1. Facilitated Recycling Pathway for RNA Polymerase III
Giorgio Dieci, André Sentenac 
Cell 
Volume 84, Issue 2, Pages (January 1996)
DOI: /S (00)

2. Figure 1
Transcription Initiation and Reinitiation on the SUP4 tRNA Gene
(A) Overall time course of initiation by pol III. A stable preinitiation complex was formed on the SUP4 tRNA gene as described in Experimental Procedures. Pol III (50 ng) was then added together with ATP (500 μM), CTP (500 μM) and [α-32P]UTP (10 μM; 40 μCi/nmol). At the indicated times, samples were taken from the mixture and transferred to tubes chilled in dry ice to stop the reaction. The reaction products were separated on a 15% polyacrylamide, 7 M urea gel. The position of the 17 nt transcript is indicated. The upper part of the figure shows a scheme of the incubation protocol.
(B) Time course of pol III recycling. A stable preinitiation complex was formed on the SUP4 tRNA gene as described in Experimental Procedures. Pol III (50 ng) was then added together with ATP (500 μM), CTP (500 μM), and [α-32P]UTP (100 μM; 4 μCi/nmol), and the incubation was continued for 10 min. Transcription was restarted by the addition of GTP (500 μM) in the presence (lanes 1 and 2) or absence (lanes 3–5) of heparin (0.3 mg/ml). At the indicated times, samples were taken from the mixtures and transferred to tubes chilled in dry ice to stop the reaction. The products were separated on a 6% polyacrylamide, 7 M urea gel. The position of the tRNA gene primary transcript (SUP4 tRNA) is indicated. S, single-round transcription; M, multiple-round transcription.
(C) UTP dependence of recycling. Transcription reactions were as described for (A), with the exception that UTP concentration was varied as indicated. To keep the specific radioactivity constant, reactions carried out at 20 μM, 100 μM, and 500 μM UTP contained, respectively, 2, 10, and 50 μCi of [α-32P]UTP. After the addition of GTP only (lanes 2, 4 and 6; M, multiple-round transcription) or GTP plus heparin (lanes 1, 3 and 5; S, single-round transcription), the incubation was continued for 3 min. Products in lanes 2, 4, and 6 corresponded to 2.5, 5.8, and 7.0 rounds of transcription, respectively.
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3. Figure 2 Heparin Inhibition of Initiation and Reinitiation
(A) Heparin sensitivity of initiation. A stable preinitiation complex was formed on the SUP4 tRNA gene as described in Experimental Procedures. Pol III (50 ng) was then added together with ATP (500 μM), CTP (500 μM), [α-32P]UTP (10 μM; 40 μCi/nmol), and variable concentrations of heparin, as indicated. Synthesis of the 17-mer was allowed to proceed for 15 min. The products were separated on a 15% polyacrylamide, 7 M urea gel. The position of the 17 nt transcript is indicated.
(B) Heparin sensitivity of reinitiation. A stable preinitiation complex was formed on the SUP4 tRNA gene as described in Experimental Procedures. Pol III (50 ng) was then added together with ATP (500 μM), CTP (500 μM) and [α-32P]UTP (100 μM; 4 μCi/nmol) and the incubation continued for 10 min. GTP was then added, together with variable concentrations of heparin, as indicated. The reactions were stopped after 5 min (equivalent to about eight rounds of transcription in the absence of heparin) and the products separated on a 6% polyacrylamide, 7 M urea gel. The position of the tRNA gene primary transcript is indicated.
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4. Figure 3 Gene Commitment by Recycling RNA Pol III
(A) The tRNALeu3 and SUP4 tRNA genes were separately preincubated with TFIIIC and reconstituted TFIIIB, to allow for the formation of stable preinitiation complexes. Pol III (50 ng) was then added to one of the two complexes (template 1) together with ATP (500 μM), CTP (500 μM), UTP (100 μM), and [α-32P]UTP. For the reaction in lane 5, pol III (50 ng) was added to a mixture of the two preinitiation complexes. The incubation was continued for 10 min, and then GTP (500 μM) was added alone (lanes 3–5), together with heparin (lanes 1 and 2), or together with the competitor template preassembled in a stable preinitiation complex (template 2; lanes 6 and 7). Transcription was allowed to proceed for 5 min, and then reactions were blocked and the products separated on a 6% polyacrylamide, 7 M urea gel. The position of the two primary transcripts is indicated. Reactions in lanes 1–4 contained 20 μCi of [α-32P]UTP in a final volume of 50 μl. Reactions in lanes 5–7 contained 40 μCi of [α-32P]UTP in a final volume of 100 μl. S, single-round transcription; M, multiple-round transcription. The ratios between SUP4 and Leu3 transcription were 1.7, 7.0, and 0.3 in lanes 5, 6, and 7, respectively.
