Figure 1. Effect of random T/A→dU/A substitutions on transcription by T7 RNAP using a 321 bp DNA transcription template ... Figure 1. Effect of random.

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Figure 1. Effect of random T/A→dU/A substitutions on transcription by T7 RNAP using a 321 bp DNA transcription template ... Figure 1. Effect of random T/A→dU/A substitutions on transcription by T7 RNAP using a 321 bp DNA transcription template (S321). (A) Representative time courses for transcription of S321 with all T/A and increasing levels of dU/A pairs. (B) Kinetics of transcription of S321 containing increasing levels of random dU/A pairs. The curves are non-linear least squares best fits to Equation (1). The reaction conditions were: [DNA] = 1–60 nM (T321 and U<sup>50</sup>321) and 5–120 nM for U321, [T7 RNAP] = 5 nM, [NTP] = 0.5 mM and 100 μCi/ml α-<sup>32</sup>P-GTP. Each measurement was repeated at least twice and the data points are the mean values with indicated standard deviations. For clarity of presentation, only a few representative error bars are shown. The average error over all the measurements was ±18%. (C) Comparison of the relative kinetic parameters of S321 with increasing levels of random dU/A pairs. Each parameter for U<sup>50</sup>321 and U321 was normalized to that of T321. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz183, https://doi.org/10.1093/nar/gkz183 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 2. Transcriptional effects of specific dU/A pairs located in a 23 bp DNA (S23) containing the T7 RNAP promoter ... Figure 2. Transcriptional effects of specific dU/A pairs located in a 23 bp DNA (S23) containing the T7 RNAP promoter and initiation sequences. (A) S23 sequences with dU/A pairs at the indicated sites (N = T or dU). The boxed sequence is the initiation region (+1 to +5) which generates a short 5 nucleotide transcript. NT and T refer to non-template and template respectively. (B) Kinetics of transcription of S23 containing T/A and dU/A pairs at specific sites. The reaction conditions were: [DNA] = 120 nM, [T7 RNAP] = 10 nM, [NTP] = 0.5 mM and 100 μCi/ml α-<sup>32</sup>P-GTP. The curves are non-linear least-squares best fits to Equation (1). The data points are the mean velocities obtained from linear plots of [P] versus time and the indicated errors are standard deviations (n = 2–4). For clarity of presentation, only a few representative error bars are shown. The average deviation in replicate measurements was ± 13%. (C) Comparison of catalytic efficiencies of T7 RNAP with the indicated substrates. The relative catalytic efficiency (k<sub>cat</sub>/K<sub>m</sub><sup>rel</sup>) is the ratio of k<sub>cat</sub>/K<sub>m</sub> for the dU/A substrate to that of T23. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz183, https://doi.org/10.1093/nar/gkz183 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 3. Combinatorial dU/A substitutions at the –1 and –3 positions of S23 rescue the damaging effect of ... Figure 3. Combinatorial dU/A substitutions at the –1 and –3 positions of S23 rescue the damaging effect of U<sup>−6</sup>. (A) Sequences of S23 with uracils at the –6 position in combination with substitutions at the –1, and –3 position (N = T or dU). NT and T refer to non-template and template respectively. (B) The inhibitory effect of U<sup>−6</sup>23 is rescued by substitutions at U<sup>−3</sup> and U<sup>−1</sup>. The reaction conditions were: [DNA] = 120 nM, [T7 RNAP] = 10 nM, [NTP] = 0.5 mM and 100 μCi/ml α-<sup>32</sup>P-GTP. The curves are non-linear least-squares best fits to Equation (1). Each measurement was repeated at least twice and the data points are the mean values with indicated standard deviations. For clarity of presentation, only a few representative error bars are shown. The average deviation in replicate measurements was ±8%. (C) Fold increases in the kinetic parameters for U<sup>−1,-6</sup>23, U<sup>−3,-6</sup>23 and U<sup>−1,-3,-6</sup>23 relative to U<sup>−6</sup>23. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz183, https://doi.org/10.1093/nar/gkz183 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 4. Transcriptional effects of dU/A pairs located in the DNA promoter (P), initiation (I) and elongation (E) ... Figure 4. Transcriptional effects of dU/A pairs located in the DNA promoter (P), initiation (I) and elongation (E) sequences for T7 RNAP. (A). Sequences of S38 PIE substrate with dU/A pairs in either the I, IE or PIE regions (N = T or dU). NT and T refer to non-template and template respectively. (B). Time course for transcription of T38 and U<sup>PIE</sup>38. (C) Concentration dependences of the rate of transcription for the four S38 substrates. The reaction conditions were: [DNA] = 20–600 nM, [T7 RNAP] = 20 nM, [NTP] = 0.5 mM and 100 μCi/ml α-<sup>32</sup>P-GTP. The curves are non-linear least-squares best fits to Equation (1). Each measurement was repeated at least twice and the data points are the mean values with indicated standard deviations. For clarity of presentation, only a few representative error bars are shown. The average error over all the measurements was ±11%. (D). Fold changes in the kinetic parameters for U<sup>I</sup>38, U<sup>IE</sup>38, and U<sup>PIE</sup>38 relative to T38. