1. Poly(A) Signal-Dependent Transcription Termination Occurs through a Conformational Change Mechanism that Does Not Require Cleavage at the Poly(A) Site 
Huimin Zhang, Frank Rigo, Harold G. Martinson 
Molecular Cell 
Volume 59, Issue 3, Pages (August 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 59, 437-448DOI: (10.1016/j.molcel.2015.06.008)
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3. Figure 1 The Basic PADT Assay See text.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 PADT Is Faithfully Reproduced in a Nuclear Extract In Vitro
(A) The dsxSV40L template drawn to scale. The PAS hexamer (mutated for lanes 5–8 of [B]) lies just to the left of the poly(A) cleavage site indicated by the arrow. The size and binding location of the anti-PAS MOE, used for (C) and (D), and of the DNA oligos used for (D), are shown. Note that almost all sequence downstream of the PAS derives from the pSP72 cloning vector and is highly unlikely to contain any functional elements related to RNA polymerase II transcription or processing.
(B) PADT by the SV40 late PAS. As shown in Figure 1, parallel transcription reactions were carried out for templates with a wild-type or a mutant PAS. At the indicated times, aliquots were withdrawn and treated as shown in Figure 1. Fragment 0 is a doublet because one end is blunt and the other a 5′ overhang, giving single strands on the gel of 282 and 286 nt. Fragment 1 is similar, but the strands are not resolved. The asterisk indicates a band produced by EcoRI star activity near the end of fragment 2. The data in lanes 5–8 are presented quantitatively in the graphs beside the gel and show band intensity (arbitrary units) as a function of time for fragments 0, 1, and 2 (not including the star band). The graph below the gel quantitates polymerase occupancy (see text) from several experiments. The values at 3, 7, 15, and 30 min are averaged from three experiments (± SD), and the values at 56 min are from two experiments (± range). When the exact time of sampling varied between experiments, the average was plotted. All lanes in the figure come from the same gel but were rearranged for ease of discussion. The original, unspliced gel is shown in Figure S4.
(C) PADT detected using a MOE directed to the PAS. Two parallel transcription reactions were carried out. In one, the PAS was inactivated using a complementary MOE. In the other a control, noncomplementary MOE was used. The graph below the gel quantitates polymerase occupancy from several experiments. The values at 2 min are averaged from two experiments (± range); the values at 5, 17, and 30 min are from four experiments (± SD), and the values at 9 and 50 min are from three experiments (± SD). The 6.5 min sample shown in the gel was averaged with one 5 min sample and two 4 min samples from additional experiments and is plotted at 5 min on the graph. All lanes in the figure come from the same gel, but were rearranged for ease of discussion. The original, unspliced gel is shown in Figure S4.
(D) PADT is blocked when the tether is cut. Transcription was carried out in the presence of either a control MOE or one targeted to the PAS, as for (C). Also included in each mixture was one of three DNA oligos: a control non-complementary oligo, and two complementary oligos that directed RNase H cutting to positions 112 and 201 nt downstream of the poly(A) site. The approximate binding locations of the complementary MOE and DNA oligos are shown in the accompanying cartoon (see Rigo and Martinson, 2009) and in (A).
(E) PADT by the BGH PAS. See Figure S2 for the gel image.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 PADT Does Not Require Poly(A) Site Cleavage
(A) Removal of CP prevents poly(A) site cleavage but not PADT. After 5 min of transcription, TECs were rinsed and resuspended in fresh transcription mixture in the presence or absence of CP. The fresh transcription mixture contained all of the normal ingredients, including extract, but lacked NTPs, DNA, and Gal4-p53. For the −CP graph, the values at 6, 35, and 73 min are averaged from three experiments (± SD), and the values at 11 and 21 min are from two experiments (± range). When the exact time of sampling varied between experiments, the average was plotted. All averages plotted in the graph include at least one wild-type/mutant experiment and one MOE-based experiment. Representative gels for this and all subsequent figures are found in Figure S2. All RNase protection lanes shown come from the same gel, but were rearranged for ease of discussion. The original, unspliced gel is shown in Figure S4.
