Integrity of the DNA Template, a Prerequisite for the Faithful Transcription of <i>Xenopus</i> rDNA <i>in vitro</i>

Abstract

RNA polymerase from Micrococcus luteus transcribes in vitro the cistrons specifying ribosomal RNA in Xenopus laevis DNA. The rDNA template is transcribed asymmetrically in the same sense as in vivo ; furthermore the rDNA spacer which is not transcribed in vivo is also poorly transcribed by the bacterial RNA polymerase. The essential conditions for such near fidelity to rDNA transcription in vivo is the use of native rDNA which exhibits a high, single‐strand molecular weight of 8–10 × 10 6 . Regardless of the cation present during the reaction it is shown by critical hybridization and hybridization‐competition studies that 90% of the RNA synthesized on this template (cRNA) is complementary to the ribosomal cistrons. It is also shown that in short incubation the M. luteus RNA polymerase initiates RNA synthesis preferentially near the 18‐S cistron and that some of the transcribed spacer DNA coding for the RNA excess of the ribosomal 40–S precursor is located adjacent to the 18‐S cistron. In longer incubations the “intact”, large molecular weight primer allows the polymerase to read through to the 28‐S cistron while “nicked” rDNA inhibits this polymerization. Both “nicked” or “intact” native rDNA produce an asymmetric cRNA but the latter exhibits a higher degree of asymmetry and loci specificity. Denatured rDNA produces a cRNA which shows no strand specificity and no loci specificity, i.e. 50% of the cRNA is complementary to each strand of rDNA and about half its sequences are complementary to the spacer DNA. In support, the cRNA is capable of competing out all labelled 28‐S and 18‐S RNA hybridization. It is also shown that the RNA sequences complementary to spacer rDNA are unique to rDNA and are not located elsewhere in the genome of Xenopus laevis.