Strong natural pausing by RNA polymerase II within 10 bases of transcription start may result in repeated slippage and reextension of the nascent RNA.

We find that immediately following transcript initiation, RNA polymerase II pauses at several locations even in the presence of relatively high (200 M) levels of nucleoside triphosphates. Strong pauses with half-lives of >30 s were observed at 7, 18/19, and about 25 on the template used in these experiments. We show that the strong pause at 7, after the synthesis of 5-ACUCUCU, leads to repeated cycles of upstream slippage of the RNA-DNA hybrid followed by re-pairing with the DNA and continued RNA synthesis. The resulting transcripts are 2, 4, and 6 bases longer than predicted by the template sequence. Slippage is efficient when transcription is primed with the 1/2 (ApC) dinucleotide, and it occurs at even higher levels with the 2/3 primer (CpU). Slippage can occur at high levels with ATP initiation, but priming with CpA (1/1) supports very little slippage. This latter result is not simply an effect of transcript length at the point of pausing. Slippage can also occur with a second template on which the polymerase can be paused after synthesizing ACUCU. Slippage is not reduced by an ATP analog that blocks promoter escape, but it is inhibited by substitution of 5Br-U for U in the RNA. Our results reveal an unexpected flexibility of RNA polymerase II ternary complexes during the very early stage of transcription, and they suggest that initiation at different locations within the same promoter gives rise to transcription complexes with different properties. An RNA polymerase II transcription cycle consists of several distinct steps: recruitment to assemble the preinitiation complex, open-complex formation, initiation, promoter escape and clearance, transcript elongation, and termination. It is now clear that regulation may be imposed at steps after initiation (reviewed in references 20, 26, 31, and 36). In particular, many transcription units contain newly initiated RNA polymerases that are stalled from 20 to 40 nucleotides downstream of transcription start (reviewed in reference 24). The molecular basis for the regulated pausing and release of RNA polymerase II from promoter-proximal locations has not been established, in part because the transition from the well-characterized preinitiation complex to the stable transcript elongation complex is poorly understood. Rate-limiting steps early in transcription should be particularly interesting, since they correspond to likely regulatory targets and could identify important structural transitions in the transcription complex. This idea is strongly supported by the recent results of Kugel and Goodrich (18), who placed the rate-limiting step for in vitro transcription from a nonregulated promoter somewhere between the formation of the 4th and 14th bonds. In light of this report, we began the current work by preparing gently washed RNA polymerase II preinitiation complexes, which were then incubated with nonlimiting (i.e., 200 M) levels of labeled nucleoside triphosphates (NTPs). This experiment revealed that transcription immediately downstream of initiation does not proceed uniformly. Instead, we observed a series of very discrete pauses with half-lives at least 20 times longer than the average bond formation rate under the conditions we employed. The first of these pauses occurs at 7, after the synthesis of the transcript 5-ACUCU CU, on the template we are studying. We were surprised to discover that the 7 pause allowed the RNA-DNA hybrid to shift upstream by 2 bp and then re-pair with the template, leading to the synthesis of a transcript 2 bases longer than that predicted by the template sequence. Multiple rounds of this upstream slippage and reextension event can take place.