Amendments to Fig 5, S8 Fig, S9 Fig and S2 Table

Panel D in Fig 5, and panels B and C in S8 Fig have been corrected to account for these changes. Panel F of S8 Fig and panels A and B of S9 Fig have been updated to include the observed phenotypes of the viable E712A, D716K and D716K-containing double mutants. S2 Table has also been updated to account for the changes. Please view the corrected Fig 5 and the corrected and updated S8 Fig, S9 Fig and S2 Table, along with the corresponding legends below.

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Fig 5. Functional contribution of TL tip and funnel helix α-21 to proper TL dynamics.

(A) Observed and predicted interactions between TL and TL-proximal domains. TL schematic is shown with residues identified by single-letter amino acid code and positions of interest annotated. Positions of GOF mutants isolated in our screen, along with the positions for a subset of previously isolated TL-proximal GOF mutants, are color coded in green. Observed TL interactions with other Rpb1 domains from structures or simulation studies are shown as grey dashed lines. (B) Maximal in vitro elongation rates (nucleotides/second) of Pol II WT and genetic GOF mutants S713P, I1327V and A1076T. (C) Observed interactions between open TL tip and TL adjacent charged residues (PDB: 5C4X). Funnel Helix refers to the Rpb1 α-21 alpha-helix. (D) Genetic interactions between the TL tip and proximal Rpb1 domains. Schematics of the TL and adjacent domains are shown in lines, with positions of interest shown in single-letter amino acid code. Substituted residues are shown in grey, with substituting amino acids shown in white or green filled circles based on single substitution phenotypes (S8F Fig). Double substitution phenotypes are shown as colored lines connecting the two relevant single substitutions. Some sets of similar interactions were grouped into nodes to reduce complexity in interaction lines.

https://doi.org/10.1371/journal.pgen.1007158.g001

These changes do not affect the main conclusion of the article, but affect the interpretation of a small subset of D716K or E712A related mutants which are reported in the Results and Discussion. The authors have added an interpretation of transcription-related phenotypes of D716K and its relevant double mutants, and corrected an interpretation of E712A-containing double mutants in comparison to the E712A single substitution mutant. The corrected text is provided below.

Paragraphs three and four under the subheading “Functional contributions of the TL tip region” in the Results

“Functional interactions among residues can be explored by the similarity between single substitution variants and the phenotypes of double mutants. We first sought evidence that variants in potential TL interaction partners could confer similar GOF or LOF phenotypes. In the simulation, K1092 switched interaction partners between two funnel helix residues D716 and E712 [18], and other charged residues were either observed or simulated to interact with S1091, K1092 or K1093 (Fig 5A). Therefore, we constructed a panel of mutants in the residues D716, E712, R1281, E1307, and D1309 for phenotypic analyses. Notably, we observed GOF phenotypes (Mn^S and MPA^S) in E1307K but not E1307A, suggesting that E1307K gained an interfering interaction to destabilize the open TL state. In contrast, mutations in the residues E712 and D716 did not confer transcription-related phenotypes.

To further dissect functional relationships, we phenotyped double mutants from potential interaction partners, and observed a number of genetic interactions (Fig 5D, S9 Fig). First, mutations in D716 (D716A/K) suppressed the S1091C and K1093M GOF phenotypes and conferred LOF phenotype (Gal^R) when combined with other S1091 alleles (D716A/S1091A, D716A/S1091E, D716K/S1091A, D716K/S1091E), although the D716A/K or S1091A/E single substitutions did not confer strong transcription-related phenotypes (Fig 5D, S8F and S9A Figs). These allele-specific genetic interactions suggested that D716A/K and S1091A/E lost putatively redundant interactions that together conferred LOF phenotypes, and loss of D716 interaction(s) might also suppress the putatively gain of interactions in GOF mutants S1091C and K1093M. Second, mutations in E712 (E712A/E712R) did not confer transcription-related phenotypes, but suppressed the K1093M GOF phenotypes (S8F and S9B Figs). Similarly, K1092A/D single substitutions did not confer transcription-related phenotypes, but were able to suppress the E1307K GOF phenotypes (S8F and S9E Figs). This observed epistasis suggested that loss of potential interacting residues (E712 and K1092, respectively) relieved putative gain of interactions in the GOF mutants (K1093M and E1307K, respectively) (discussed above). Taken together, the observed allele-specific and epistatic interactions between TL tip and proximal residues suggest a highly complex genetic network of residues controlling TL dynamics, and illustrate how individual residues might constrain or allow diversification of the TL through evolution.”

Fourth to last sentence of the third paragraph in the Discussion

“In addition to the three previously identified mutants, we utilized a new set of TL mutants to assess genetic interactions between the TL and the funnel helix α-21, and discover epistasis between K1093M (TL) and phenotypically inert E712A/R (funnel helix), along with multiple allele specific genetic interactions (Fig 5D)”.

Supporting information