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.

Genome-wide analysis of the plant-specific PLATZ proteins in maize and identification of their general role in interaction with RNA polymerase III complex.

BACKGROUND: PLATZ proteins are a novel class of plant-specific zinc-dependent DNA-binding proteins that are classified as transcription factors (TFs). However, their common biochemical features and functions are poorly understood. RESULT: Here, we identified and cloned 17 PLATZ genes in the maize (Zea mays) genome. All ZmPLATZs were located in nuclei, consistent with their predicted role as TFs. However, none of ZmPLATZs was found to have intrinsic activation properties in yeast. Our recent work shows that FL3 (ZmPLATZ12) interacts with RPC53 and TFC1, two critical factors in the RNA polymerase III (RNAPIII) transcription complex. Using the yeast two-hybrid assay, we determined that seven other PLATZs interacted with both RPC53 and TFC1, whereas three had no protein-protein interaction with these two factors. The other six PLATZs interacted with either RPC53 or TFC1. These findings indicate that ZmPLATZ proteins are generally involved in the modulation of RNAPIII-mediated small non-coding RNA transcription. We also identified all of the PLATZ members in rice (Oryza sativa) and Arabidopsis thaliana and constructed a Maximum likelihood phylogenetic tree for ZmPLATZs. The resulting tree included 44 members and 5 subfamilies. CONCLUSIONS: This study provides insight into understanding of the phylogenetic relationship, protein structure, expression pattern and cellular localization of PLATZs in maize. We identified nine and thirteen ZmPLATZs that have protein-protein interaction with RPC53 and TFC1 in the current study, respectively. Overall, the characterization and functional analysis of the PLATZ family in maize will pave the way to understanding RNAPIII-mediated regulation in plant development.

A high density of cis-information terminates RNA Polymerase III on a 2-rail track.

Transcription termination delineates the 3' ends of transcripts, prevents otherwise runaway RNA polymerase (RNAP) from intruding into downstream genes and regulatory elements, and enables release of the RNAP for recycling. While other eukaryotic RNAPs require complex cis-signals and/or accessory factors to achieve these activities, RNAP III does so autonomously with high efficiency and precision at a simple oligo(dT) stretch of 5-6 bp. A basis for this high density cis-information is that both template and nontemplate strands of the RNAP III terminator carry distinct signals for different stages of termination. High-density cis-information is a feature of the RNAP III system that is also reflected by dual functionalities of the tRNA promoters as both DNA and RNA elements. We review emerging developments in RNAP III termination and single strand nontemplate DNA use by other RNAPs. Use of nontemplate signals by RNAPs and associated transcription factors may be prevalent in gene regulation.

A methods review on use of nonsense suppression to study 3' end formation and other aspects of tRNA biogenesis.

Suppressor tRNAs bear anticodon mutations that allow them to decode premature stop codons in metabolic marker gene mRNAs, that can be used as in vivo reporters of functional tRNA biogenesis. Here, we review key components of a suppressor tRNA system specific to Schizosaccharomyces pombe and its adaptations for use to study specific steps in tRNA biogenesis. Eukaryotic tRNA biogenesis begins with transcription initiation by RNA polymerase (pol) III. The nascent pre-tRNAs must undergo folding, 5' and 3' processing to remove the leader and trailer, nuclear export, and splicing if applicable, while multiple complex chemical modifications occur throughout the process. We review evidence that precursor-tRNA processing begins with transcription termination at the oligo(T) terminator element, which forms a 3' oligo(U) tract on the nascent RNA, a sequence-specific binding site for the RNA chaperone, La protein. The processing pathway bifurcates depending on a poorly understood property of pol III termination that determines the 3' oligo(U) length and therefore the affinity for La. We thus review the pol III termination process and the factors involved including advances using gene-specific random mutagenesis by dNTP analogs that identify key residues important for transcription termination in certain pol III subunits. The review ends with a 'technical approaches' section that includes a parts lists of suppressor-tRNA alleles, strains and plasmids, and graphic examples of its diverse uses.