(B) Lanes 1 and 3, single-round transcription reactions done as in lanes 1 and 2 of (A), respectively. Lanes 2 and 4, SUP4 tDNA or tDNALeu3 were preincubated with TFIIIC and TFIIIB to form stable preinitiation complexes, then added to a mixture containing pol III (50 ng) and all four NTPs, at the same concentrations as in (A), and multiple rounds of transcription were allowed for 5 min. The ratios between the signals in lanes 2 and 4 and the signals of corresponding single-round transcription reactions were 5.6 and 5.0 for the SUP4 and Leu3 genes, respectively.
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5. Figure 4
Time Course of 17-mer Synthesis by a Complete Initiation Complex
The experiment was as in Figure 1A, but pol III alone was preincubated 10 min with the preinitiation complex before the addition of ATP, CTP, and UTP. A scheme of the incubation protocol is shown at the top.
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6. Figure 5
Time Course of Initiation on a Previously Transcribed Template
A stable preinitiation complex was formed on the SUP4 tRNA gene as described in Experimental Procedures. The incubation mixture was split into two parts. One part received pol III (50 ng) together with ATP (500 μM), CTP (500 μM), and UTP (100 μM); after a 10 min incubation, GTP (500 μM) was added together with heparin (0.25 mg/ml), to generate preinitiation complexes transcribed once (Kassavetis et al. 1990). The other half of the mixture, containing never-transcribed complexes, received only heparin. Each of the two mixtures (110 μl) was loaded onto a 1 ml Sepharose CL-2B gel filtration column. Fractions 9–11 from each column, containing stable preinitiation complexes but not free RNA polymerase (data not shown), were collected and pooled. Pol III (50 ng) was then added to each pool, together with ATP (500 μM), CTP (500 μM), and [α-32P]UTP (10 μM; 40 μCi/nmol), and samples of the reactions were blocked at the indicated times. The reaction products were separated on a 15% polyacrylamide, 7 M urea gel. The position of the 17 nt transcript is indicated.
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7. Figure 6
Requirement of the Terminator Element for Efficient Reinitiation on the U6 Gene
(A) Decreased reinitiation frequency after runoff termination. TFIIIB was assembled on the truncated version of the yeast U6 gene (contained in pTaq6 plasmid) as described in Experimental Procedures. Reactions in lanes 1 and 2 contained the uncut, circular plasmid. For reactions in lanes 3 and 4, the plasmid was linearized at the polylinker SmaI site, downstream of the U6 terminator sequence. For reactions in lanes 5 and 6, the plasmid was linearized at the EcoNI site in the coding region, 30 bp upstream of the terminator (Moenne et al. 1990). Pol III (50 ng) was added together with CTP (500 μM), GTP (500 μM), and [α-32P]UTP (200 μM; 2 μCi/nmol), and the incubation was continued for 15 min to allow for the formation of a ternary transcription complex containing a 7 nt nascent transcript (Brow and Guthrie 1988). ATP was then added in the presence (lanes 1, 3, and 5) or the absence (lanes 2, 4, and 6) of heparin to inhibit reinitiation, and the incubation was continued for 6 min. Reaction products were separated on a 6% polyacrylamide, 7 M urea gel. The positions of the complete and runoff U6 RNA gene products are indicated. S, single-round transcription; M, multiple-round transcription.
(B) The terminator sequence does not influence the affinity of the preinitiation complex for RNA pol III. TFIIIB was separately assembled on SmaI- and EcoNI-linearized pTaq6, and then pol III was added to a mixture of the two templates, together with CTP (500 μM), GTP (500 μM), and [α-32P]UTP (200 μM; 2 μCi/nmol). After 15 min, ATP (500 μM) was added with (lane 1) or without (lane 2) heparin, and transcription was allowed to proceed for 6 min in a final volume of 100 μl. Reaction products were separated on a 6% polyacrylamide, 7 M urea gel. The positions of the complete and runoff U6 RNA gene products are indicated. S, single-round transcription; M, multiple-round transcription.
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8. Figure 7
Model for the Pathways of Transcription Reinitiation by RNA Pol III
For simplicity, TFIIIC has been omitted from the scheme. Steps 1 and 2 summarize a simple transcription cycle, from initiation by free pol III to termination. Step 1 can be inhibited by 2 μg/ml heparin. Concomitant with the termination step, two alternative pathways can be followed by pol III: it can either dissociate (step 3b) and then slowly reinitiate a new cycle from step 1, or undergo a termination-coupled, fast reinitiation event without being released from the template (steps 3a and 4). The latter pathway can take place in the presence of 2 μg/ml heparin and can be inhibited by 50–100 μg/ml heparin. A single arrow in the figure does not imply that there is only one biochemical step involved. For details, see Discussion.
Cell  , DOI: ( /S (00) )