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Molecular and Cellular Biology, January 2002, p. 30-40, Vol. 22, No. 1
0270-7306/01/$04.00+0     DOI: 10.1128/MCB.22.1.30-40.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Strong Natural Pausing by RNA Polymerase II within 10 Bases of Transcription Start May Result in Repeated Slippage and Reextension of the Nascent RNA

Mahadeb Pal and Donal S. Luse*

Department of Molecular Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195

Received 25 June 2001/ Returned for modification 9 August 2001/ Accepted 17 September 2001

We find that immediately following transcript initiation, RNA polymerase II pauses at several locations even in the presence of relatively high (200 µM) 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.

* Corresponding author. Mailing address: Department of Molecular Biology, NC20, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland OH 44195. Phone: (216) 445-7688. Fax: (216) 444-0512. E-mail: lused@ccf.org.

Molecular and Cellular Biology, January 2002, p. 30-40, Vol. 22, No. 1
0022-538X/01/$04.00+0     DOI: 10.1128/MCB.22.1.30-40.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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