Figure 2.

Twelve nucleotides of nascent RNA are protected within the EC. A startup complex that extends to +14 was formed on a template derived from pPK12 (11), and the transcripts were extended by incubation with UTP, [α-³²P]ATP, and [α-³²P]GTP (bold). The sample was divided into three portions that were exposed to 0.1% SDS and/or RNase T1 (0.1 unit, 15 min at 25°C) as indicated, and analyzed by PAGE in a 20% gel in the presence of 6 M urea. An intact transcript of the expected length (17 nt) was observed in the untreated sample. This transcript was reduced to a limit size of 12 nt (shaded box) in undissociated complexes (−SDS) but was degraded in complexes that had been disrupted by exposure to SDS. The size of the limit digest was determined from an overexposed film in which a complete ladder of transcripts extending to +17 was observed (data not shown).