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz183, https://doi.org/10.1093/nar/gkz183 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 5. Transcriptional mutations observed with T7 RNAP transcription from the uniformly uracilated DNA template. (A) ... Figure 5. Transcriptional mutations observed with T7 RNAP transcription from the uniformly uracilated DNA template. (A) Sequence context of the C→A transversion. Red letters refer to the position of the substitution and the numbers on the top of the sequence correspond to the positions on the original psiCHECK2 plasmid sequence. (B) Sequence context analysis of the U insertion mutation in the homopolymer run AAA on the template strand. These mutations were observed on both strands of the Sanger sequencing reads. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz183, https://doi.org/10.1093/nar/gkz183 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 6. Effects of template dUMP on transcription and protein expression in human cells. (A) DNA constructs ... Figure 6. Effects of template dUMP on transcription and protein expression in human cells. (A) DNA constructs containing a CMV promoter to drive an eGFP expression cassette and increasing levels of dU/A pairs were transfected into the human HAP1<sup>ΔUNG</sup> cell line that has no uracil excision activity. The relative copy number of the intracellular transfected DNA was determined by qPCR 24h after transfection. ΔΔC<sub>t</sub> is defined in the Methods and quantifies the efficiency of each transfection relative to the all T DNA and normalizing to the genomic copies of the RPP30 gene. No transfection control is denoted as (–) (B) The mRNA transcripts produced 24 h after transfection with the uracilated DNA templates were quantified using RT-qPCR. ΔΔCt is defined in the Methods and quantifies the mRNA produced relative to the all T DNA, while normalizing to cellular GAPDH expression. No transfection control is denoted as (–) (C) The fraction of eGFP positive cells 24 h after transfection was determined by flow cytometry. Transfections and flow cytometry were performed on HAP1<sup>wt</sup> or HAP1<sup>ΔUNG</sup> cells and the relative eGFP expression levels were normalized to the number of GFP positive cells obtained with the all T DNA. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz183, https://doi.org/10.1093/nar/gkz183 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 7. Interactions between T7 promotor DNA and T7RNAP Figure 7. Interactions between T7 promotor DNA and T7RNAP. Formation of the transcriptional bubble begins at the –7 to ... Figure 7. Interactions between T7 promotor DNA and T7RNAP. Formation of the transcriptional bubble begins at the –7 to –5 region of the template strand of DNA, where base pairs –4 through –1 are subsequently melted by T7RNAP and the template strand is directed into the active site of the polymerase. The key thymidine substitutions that produced effects on transcription are indicated (T-6, T-3, T-1, see text). The expanded inset shows the interactions involving T<sup>−6</sup>. PDB accession number 1CEZ. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz183, https://doi.org/10.1093/nar/gkz183 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 8. Possible mutational mechanisms with T7 RNAP arising from uracilated DNA templates. (A) Substitutions by ... Figure 8. Possible mutational mechanisms with T7 RNAP arising from uracilated DNA templates. (A) Substitutions by misalignment. (B) Insertion mutation by slippage of the extending mRNA. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz183, https://doi.org/10.1093/nar/gkz183 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 9. Mutational spectrum of human RNA pol II using all T and partially uracilated transcriptional templates and ... Figure 9. Mutational spectrum of human RNA pol II using all T and partially uracilated transcriptional templates and effects on protein sequence. (A) Mutation analysis of RNA pol II on eGFP-T template. There are seven clones containing seven mutations, no multiple mutations observed in any of the clones. (B) Mutation analysis of RNA pol II on eGFP-U<sup>50</sup> template. There are six clones containing eleven mutations, three clones contain multiple mutations. Black boxes refer to the codon and corresponding amino acid of the reference sequence. Red boxes refer to the codon and corresponding amino acid of the clonal sequences. Blue letters refer to the codon where mutations occur. Red letters refer to the position of the substitution and the numbers on the top of the sequence correspond to the positions on the original pCMV-GFP plasmid sequence. Amino acid mutations are highlighted with yellow color. These mutations were observed on both strands of the Sanger sequencing reads. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, gkz183, https://doi.org/10.1093/nar/gkz183 The content of this slide may be subject to copyright: please see the slide notes for details.