Evidence that the Tfg1/Tfg2 dimer interface of TFIIF lies near the active center of the RNA polymerase II initiation complex

Abstract

The ssu71 alleles of the TFG1 gene, which encodes the largest subunit of TFIIF, were isolated as suppressors of a TFIIB defect that affects the accuracy of transcription start site selection in the yeast Saccharomyces cerevisiae. Here we report that ssu71-1 also suppresses the cell growth and start site defects associated with an altered form of the Rpb1 subunit of RNA polymerase II (RNAP II). The ssu71-1 and ssu71-2 alleles were cloned and found to encode single amino acid replacements of glycine-363, either glycine to aspartic acid (G363D) or glycine to arginine (G363R). Two other charged replacements, G363E and G363K, were constructed by site-directed mutagenesis and suppress both TFIIB E62K and Rpb1 N445S, whereas neither G363A nor G363P exhibited any effect. G363 is phylogenetically conserved and its counterpart in human TFIIF (RAP74 G112) is located within the RAP74/RAP30 dimerization domain. We propose that the TFIIF dimerization domain is located in proximity to the B-finger of TFIIB near the active center of RNAP II where the TFIIB-TFIIF-RNAP II interface plays a key role in start site selection.

Figure 1

Depiction of the ssu71 suppressor mutations. (A) The ssu71-1 and ssu71-2 alleles encode G363D and G363R replacements, respectively. The gray box denotes the conserved sequence (residues 318–416) shown in B. (B) Sequence alignment of the conserved region of the Tfg1/RAP74 subunit of TFIIF. Hs, human; Dm, Drososphila melanogaster; Ce, Caenorhabditis elegans; At, Arabidopsis thaliana; Sp, S.pombe; and Sc, S.cerevisiae. Secondary structures from the human RAP74 protein are indicated below the sequences. The conserved G363 residue of Tfg1 lies within the β7-sheet and is denoted by the black oval. Black shading indicates identity with the human sequence; gray shading denotes similarity. This sequence was reproduced from Figure 1 of Funk et al. (33), with permission. (C) RAP74 G112 (Tfg1 G363) lies within the triple barrel structure of the RAP74–RAP30 interaction domain. RAP30 (residues 2–219) is depicted in red; RAP74 (residues 2–172) in green; residue G112 is highlighted in yellow and marked by the black arrow. This structure was created using PyMol based on the X-ray structure of human RAP74/RAP30 (34).

Figure 2

Phenotypes associated with the ssu71-1 suppressor of sua7-1 and sua8-1. The top three panels depict cell growth on YPD medium at the indicated temperatures. The bottom panels depict growth on SC medium either in the presence (+Ino) or absence (−Ino) of inositol at 30°C. The 10-fold serial dilutions of each strain were spotted onto culture plates and photographed after 2 (30°C), 3 (37°C) or 5 (16°C) days of incubation. Strains: 1, YZS14 (sua7-1 ssu71-1); 2, YDW546 (sua7-1); 3, T16 (wt); 4, YDW383 (sua8-1); and 5, YZS96 (sua8-1 ssu71-1).

Figure 3

Growth phenotypes associated with Tfg1 G363 amino acid replacements in the WT, sua7-1 and sua8-1 backgrounds. All strains (Table 1) are isogenic, differing only by the indicated sua7-1 or sua8-1 alleles and Tfg1 G363 amino acid replacements, D, E, K or R.

Figure 4

Primer extension analysis of ADH1 transcription start sites. All strains (Table 1) are identical to those defined in Figure 3. In the wild-type strain (lane 1), transcription initiates at two sites, −37 and −27 (A of ATG is denoted +1), indicated by the arrows. Start sites that are enhanced in the mutant and suppressor strains upstream of −37 and downstream of −27 are highlighted by arrow heads. The ratio indicates the amount of transcript at −37 relative to −27. Quantification was performed using software from the Alpha Imager (Alpha Innotech). All values were normalized to the −37:−27 ratio for the WT strain, which was defined as 1.0.

Figure 5

Model depicting interactions among TFIIB, TFIIF and Rpb1 at the active center of RNAP II. The sua7-1 (TFIIB E62K) and sua8-1 (Rpb1 N445S) alleles confer similar growth defects and altered patterns of transcription start site selection at ADH1. The ssu71-1 and ssu71-1 alleles of TFG1 encode charged residue replacements of Tfg1 G363, which is predicted to lie at the Tfg1–Tfg2 dimer interface of TFIIF (Figure 1). Based on suppression by Tfg1 G363 replacements of the cell growth and start site defects associated with the TFIIB E62K and Rpb1 N445S replacements (Figures 3 and 4), we place the TFIIF dimer interface at the active center (purple dot) of RNAP II. The dotted black triangle denotes genetic, and presumably physical (Discussion), interactions among Tfg1, TFIIB and Rpb1. This model is consistent with earlier protein–DNA crosslinking data showing Tfg1 and Tfg2 contact points at the transcription start site (36).