Quantification of hepatic transcribing RNA polymerase molecules, polyribonucleotide elongation rates and messenger RNA complexity in fed and fasted rats.

Abstract

We have investigated the mechanisms by which the rate of preribosomal RNA and premessenger RNA synthesis are repressed during starvation of rats by a quantitative study of the transcriptional events in isolated nuclei and by complexity analysis of the cytoplasmic polyadenylated RNA. The transcriptional activities of form A and B DNA-dependent RNA polymerases, which produce preribosomal and premessenger RNA respectively, were distinguished by the use of α-amanitin. Form C activity was shown to be absent from nuclei as isolated here. Polyribonucleotide elongation rates and the number of transcribing RNA polymerase molecules per diploid genome were determined by UMP/uridine analysis of the alkali-digested, nascent RNA labelled from [3H]UTP during 1-min incubations at 22 °C in the presence and absence of α-amanitin. The inhibition of form-A-mediated preribosomal RNA synthesis in response to starvation and cycloheximide treatment could be entirely accounted for by a decrease in the polyribonucleotide elongation rate. No significant change in the number of transcribing enzyme molecules (about 20 × 103 per diploid genome) was detected. However on refeeding, the elongation rate was restored to normal and the number of transcribing form A molecules was elevated over the normal level. These results suggest that there are at least two modes of control acting on preribosomal RNA synthesis, one linked to protein synthesis and affecting the elongation rate, while a second regulates the number of form A RNA polymerase molecules transcribing the template. Form-B-mediated premessenger RNA synthesis responded to starvation by a decrease in the number of transcribing enzyme molecules from approximately 7 × 103 to 3 × 103 per diploid genome. This change was reversed on refeeding. Cycloheximide treatment did not affect premessenger RNA synthesis either via elongation or the number of transcribing enzyme molecules. The data indicate that premessenger RNA synthesis is controlled in this system at the level of RNA polymerase initiation by a mechanism not linked to protein synthesis. Hybridization analysis of the complexity of liver cytoplasmic polyadenylated mRNA from normal, starved and refed animals showed that three or four abundance classes were present in each case. No significant difference in mRNA populations between the dietary conditions was detected. More detailed examination of the most abundant class of mRNA confirmed this conclusion. The data show that although the number of transcribing form B molecules is reduced during starvation, the complexity and relative abundance of the mRNA synthesised does not change. The simplest explanation of these results is that, whilst the number of active genes does not change significantly during starvation, on average there are fewer form B enzyme molecules transcribing each gene in the starved animal.