Transcription of Xenopus ribosomal RNA genes by RNA polymerase I in vitro

Abstract

We have developed an in vitro system in which RNA polymerase I accurately transcribes Xenopus ribosomal RNA (rRNA) genes. Cloned Xenopus laevis ribosomal DNA (rDNA) is incubated in a homogenate of manually isolated Xenopus borealis oocyte nuclei. The resultant RNA is analyzed by an S1 nuclease mapping procedure specific for the initiation region of X. laevis rRNA. We show that transcription initiates in vitro on the cloned X. laevis rDNA at the in vivo initiation site. In addition, the dinucleotide ApG significantly stimulates transcription in vitro by acting as a sequence-specific primer. The ApG stimulation is dependent on the template concentration and independent of the ribonucleoside triphosphate levels suggesting that the primer circumvents or augments an initiation-specific factor. We have also investigated transcription of Xenopus rDNA in a mouse cell extract. This heterologous extract can catalyze accurate initiation at a low level. However, under different ionic conditions, this extract transcribes X. laevis rDNA approximately 100-fold more efficiently, but synthesis initiates 4 nucleotides upstream from the in vivo initiation site. There is strong sequence conservation between this start site, the in vivo X. laevis start site, and the in vivo mouse start site suggesting that these sequences are important in RNA polymerase I initiation and that species specificity for RNA polymerase I transcription may not be as great as suggested earlier.