The C-terminal tails of the mitochondrial transcription factors Mtf1 and TFB2M are part of an autoinhibitory mechanism that regulates DNA binding

Urmimala Basu,Nandini Mishra,Mohammed Farooqui,Jiayu Shen,Laura C Johnson,Smita S Patel

doi:10.1074/jbc.ra120.013338

Abstract

The structurally homologous Mtf1 and TFB2M proteins serve as transcription initiation factors of mitochondrial RNA polymerases in Saccharomyces cerevisiae and humans, respectively. These transcription factors directly interact with the nontemplate strand of the transcription bubble to drive promoter melting. Given the key roles of Mtf1 and TFB2M in promoter-specific transcription initiation, it can be expected that the DNA binding activity of the mitochondrial transcription factors is regulated to prevent DNA binding at inappropriate times. However, little information is available on how mitochondrial DNA transcription is regulated. While studying C-terminal (C-tail) deletion mutants of Mtf1 and TFB2M, we stumbled upon a finding that suggested that the flexible C-tail region of these factors autoregulates their DNA binding activity. Quantitative DNA binding studies with fluorescence anisotropy-based titrations revealed that Mtf1 with an intact C-tail has no affinity for DNA but deletion of the C-tail greatly increases Mtf1's DNA binding affinity. Similar observations were made with TFB2M, although autoinhibition by the C-tail of TFB2M was not as complete as in Mtf1. Analysis of available TFB2M structures disclosed that the C-tail engages in intramolecular interactions with the DNA binding groove in the free factor, which, we propose, inhibits its DNA binding activity. Further experiments showed that RNA polymerase relieves this autoinhibition by interacting with the C-tail and engaging it in complex formation. In conclusion, our biochemical and structural analyses reveal autoinhibitory and activation mechanisms of mitochondrial transcription factors that regulate their DNA binding activities and aid in specific assembly of transcription initiation complexes.

Highlights

Mitochondrial RNA polymerases (RNAPs) are evolutionarily related to singlesubunit bacteriophage T7/T3 RNAP [1]
To catalyze promoter-specific transcription initiation, the core subunit of Saccharomyces cerevisiae mitochondrial RNAP, Rpo41, requires Mtf1 (Mitochondrial Transcription Factor 1) [2,3] and the human mitochondrial RNAP, POLRMT, requires TFB2M (Transcription Factor B2 Mitochondrial) and TFAM (Transcription Factor A Mitochondrial) [3,4,5,6,7] These factors play a key role in stabilizing the transcription initiation bubble
The binding data of Mtf1-Δ20 fit well to a hyperbola and provided a Kd of ~870 nM for the 15S NT complex. These results indicate that the presence of the full-length or even partial C-tail region inhibits the DNA binding activity of Mtf1

Summary

Introduction

Mitochondrial RNA polymerases (RNAPs) are evolutionarily related to singlesubunit bacteriophage T7/T3 RNAP [1]. During our studies of the C-terminal deletion mutants of Mtf1 [3], we stumbled upon a new finding that suggested that the flexible C-terminal tail of Mtf has an autoinhibitory function in DNA binding Both Mtf and TFB2M contain a flexible Ctail region consisting of 16 to 20 aa, which we recently showed plays a critical role in template strand alignment including supporting high affinity binding of the initiating nucleotide for efficient RNA priming reaction [3]. Based on our biochemical data and analysis of available structures, we propose a model that explains the autoinhibition and activation of DNA binding involving the C-tail of the mitochondrial transcription factors This model provides new insights into the assembly and regulation of the initiation complex

Results

Discussion

Experimental procedures