Fig 1.
Uncovering the role of TFIIIA-7ZF and Pol II in transcribing (-) PSTVd oligomers.
(A) Reconstituted in vitro transcription (IVT) system using partially purified Pol II (from wheat germ, 5.4 nM), (-) PSTVd dimer RNA (7.8 nM), and various amounts of TFIIIA-7ZF. Sequence-specific riboprobes were used to detect (-) PSTVd templates and (+) PSTVd products (at the position close to dimer PSTVd). Quantification of product intensities was performed using ImageJ. The first lane signal was set as 0 and the second lane signal was set as 1. Data from three replicates were used for graphing the fold increases induced by various amounts of TFIIIA-7ZF. The uncropped gel images are shown in S4 Fig. (B) Schematic presentation for RNA-based affinity purification followed by nLC-MS/MS identifying protein factors in partially purified Pol II and remodeled Pol II. The presence of Rpb12 in the partially purified Pol II is based on literature. It remains to be determined whether Rpb12 is in the remodeled Pol II. (C) Peptide counts for each Pol II subunit in all nLC-MS/MS replicates. The summarized nLC-MS/MS data are listed in S1 Table. The original data can be found in S1–S6 Datasets. (D) IVT assay demonstrating the activity of remodeled Pol II. The first lane contains free RNA as template, while the second lane contains mixed free RNA and desthiobiothnylated RNA as template. The reaction condition is described in Methods in detail.
Fig 2.
Analyses on transcription elongation factors.
(A) Analyses of peptide counts of the FACT complex (SSRP1-B, SPT16), SPT6, PAF1-C, and TFIIF in partially purified Pol II and remodeled Pol II. Y-axis lists numbers of identified peptides corresponding to each gene. P values were calculated via a two-tailed T-test, a built-in function in Prism. The summarized nLC-MS/MS data are listed in S1 Table. The original data can be found in S1–S6 Datasets. (B) Schematic presentation of Pol II interactions with SPT6 and PAF1-C during DNA-dependent transcription, based on a reference [16]. P, PAF1. L, LEO1. R, RTF1. Purple line, CTR9.
Fig 3.
Analyses on the role of TFIIIA-7ZF zinc finger domains in aiding Pol II activity on RNA templates.
Reconstituted in vitro transcription (IVT) system using partially purified Pol II (5.4 nM), (-) PSTVd dimer RNA (7.8 nM), and various amounts of TFIIIA-7ZF mutants. Sequence-specific riboprobes were used to detect (-) PSTVd templates and (+) PSTVd products. Arrowheads indicate the position of (-) PSTVd dimer template, based on the extremely low cross-reaction signals that were only visible after over-exposure. Fold changes were analyzed as described in Fig 1. P values for significant fold changes as compared with Pol II only samples were listed. n.s., no significant change was identified. Uncropped gel images were shown in S4 Fig.
Fig 4.
Analyses of the binding ability of TFIIIA-7ZF mutants.
WT and mutants TFIIIA-7ZF proteins were used for RNA-based affinity purification in the presence of partially purified Pol II. (A) Immunoblots for input and RNA-bound TFIIIA-7ZF (anti-TFIIIA) and the Rpb1 subunit of Pol II (8WG16). Values from the samples with WT TFIIIA-7ZF were set as 100%. Data from samples with mutant TFIIIA-7ZF were normalized to the values of samples with WT TFIIIA-7ZF. None, no TFIIIA-7ZF protein was supplied. (B) Quantification of immunoblotting results in (A). Protein signals in Pull-down blots were normalized to the corresponding signals in the Input blots. The normalized signals in WT (TFIIIA and Rpb1) were set as 100%. Three replicates were performed for quantification and statistical analyses. A two-tailed T-test was used to calculate P values (listed in tables), by using the built-in function in Prism.