Repeat-Specific Functions for the C-Terminal Domain of RNA Polymerase II in Budding Yeast

Abstract

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) is required to regulate transcription and to integrate it with other essential cellular processes. In the budding yeast Saccharomyces cerevisiae, the CTD of Rpb1p consists of 26 conserved heptad repeats that are post-translationally modified to orchestrate protein factor binding at different stages of the transcription cycle. A long-standing question in the study of the CTD is if there are any functional differences between the 26 repeats. In this study, we present evidence that repeats of identical sequence have different functions based on their position within the CTD. We assembled plasmids expressing Rpb1p with serine to alanine substitutions in three defined regions of the CTD and measured a range of phenotypes for yeast expressing these constructs. Mutations in the beginning and middle regions of the CTD had drastic, and region-specific effects, while mutating the distal region had no observable phenotype. Further mutational analysis determined that Ser5 within the first region of repeats was solely responsible for the observed growth differences and sequencing fast-growing suppressors allowed us to further define the functional regions of the CTD. This mutational analysis is consistent with current structural models for how the RNAPII holoenzyme and the CTD specifically would reside in complex with Mediator and establishes a foundation for studying regioselective binding along the repetitive RNAPII CTD.

Keywords: RNA polymerase; tandem repeat domains; transcriptional regulation.

Figure 1

Length dependence of RNAPII CTD in TET-off system. (A) CTD truncation mutants tested in this study. Each block represents a single seven amino acid heptad repeat sequence. Constructs are labeled based on the number of total CTD repeats. (B) Spotting assay measuring the dependence of CTD length on yeast viability. In the absence of doxycycline (DOX) both the genomic copy of RPB1 and the LEU2 plasmid copy of RPB1 harboring different length CTD regions are expressed. When DOX is present, only the plasmid copy is transcribed (malagon et al. 2006; morrill et al. 2016). (C) Spotting assay measuring the dependence of CTD length on yeast viability in media lacking inositol (INO).

Figure 2

Position-specific phenotypes of CTD mutants. (A) CTD mutants were created that harbored Ser>Ala substitutions at precise positions within the CTD sequence as noted by the subscripts in the name. Repeats with wildtype sequence are colored in green with mutant repeats in orange. Spotting assay measuring the dependence of CTD position on yeast viability (B) and inositol auxotrophy (C).

Figure 3

Additional phenotypes of position-specific CTD mutants. Preparation of the spotting assay and ordering of the mutants is the same as in Figure 2. CTD constructs were assayed on additional stresses including: 50 μg/mL of 6-Azauracil (6AU), plates with galactose as the only sugar (SC-GAL) and osmotic stress in the form of 1M NaCl and 1M sorbitol.

Figure 4

Effect of CTD position on INO1 expression. (A) Representative agarose gels of RT-PCR reactions using primers specific for INO1 and ACT1 as a loading control. (B) Quantification of the effects of CTD mutation on INO1 expression. Signal from agarose gels was quantified by densitometry using ImageJ and data are plotted as the ratio of the INO1 band intensity to the ACT1 band intensity. Two-way ANOVA was used to measure significance of interactions, and a subset of significant interactions are indicated as (**, adjusted P-value < 0.05; ***, adjusted P-value <0.01).

Figure 5

Influence of Ser>Ala substitutions on inositol auxotrophy in proximal CTD repeats. (A) CTD mutants expressing one or more Ser>Ala substitutions at discrete positions within repeats 2–9 of the RNAPII CTD. The position of the Ser>Ala substitution is marked in pink, noted in the name, and is carried by all 8 repeats within this region. Spotting assays measured the dependence of Ser position on yeast viability (B) and inositol auxotrophy (C).

Figure 6

Mapping functional regions of the yeast CTD. (A) Summary of plasmid-based spontaneous suppressor mutants and their growth characteristics on general growth (+DOX) and inositol deficient (–INO+DOX) media. No growth is scored as (–) with poor, moderate, and unimpaired growth scored as +, ++, and +++, respectively. Representative spotting assays are available in Figure S4 in File S1. (B) Based on the growth of different constructs in (A), Regions essential for general growth (purple), and important for growth in –INO media (blue) were mapped to the 26 repeats of the yeast CTD. Intensity of the color correlates with importance of a repeat for a particular phenotype. The scale bar represents approximate length of the CTD tail in a fully extended conformation, assuming a length of 2 Å/ amino acid for an extended peptide lacking secondary structure. (C) A model based on existing structures for the RNAPII in complex with Mediator proposing that Mediator interacts with repeats in the proximal region of the CTD, most likely repeats 5–9 (blue). This is spatially consistent with the binding of additional CTD associating factors (yellow) to the region defined as important for general viability (purple).