Distinct roles of transcription factors TFIIIB and TFIIIC in RNA polymerase III transcription reinitiation

Abstract

Eukaryotic RNA polymerase (Pol) III is recruited to target promoters by a stable preinitiation complex containing transcription factors TFIIIC and TFIIIB. After the first transcription cycle, reinitiation proceeds through facilitated recycling, a process by which the terminating Pol III rapidly reloads onto the same transcription unit. Here, we show that Pol III is repeatedly recaptured in vitro by the first transcribed gene, even in the presence of a juxtaposed competitor promoter complex, thus suggesting that facilitated recycling is not merely due to a stochastic reassociation process favored by the small size of class III genes. The transcription factor requirements for facilitated reinitiation were investigated by taking advantage of Pol III templates that support both TFIIIC-dependent and TFIIIC-independent transcription. A TFIIIC-less transcription system, in which TFIIIB was reconstituted from recombinant TATA box-binding protein and Brf1 proteins and a crude fraction containing the Bdp1 component, was sufficient to direct efficient Pol III recycling on short ( approximately 100 bp) class III genes. Unexpectedly, however, on longer (>300 bp) transcription units, reinitiation in the presence of TFIIIB alone was compromised, and TFIIIC was further required to reestablish a high reinitiation rate. Transcription reinitiation was also severely impaired when recombinant Bdp1 protein replaced the corresponding crude fraction in reconstituted TFIIIB. The data reveal an unexpected complexity in the Pol III reinitiation mechanism and suggest the existence of a handing-back network between Pol III, TFIIIC, and TFIIIB on actively transcribed class III genes.

Fig. 1.

Facilitated transcription reinitiation on tRNA and U6 RNA genes. (A) Stable PICs, containing both TFIIIB (reconstituted from rTBP, rBrf1, and crude B′′) and TFIIIC (+IIIC) or TFIIIB alone (–IIIC) were formed on tDNA^Gly(TCC) (lanes 1 and 2), SNR6 (lanes 3–6), or U6ΔB (lanes 7 and 8) templates. Pol III (10 ng) was then added together with NTP mixtures lacking CTP (lanes 1 and 2) or ATP (lanes 3–8), and the incubation continued for 10 min. Transcription was resumed by the addition of the missing nucleotide, with (lanes 2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) heparin, and was run for 5 min. The migration positions of U6 RNA and pre-tRNA^Gly are indicated. The ratios between the amounts of single- and multiple-round transcription products, derived from PhosphorImager counting, are reported below the lanes with the SE of three independent experiments. (B) Stable PICs were assembled on the U6ΔB template for 20 min in the absence of TFIIIC. Limiting (10 ng, upper gel) or saturating (100 ng, lower gel) amounts of Pol III were then added together with an NTP mixture lacking ATP. After the indicated time periods, the missing nucleotide was added in association with heparin, to allow for completion of the transcription cycle by heparin-resistant complexes. The plot shown is derived from quantification of the shown gels. (C) Stable PICs containing both TFIIIB and TFIIIC were assembled for 20 min on either tDNA^Gly(TCC) (upper gel) or on the wt SNR6 template (lower gel). Limiting (10 ng) amounts of Pol III were then added together with NTP mixtures lacking either CTP (for the tDNA) or ATP (for SNR6). After the indicated time periods, the missing nucleotides were added in association with heparin, to allow for completion of the transcription cycle by heparin-resistant complexes. The plot shown is derived from quantification of the shown gels.

Fig. 2.

Template exclusion experiments with physically linked transcription units. (A) The schemes illustrate the control (Upper) and competition (Lower) reaction protocols, respectively (see text for details). (B–D) In vitro transcription was performed after the control (lanes 1–4) or the competition (lanes 5–9 in B and C and lanes 6–10 in D) reaction schemes using the templates illustrated above each gel. Filled and empty arrows indicate the direction of transcription and represent the U6ΔB and the tDNA^Gly templates, respectively. Samples of the reaction mixtures were stopped at the indicated times. Lane 5 in D refers to a control reaction in which Pol III and the four NTPs were added simultaneously to PICs preassembled on both templates, and transcription was then run for 5 min. Lane 5 in B and C and lane 6 in D show the products of reactions (competition protocol) in which transcription was limited to a single round by the addition of heparin (H). The positions of U6 RNA and pre-tRNA^Gly are indicated on the left. The bar plots report the ratios between the amount of U6ΔB and tDNA^Gly transcription products accumulated at given times with the control (white bars) or the competition (hatched bars) reaction protocol.

Fig. 3.

Reinitiation properties of all-rTFIIIB. PICs were assembled for 30 min on tDNA^Ile(TAT) (Upper) or tDNA^Gly(TCC) (Lower) by using TFIIIC and different types of reconstituted TFIIIB. In lanes 1–8 and 17–24, TFIIIB was reconstituted from rTBP and rBrf1 proteins plus crude B′′. In lanes 9–16 and 25–32, pure rBdp1 protein from insect cells (100 ng) was used instead of B′′. Pol III (10 ng) was then added together with an NTP mixture lacking CTP, and the incubation was continued for 15 min. Transcription was resumed by the addition of CTP, either in the presence or in the absence of heparin, and the incubation continued for the indicated times. The ratios between the amounts of single- and multiple-round transcription products (MR/SR) are reported below the lanes.

Fig. 4.

Reinitiation properties of Pol III on long-size derivatives of the U6 RNA gene. Stable PICs containing TFIIIB alone, reconstituted from rTBP, rBrf1, and crude B′′ (–IIIC), or both TFIIIB and TFIIIC (+IIIC), were formed for 20 min on the U6ΔB_520 (lanes 1–4), U6ΔB_300 (lanes 5–8), SNR6_520 (lanes 9–12), or SNR6_300 (lanes 13–16) templates. Pol III (10 ng) was then added together with GTP, CTP, and UTP, and the incubation was continued for 10 min. Transcription was resumed by the addition of ATP, either with or without heparin, and was run for 5 min. The ratios between the amounts of multiple- and single-round transcription products (MR/SR) are reported below the gel.

Fig. 5.

Reinitiation on the SCR1 gene. Stable PICs, containing TFIIIB alone, reconstituted from rTBP, rBrf1, and crude B′′ (–IIIC) or both TFIIIB and TFIIIC (+IIIC) were formed for 20 min on the SCR1-C4T template (ref. ; lanes 1–4), the U6-SCR1 template, which consisted of the 5′-flanking region of SNR6 fused to SCR1 (lanes 5–8), or the U6-SCR1 mini template, and was generated by inserting a terminator at position +90 of U6-SCR1. After PIC assembly, Pol III (10 ng) was added together with an NTP mixture lacking CTP. Transcription was resumed 10 min later by adding CTP, either with (lanes 2, 4, 6, 8, 10, and 12) or without (lanes 1, 3, 5, 7, 9, and 11) heparin, and was run for 5 min. The positions of the SCR1-derived transcription products are indicated by arrow-heads. The ratios between the amounts of multiple- and single-round transcription products (MR/SR) are reported below with the SE of three independent experiments.