DNA was prepared by equilibrium centrifugation in alkaline cesium chloride. It was shown by electron microscopy that at least 20% of the single-stranded DNA molecules present in H and L fractions consisted of full-length 5 μ linear or circular forms. It was found that mitochondrial leucyl- and phenylalanyl-tRNA hybridized exclusively with the H strand, whereas tyrosyl- and seryl-tRNA hybridized exclusively with the L strand of mitochondrial DNA. These studies show (1) that at least four mitochondrial tRNA's are potentially transcribed from mitochondrial DNA; (2) these mitochondrial tRNA's differ in base sequence from their cytoplasmic counterparts; and (3) some species of mitochondrial tRNA may be transcribed in vivo from one strand and others from the complementary strand of mitochondrial DNA.