A. Cartoon representation of “closed” Pol II TL in relation to nucleic acids, Rpb1 bridge helix and a matched GTP substrate from structure PDB 2E2H [1] overlaid with TL constrained in open conformation by TFIIS (not shown) from structure PDB 1Y1V [69]. Amino acids (all derived from Rpb1) adjacent to the matched GTP substrate are indicated by numbers and single-letter amino acid codes. This figure was created with Pymol [111]. B. Schematic of TL showing amino acid sequence in single-letter code, with positions of interest numbered, residues with direct contact to GTP substrate in structure 2E2H shown in green, and position 1103 shown in yellow. Two hinge regions, about which the TL appears to change conformation from the open to closed positions are indicated.

A. 10-fold serial dilutions of saturated cultures of Pol II TL mutant strains plated on different media. YPD is rich medium with dextrose as a carbon source. YPRaf is rich medium with raffinose as a carbon source. YPRaf/Gal has both raffinose and galactose, allowing assay of gal10Δ56-dependent galactose toxicity phenotypes. SC-Leu is defined, complete medium lacking leucine. MPA was added to this medium (SC-Leu+MPA) to 20 µg/ml final concentration, showing that a number of Pol II mutants are sensitive to this drug. SC-Lys is defined, complete medium lacking lysine, and detects the Spt⁻ phenotype (Lys⁺) for strains containing lys2-128∂. WT strains grow robustly on most media, but will not grow on SC-Lys when lys2-128∂ is present and grow very poorly on YPRaf/Gal when gal10Δ56 is present. Mutant-dependent transcriptional phenotypes allow modulation of these specific growth defects. B. Schematic of TL (as diagrammed in Figure 1B) showing distribution of viable substitutions with their growth defects on YPD as well as lethal substitutions. All mutants were examined in the background of an Rpb1 T69 change, which is the allele present in the S288C reference genome as well as being normally found in our strains, representing a distinction for the subset of mutants described previously [2](see Materials and Methods, Text S1 and note concerning viability of Q1078S). Scoring of phenotypic strength is based on visual inspection of (A). C. Distribution of gal10Δ56 suppression (Gal resistance) or enhancement (Gal super-sensitivity) among TL substitutions. Scoring of phenotypic strength is based on visual inspection of (A). *Indicates strength of gal10Δ56 suppression is likely underestimated due to confounding generic growth defects. D. Distribution of MPA phenotypes among TL substitutions. Scoring of phenotypic strength is based on visual inspection of (A). E. Distribution of lys2-128∂ suppression (Spt⁻ phenotype scored as Lys⁺) among TL substitutions. Scoring of phenotypic strength is based on visual inspection of (A).

Pol II TL single substitution elongation rates as determined in vitro using reconstituted Pol II elongation complexes with synthetic oligonucleotides and an RNA primer (Materials and Methods). Raw data for some mutants (*) are from [2] and are shown for comparison. Plotted data are maximal elongation rates as determined by non-linear regression of elongation rates determined for different NTP substrate concentrations, and error bars indicate the range of the 95% confidence intervals (Figures S4, S5). Growth on rich medium of mutants is shown in the right panels for comparison with elongation defects (from Figure 2).

A. GOF-GOF and GOF-LOF genetic interactions among rpb1 TL and other substitutions. GOF (yellow) and LOF (blue) classifications were determined by biochemical and genetic phenotypes of single substitution mutants, with lethal single substitutions shown in black. Double mutant phenotypes are illustrated as the colored lines connecting colored nodes of particular single mutants. Schematic of TL sequence and orientation is as in Figure 1B. Data were compiled from Figure S2, Figure S3 and Table 1. N1082S/F1084I shows a complex interaction with exacerbation of growth defects on rich medium but mutual suppression of other conditional plate phenotypes. B. In vitro elongation rates determined as in Figure 3 for select Pol II TL double mutant enzymes. Single mutants and their relevant E1103G double mutants are indicated by color-coding: F1086 and F1086/E1103G, yellow; H1085Q and H1085Q/E1103G, green; H1085Y and H1085Y/E1103G, blue. Black bars indicate double mutants with E1103G for which the counterpart single substitution mutant is inviable (Q1078A, N1082A, H1085A). Single mutant data are from Figure 3. Raw data for some mutants (*) are from [2] and are shown for comparison. Error bars indicate the ranges of the 95% confidence intervals. Note that x-axis is logarithmic scale. Growth on rich medium of mutants is shown in the right panels for comparison with elongation defects (from Figure 2, Figure S2). C. Interaction diagram showing phenotypes of pairwise combinations of rpb1 LOF alleles (legend shown in (A)). D. Interaction diagram illustrating genetic interactions between pairs of Rpb1 substitution mutants each in the presence of the E1103G substitution (yellow shading encompassing all relevant residues, legend shown in (A)).

