Regions upstream from the core promoter of the rat ribosomal gene are required for the formation of a stable transcription initiation complex by RNA polymerase I in vitro

Abstract

The sites required for the formation of a stable transcription initiation complex and for the initiation of transcription of rat rDNA in vitro were examined. A series of 5' deletion mutants of the rat transcription initiation region (-167 through +638) were constructed. These mutants were examined for their ability to support the faithful initiation of transcription in vitro. Mutants which contain less than 31 nucleotides upstream of the initiation site (+1) were unable to support detectable initiation of transcription. In this transcription system a series of deletion mutants from -167 to -31 were transcribed with equal efficiency when assayed individually. On the other hand, when the wild-type and mutant templates were compared in order-of-addition assays, they were found to be unequal. The incubation of an extract with a wild-type template, prior to the addition of nucleotides, precluded transcription of any second template added after the preincubation step. However, the preincubation of extract with mutants of the region upstream of the core promoter, from -122 to -31, did not preclude transcription of a wild-type template added after the preincubation step. Formation of the stable preinitiation complex was found to require the region between and -167.