Positionally exact initiation is required for the formation of a stable RNA polymerase II transcription complex in vivo

The requirements for the formation of a stable transcription complex on the RNA polymerase II-transcribed Xenopus U2 snRNA gene have been analysed in vivo by oocyte microinjection experiments. The two elements of the U2 promoter which are located in the 5' flanking region of the gene, the DSE and the PSE, are shown to be essential but not sufficient for stable complex formation. Two additional elements are required. The first is a short gene-internal sequence; the second is the nucleotide at the normal point of initiation, which must be a purine. If this nucleotide is changed to a pyrimidine the site of initiation is altered and, concomitantly, the transcription complex formed on the mutant template remains unstable. These results suggest that there is a distinct topological requirement for complex formation which may involve an exact stereospecific alignment of RNA polymerase II with transcription factors bound to the promoter. Despite the apparent involvement of RNA polymerase, transcription per se is not required for complex stability.