Ribosomal RNA Synthesis In Vitro

Abstract

INTRODUCTION In vitro studies of ribosomal RNA transcription aim to reproduce as closely as possible the in vivo synthesis of rRNA and hence to provide a molecular basis for the phenomena of regulation of rRNA synthesis in vivo. An extreme example of the regulation of rRNA accumulation in vivo is the stringent response, defined genetically by the rel locus. The product of the rel gene is a protein which is involved in the synthesis of the nucleotide guanosine 5′-diphosphate 3′-diphosphate or ppGpp (see Block and Haseltine, this volume). When rel + strains are functionally starved for an amino acid, the rate of net rRNA synthesis is drastically reduced and the intracellular concentration of ppGpp rapidly rises, attaining and usually exceeding that of guanosine 5′-triphosphate (Cashel 1969; Gallant et al. 1970). Two classes of mutant fail to restrict net rRNA synthesis in a normal manner. One, a mutation to rel − , fails to synthesize ppGpp on amino acid starvation (Cashel 1969) and is recessive to rel + (Fiil 1969). This observation has led to the suggestion that ppGpp acts as a direct inhibitor of rRNA synthesis in vivo (Cashel 1969). A second class of mutant, of which ts103 is an example, contains an altered RNA polymerase (Jacobson and Gillespie, unpublished observations) which is defective in vitro for pppA initiations (Jacobson and Gillespie 1970). This mutation is dominant to rel + . This observation suggests that RNA polymerase itself is the target of stringent control and that the net synthesis of rRNA is controlled primarily at the...