Mutational and biophysical analyses reveal a TFIIIC binding region in the TFIIF-related Rpc53 subunit of RNA polymerase III.

The TFIIF-like Rpc53/Rpc37 heterodimer of RNA polymerase (pol) III is involved in various stages of transcription. The C-terminal region of Rpc53 dimerizes with Rpc37 to anchor on the lobe domain of the pol III cleft. However, structural and functional features of the Rpc53 N-terminal region had not been characterized previously. Here, we conducted site-directed alanine replacement mutagenesis on the Rpc53 N-terminus, generating yeast strains that exhibited a cold-sensitive growth defect and severely compromised pol III transcriptional activity. Circular dichroism and NMR spectroscopy revealed a highly disordered 57-amino acid polypeptide in the Rpc53 N-terminus. This polypeptide is a versatile protein-binding module displaying nanomolar-level binding affinities for Rpc37 and the Tfc4 subunit of the transcription initiation factor TFIIIC. Accordingly, we denote this Rpc53 N-terminus polypeptide as the TFIIIC-binding region or CBR. Alanine replacements in the CBR significantly reduced its binding affinity for Tfc4, highlighting its functional importance to cell growth and transcription in vitro. Our study reveals the functional basis for Rpc53's CBR in assembly of the pol III transcription initiation complex.

Site-directed biochemical analyses reveal that the switchable C-terminus of Rpc31 contributes to RNA polymerase III transcription initiation.

Rpc31 is a subunit in the TFIIE-related Rpc82/34/31 heterotrimeric subcomplex of Saccharomyces cerevisiae RNA polymerase III (pol III). Structural analyses of pol III have indicated that the N-terminal region of Rpc31 anchors on Rpc82 and further interacts with the polymerase core and stalk subcomplex. However, structural and functional information for the C-terminal region of Rpc31 is sparse. We conducted a mutational analysis on Rpc31, which uncovered a functional peptide adjacent to the highly conserved Asp-Glu-rich acidic C-terminus. This C-terminal peptide region, termed 'pre-acidic', is important for optimal cell growth, tRNA synthesis, and stable association of Rpc31 in the pre-initiation complex (PIC). Our site-directed photo-cross-linking to map protein interactions within the PIC reveal that this pre-acidic region specifically targets Rpc34 during transcription initiation, but also interacts with the DNA entry surface in free pol III. Thus, we have uncovered a switchable Rpc31 C-terminal region that functions in an initiation-specific protein interaction for pol III transcription.