Characterization of the processivity determinants in eukaryotic RNA polymeraseⅡ is crucial for understanding both the mechanisms of eukaryotic gene expression at the level of promoter escape, pausing, release of the RNA from transcription terminator regions, and the mechanisms of transcription-coupled repair of DNA damage (TCR). The sliding clamp model of transcription processivity suggests the formation of a highly stable elongation complex (EC), in which RNA polymerase is tightly bound to the nascent transcript and template forming the characteristic transcription “bubble”. Here, an in vitro system for promoter-independent assembly of a functional mammalian RNA polymeraseⅡ elongation complex using highly purified polymerases and synthetic RNA and DNA oligonucleotides was presented. It was shown that the 9-nucleotide RNA∶DNA template hybrid is necessary and sufficient for the formation of a stable elongation complex with human RNA polymeraseⅡ, while further inclusion of the non-template DNA strand to form the complete transcription “bubble” actually destabilizes the already formed RNA:DNA:RNA polymeraseⅡ complex. In addition, by introducing a DNA damage at a specific site in the template DNA, this assay can potentially be used for characterizing the processes of transcription-coupled repair of DNA damages.