Selective in Vitro Transcription of the 5S RNA Genes of a DNA Template

RNA polymerase III is responsible in vivo for the synthesis of 5S ribosomal RNA. In the present study, transcriptional specificity of partially purified RNA polymerase III in a homologous system containing calf thymus enzyme and calf thymus DNA has been investigated by using DNA complementary to 5S ribosomal RNA (5S cDNA) as a probe and hybridizing RNA synthesized enzymically to this probe. The 5S cDNA probe hybridizes to 5S RNA with a R0t1/2 of 10-3 and to calf thymus DNA (in which there are approximately 1500-3000 5S ribosomal RNA genes per haploid genome, 0.005% of the genome) with a C0t1/2 of 2.1. Hybridization of the probe to RNA transcribed by RNA polymerase III from large (107 daltons) double-stranded DNA is observed at R0t1/2‘s between 0.03 and 0.06, indicating that between 1.6% and 3.5% (2.6 ± 0.88%) of the transcripts are 5S ribosomal RNA and that transcription of the 5S ribosomal RNA genes is up to 700-fold greater than random transcription of the genome. The percentage of 5S ribosomal RNA transcripts is less with smaller DNA as template. Selective transcription is also observed when denatured DNA is used as template. Concordant results are obtained when RNA is synthesized with either Mg2+ and/or Mn2+ as metal cofactor. Selective transcription of 5S ribosomal RNA genes by calf thymus RNA polymerase III is also observed with salmon sperm DNA as template. RNA transcribed by Escherichia coli RNA polymerase in the presence of Mg2+ and Mn2+ contains respectively 0.3% and 0.02% 5S rRNA. These results suggest that (1) 5S rRNA genes are preferentially transcribed by heterologous (bacterial) as well as homologous polymerase and (2) partially purified calf thymus RNA polymerase III possesses an intrinsic specificity for transcription of 5S ribosomal genes of calf thymus and salmon sperm DNAs.