Promoter-proximal pausing by RNA polymerase II in vitro: transcripts shorter than 20 nucleotides are not capped.

Abstract

RNA was synthesized from cloned adenovirus 2 late promoter DNA in an in vitro transcription extract under UTP-limiting conditions. Under these circumstances, almost all of the .alpha.-amanitin-sensitive RNA produced is shorter than 20 nucleotides; most of these short transcripts are present in 4 species, 6, 7, 13 and 17 nucleotides. These short RNA are initiated at the adenovirus 2 promoter, as judged by partial sequence analysis and by the abolition of their synthesis upon cleavage of the template DNA at sites which also abolish the production of full-length transcripts. All of the short transcripts can be chased, with excess UTP, into 197-base run-off transcripts; thus, these RNA are precursors of full-length transcripts and not synthetic dead ends. None of these short RNA is capped or 2''-O-metylated. However, 79-base run-off transcripts synthesized from this promoter with nonlimiting NTP [nucleoside triphosphate] levels are fully capped.