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Molecular and Cellular Biology, September 2001, p. 5815-5825, Vol. 21, No. 17
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.17.5815-5825.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Promoter Clearance by RNA Polymerase II Is an Extended, Multistep Process Strongly Affected by Sequence

Mahadeb Pal,1 David McKean,1,2 and Donal S. Luse1,2,*

Department of Molecular Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195,1 and Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 441062

Received 16 April 2001/Returned for modification 16 May 2001/Accepted 31 May 2001

We have characterized RNA polymerase II complexes halted from +16 to +49 on two templates which differ in the initial 20 nucleotides (nt) of the transcribed region. On a template with a purine-rich initial transcript, most complexes halted between +20 and +32 become arrested and cannot resume RNA synthesis without the SII elongation factor. These arrested complexes all translocate upstream to the same location, such that about 12 to 13 bases of RNA remain in each of the complexes after SII-mediated transcript cleavage. Much less arrest is observed over this same region with a second template in which the initially transcribed region is pyrimidine rich, but those complexes which do arrest on the second template also translocate upstream to the same location observed with the first template. Complexes stalled at +16 to +18 on either template do not become arrested. Complexes stalled at several locations downstream of +35 become partially arrested, but these more promoter-distal arrested complexes translocate upstream by less than 10 nt; that is, they do not translocate to a common, far-upstream location. Kinetic studies with nonlimiting levels of nucleoside triphosphates reveal strong pausing between +20 and +30 on both templates. These results indicate that promoter clearance by RNA polymerase II is at least a two-step process: a preclearance escape phase extending up to about +18 followed by an unstable clearance phase which extends over the formation of 9 to 17 more bonds. Polymerases halted during the clearance phase translocate upstream to the preclearance location and arrest in at least one sequence context.

* 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{at}ccf.org.

Molecular and Cellular Biology, September 2001, p. 5815-5825, Vol. 21, No. 17
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.17.5815-5825.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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