A. Northern blotting for different Pol II mutants in the presence or absence of 20 µg/ml MPA (2 hours treatment) for expression of IMD gene(s) using IMD2 DNA probe (top). SED1 was probed as a loading control. Values for IMD normalized to the WT ratio of IMD/SED1 are shown in the graph and represent the mean relative expression for IMD transcript(s) +/− the standard deviation for at least three independent experiments. B. Northern blotting for URA2 expression in the presence or absence of 20 µg/ml MPA (2 hours treatment) (top). SED1 was probed as a loading control. Values for URA2 normalized to the WT ratio of URA2/SED1 are shown in the graph and represent the mean relative expression for URA2 +/− the standard deviation for at least three independent experiments. C. Schematic of IMD2 gene transcription in the absence or presence of MPA. IMD2 is not functionally expressed in the absence of MPA or low GTP because upstream transcriptional starts are terminated at a terminator element (stop sign) that can be bypassed by utilization of downstream start sites. D. Primer extension analysis for downstream IMD2 start site usage in Pol II TL mutants in presence or absence of 20 µg/ml MPA (2 hours treatment). Numbers on right indicate position of RNA terminus relative to the A of the IMD2 ATG codon. Sequence ladder on right is derived from the primer used in (E) and has been cross-referenced with the primer used here. E. Primer extension analysis for upstream IMD2 start site usage in Pol II TL mutants as in (D). Numbers on right indicate position of RNA terminus relative to the A of the IMD2 ATG codon. Sequence ladder on right is derived from the same primer used. The examples in D and E are representative of at least three independent experiments.

A. Primer extension analysis of RNA 5′-ends at ADH1 in Pol II TL mutants. Sequence ladder at left is derived from primer used in Figure 5E but has been cross-referenced with ADH1 primer used for primer extension here. Numbers indicate positions of putative start sites in relation to the ADH1 ATG (A is +1). Right panel quantifies start site usage at ADH1 as determined by primer extension with a radiolabeled oligo. Signals in each lane were divided into bins based on positions of start sites relative to ADH1 ATG sequence (A is +1) and normalized to total signal per lane. WT start site fractions were subtracted from mutant start site fractions for particular regions to determine the relative alteration in start site distribution in Pol II mutant strains. A negative value indicates that the mutant has relatively lower usage for that particular group of start sites and a positive value indicates a relatively higher usage for that particular group of start sites. Values shown are the average of at least four independent experiments +/− standard deviation. B. Primer extension analysis of RNA 5′-ends at HIS4 and PMA1 in Pol II TL mutants. C. Primer extension analysis of RNA 5′-ends at GAL1 in Pol II TL mutants. Cells are grown in medium lacking raffinose and then GAL1 is induced by addition of galactose to 2% for two hours. Sequence ladder at left is derived from the same primer used for primer extension. Numbers indicate positions of putative start sites in relation to the GAL1 ATG (A is +1).

A. Structural cartoon showing overlay of TLs and Rpb1 N479 from a number of Pol II elongation complex/initial transcribing complex crystal structures with TLs in different states of folding/NTP interaction. Matched GTP nucleotide (orange) and TL and N479 (magenta) are from PDP 2E2H [1], which shows TL in a closed conformation. Partially folded TL and N479 (yellow) from PDB 4A3F [13], which contains a matched NTP analog (not shown) and short RNA (not shown). Related structure to PDB 4A3F from PDB 4A3D [13], but without matched NTP analog is shown in gray. Partially folded TL from PDB 1R9T, containing a mismatched NTP (not shown) is shown in pink. B. Same figure as in (A) but with GTP omitted. Certain residues are relatively closely positioned in all structures overlaid (N479, Q1078), while other residues are observed stabilized in partially folded structures directly adjacent to the active site (L1081, N1082). H1085 is observed positioned adjacent to the active site only upon complete folding/stabilization of the TL. Dashed oval indicates likely location of TL NIR residues when in out conformation where the TL is either mobile or unfolded. This figure was created with Pymol [111].

Cartoon showing predicted start site distribution for an ADH1-like gene in WT cells (top) or alteration in distribution in the presence of LOF (middle) or GOF (bottom) Pol II TL mutants.