New model for the yeast RNA polymerase I transcription cycle

Abstract

Using an immobilized template assay, we observed two steps in assembly of the yeast RNA polymerase I (Pol I) preinitiation complex: stable binding of upstream activating factor (UAF) followed by recruitment of Pol I-Rrn3p and core factor (CF). Pol I is required for stable association of CF with the promoter and can be recruited in the absence of Rrn3p. Upon transcription initiation, Pol I-Rrn3p and CF dissociate from the promoter while UAF remains behind. These findings support a novel model in which the Pol I basal machinery cycles on and off the promoter with each round of transcription. This model accounts for previous observations that rRNA synthesis may be controlled by regulating both promoter accessibility and polymerase activity.

FIG. 1

Immobilized templates and experimental design. The WT template contains yeast rDNA sequence from −200 to +41 relative to the site of transcription initiation (indicated by an arrow). The upstream promoter element (upe) and core promoter element (core) are shown by open boxes; vector DNA is shown as a thick line. Template Δ−42 has a deletion extending from −200 to −42, while template Δ−2 has a further deletion extending to −2. All templates are shown attached to a Dynal magnetic bead. In control experiments (data not shown), the Δ−42 template had the same transcription activity whether or not it was beaded. Thus, at this distance the bead does not interfere with factor binding.

FIG. 2

PIC formation is promoter dependent. PICs were formed by incubation of WT whole-cell extracts with immobilized templates (either WT, Δ−42, or Δ−2) for 45 min. PICs were then washed and analyzed either for specific Pol I transcription (by addition of NTPs followed by primer extension) or for bound proteins (by Western blotting).

FIG. 3

Pol I is recruited in the absence of Rrn3p. (A) PIC formation in the absence of Rrn3p. PICs were formed on either WT or Δ−2 templates in extract containing WT Rrn3p (RRN3, lanes 1 and 2), temperature-sensitive Rrn3p (RRN3ts, lanes 3 and 4), or in an extract lacking Rrn3p (ΔRRN3, lanes 5 and 6). After washing, bound proteins were detected by Western blotting. (B) Rescue of transcription activity on PICs lacking Rrn3p. PICs were formed on WT template by incubation with 2 μl of the RRN3ts extract for 45 min. After washing, PICs were incubated either with buffer alone (lanes 2 and 11), a WT extract (2 μl; lane 1), an RPA190ts extract (1, 2, and 4 μl; lanes 3 to 5), affinity-purified yeast Rrn3p (1, 2, and 4 μl; lanes 6 to 8), a ΔRPA190 extract (0.5, 1, and 2 μl; lanes 12 to 14), a ΔRRN7 extract (2 μl; lanes 9 and 15) or a ΔRRN5 extract (5 μl; lanes 10 and 16). Second incubations were also for 45 min followed by washing and resuspension in YTB with NTPs. After 15 min of incubation, transcripts were assayed by primer extension. Partial rescue was obtained with a second extract containing both Pol I and Rrn3p (ΔRRN7; lanes 9 and 15), while an extract containing Pol I, Rrn3p, and CF (ΔRRN5; lanes 10 and 16) rescued fully. (C) Complementation activity of mutant extracts. Extracts were premixed and incubated for 10 min before addition of template. The WT template was added and incubation continued for 45 min followed by NTP addition. Transcription was stopped after 30 min and assayed by primer extension. Volumes of extracts tested were as follows: ΔRPA190, 0.5 μl; RRN3ts, 2 μl; ΔRRN7, 2 μl; ΔRRN5, 5 μl; yRrn3p, 2 μl.

FIG. 4

CF is not recruited in the absence of Pol I. (A) PIC formation in the absence of Pol I. PICs were formed on either the WT or Δ−2 template (lanes 2 and 3) using ΔRPA190 extracts where either RRN9, RRN7, or RRN3 were epitope tagged. After washing, proteins were detected by Western blotting. The presence of intact proteins in each extract is shown in lane 1. In the absence of Pol I, UAF binds to the promoter but not to CF or Rrn3p (lane 2). (B) Complementation of Pol I-free PICs with mutant extracts. WT template was first incubated either with buffer (lanes 1, 3, 5, and 7) or ΔRPA190 extract (lanes 2, 4, 6, 8, and 9) for 45 min and washed. Templates were then incubated for 45 min with a second extract, either WT (lanes 1 and 2), ΔRRN5 (lanes 3 and 4), ΔRRN7 (lanes 5 and 6), RRN3ts (lanes 7 and 8), or buffer alone (lane 9), washed, and incubated in YTB with NTPs. After a 15-min incubation, transcripts were assayed by primer extension. PICs formed in the absence of Pol I were only rescued by a second extract lacking UAF (ΔRRN5; lane 4) or the WT extract (lane 2).

FIG. 5

Release of Pol I factors during transcription. PICs were formed for 45 min on either the WT or Δ−2 template using WT extract. After washing, PICs were resuspended in YTB with NTPs for 15 min. Factors that remain bound to the immobilized template were separated from released factors with a magnetic separator, resolved on an SDS-polyacrylamide gel, and identified by using Western blotting. Note that essentially all of Rrn3p releases from the WT template, a small fraction of Pol I (RPA34, RPA43), CF (RRN7, RRN11) and TBP release, and no UAF releases during transcription.

FIG. 6

Nucleotide requirements for factor release. PICs were formed on the WT template, washed, and incubated for 15 min in YTB with either all four NTPs (AUGC), triphosphates corresponding to the first three nucleotides of the transcript (AUG), GTP alone, a nonhydrolyzable ATP analog (AMPPCP), or buffer alone. After separation into bound and released fractions, factors were identified by Western blotting. As expected from Fig. 5, UAF does not release under any conditions. Pol I, Rrn3p, and CF release only in the presence of all four NTPs (AUGC) except for a small nonspecific release of Pol I that occurs in buffer alone. TBP releases in the presence of only AUG as well as in the presence of AUGC.

FIG. 7

Model of the Pol I transcription cycle. We define two steps in formation of the Pol I PIC. In the first step, UAF locates the promoter and binds stably in a sequence-specific manner. In the second step, Pol I-Rrn3p and CF are recruited by UAF. TBP is required for this recruitment, but whether it enters with CF and Pol I-Rrn3p or is separately recruited by UAF is not yet determined. Upon initiation of transcription, CF, Pol I/Rrn3p, and TBP all leave the promoter. UAF remains bound as a scaffold for further rounds of recruitment and transcription.