Ultrastructural analysis of ribosomal gene transcription in vitro

Xenopus laevis oocyte nuclei were manually isolated and incubated in vitro to synthesize RNA. Aliquots were removed before and during the reaction and prepared for electron microscopy, in order to observe the amplified ribosomal RNA (rRNA) genes. Before in vitro incubation the rRNA transcription units are covered with close-packed RNA polymerase molecules and attached rRNP fibers, which are interpreted to have initiated in vivo. With incubation, these close-packed transcription complexes move down the gene at a rate of 2.2 nucleotides per s. Behind this close-packed region we see RNA polymerase x nascent chain complexes at about one-fifth the in vivo frequency. These are interpreted to be in vitro initiation events. After 45 min of incubation, when most of the in vivo-initiated transcripts have terminated transcription, the in vitro-initiated transcripts define the same length transcription unit seen prior to incubation. These observations are consistent with correct in vitro initiation and termination by RNA polymerases on the endogenous amplified ribosomal DNA of Xenopus laevis.