(B) Addition of ATP restores efficient PADT but not poly(A) site cleavage. At 4 min, TECs were rinsed and resuspended as above in the indicated concentration of ATP and CP. For lane 6 of the RNA gel, the sample was taken before the TECs were rinsed. For a duplicate of the 250 μM ATP portion of the experiment, see Figure S3. All RNase protection lanes shown come from the same gel, but were rearranged for ease of discussion. The original, unspliced gel is shown in Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
A Conformational Change of the TEC Drives PADT, Not Cleavage at the Poly(A) Site
(A) PADT, but not cleavage, can occur in the absence of extract. At 4 min, TECs were rinsed and resuspended. Resuspension was either in normal transcription mixture, including extract (but lacking NTPs, DNA, and Gal4-p53), or else simply in buffer D (no extract). For lane 1 of the gel, the sample was taken before the TECs were rinsed. All RNase protection lanes shown come from the same gel, but were rearranged for ease of discussion. The original, unspliced gel is shown in Figure S4. The graph on the right shows a PADT time course for rinsed TECs that have been resuspended in buffer without extract or NTPs. Values (except at 5 min) are averaged from two experiments (± range).
(B) Poly(A) site cleavage but not PADT can be rescued after stripping TECs. After 5 or 8 min of transcription (in different experiments), TECs were either rinsed or stripped (1 M KCl and 1% sarkosyl) and then resuspended in normal transcription mixture, including extract (but lacking NTPs, DNA, and Gal4-p53). An aliquot was then analyzed immediately to determine cleavage and PADT, while the remainder was incubated for 50 min and then analyzed. Bars 8, 9, 11, and 12 of the histogram are averaged from two experiments (± range). All RNase protection lanes shown come from the same gel, but lanes 1 and 3 were enhanced to show clearly the lack of poly(A) site cleavage prior to incubation. The original, unmanipulated gel is shown in Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5 PADT Is Inhibited by α-Amanitin
(A) PADT by the SV40 late PAS is inhibited by α-amanitin. Rinsed TECs were resuspended in the absence of extract as for Figure 4A, but with or without α-amanitin. Data are averaged from seven (− α-ama) or four (+ α-ama) experiments (± SD).
(B) PADT by the SV40 early PAS.
(C) PADT by the SV40 early PAS is inhibited by α-amanitin (continuous transcription protocol). Bars 1–3 are from the 45 min data of (B), and bars 4–6 show the data for α-amanitin added at 5 min.
(D) PADT by the SV40 early PAS is inhibited by α-amanitin (rinse and resuspend protocol). Rinsed TECs were resuspended in the presence of extract as for Figure 4A, but with or without α-amanitin. In these experiments, 25 μM ATP, which enhances PADT by the SV40 early PAS, was also present. Data are averaged from two experiments (± range).
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8. Figure 6 Degradation of Downstream RNA Not Required for PADT
Addition of pAp efficiently blocks degradation of poly(A) site 3′ flanking RNA but only delays PADT. Transcription was carried out in the presence or absence of pAp for 30 or 60 min, at which times samples were taken and split for RNase protection or PADT assays. The two graphs in the figure summarize the results of several PADT assays, including the split sample assay described above. For the left hand graph, the 30 and 60 min values are averaged from either three experiments (± SD) or two experiments (± range), respectively. For the graphs on the right, the 6 min value is from two experiments (± range), and the 30 and 60 min values are from four or three experiments (± SD), respectively.
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9. Figure 7
The PADT Model
After crossing the PAS, assembly of the cleavage/PADT apparatus proceeds stochastically, with commitment to cleavage-independent PADT (ciPADT) for any transcript occurring earlier than commitment to cleavage. Enhancing elements such as MAZ accelerates assembly. Following commitment, strong PASs cleave rapidly, leading to torpedo-style acceleration of the PADT. Very weak PASs cleave very slowly, making them vulnerable to step 2 of ciPADT, which delivers the 3′ end of the released RNA to the exosome.
Molecular Cell  , DOI: ( /j.molcel )
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