The N-terminal Domain of the Yeast Mitochondrial RNA Polymerase Regulates Multiple Steps of Transcription

Abstract

Transcription of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) genome is catalyzed by nuclear-encoded proteins that include the core RNA polymerase (RNAP) subunit Rpo41 and the transcription factor Mtf1. Rpo41 is homologous to the single-subunit bacteriophage T7/T3 RNAP. Its ϳ80-kDa C-terminal domain is highly conserved among mt RNAPs, but its ϳ50-kDa N-terminal domain (NTD) is less conserved and not present in T7/T3 RNAP. To understand the role of the NTD, we have biochemically characterized a series of NTD deletion mutants of Rpo41. Our studies show that NTD regulates multiple steps of transcription initiation. Interestingly, NTD functions in an autoinhibitory manner during initiation, and its partial deletion increases the efficiency of RNA synthesis. Deletion of 1-270 amino acids (DN270) reduces abortive synthesis and increases full-length to abortive RNA ratio relative to full-length (FL) Rpo41. A larger deletion of 1-380 amino acids (DN380), decreases RNA synthesis on duplex but not on premelted promoter. We show that DN380 is defective in promoter opening near the transcription start site. Most strikingly, both DN270 and DN380 catalyze highly processive RNA synthesis on the premelted promoter, and unlike the FL Rpo41, the mutants are not inhibited by Mtf1. Both mutants show weaker interactions with Mtf1, which explains many of our results, and particularly the ability of the mutants to efficiently transition from initiation to elongation. We propose that in vivo the accessory proteins that bind NTD may modulate interactions of Rpo41 with the promoter/Mtf1 to activate and allow timely release from Mtf1 for transition into elongation.The mitochondrial (mt) 2 genome of the yeast (Saccharomyces cerevisiae) is transcribed by nuclear DNA-encoded RNA polymerase (RNAP) subunits that produce ribosomal RNAs, transfer RNA, and mRNAs of the proteins of the oxidative phosphorylation machinery (1). The yeast transcription machinery consists of the core RNAP subunit, a 153-kDa protein called Rpo41, and the transcription factor, a 40-kDa protein called Mtf1 (2-5). Rpo41 is evolutionarily related to the single-subunit phage T7/T3 RNAP (6). Although the ϳ80-kDa C-terminal domain of Rpo41 is highly homologous to the phage T7/T3 RNAPs and conserved across the mitochondrial RNAPs of yeasts, plants, animals, and humans, the ϳ50-kDa N-terminal domain (NTD) is only conserved in few yeasts and absent in single subunit T7/T3 RNAPs (supplemental Table 1) (6, 7).The yeast Rpo41 NTD has been implicated previously to play a role in RNA processing and translation (8). However, whether the NTD of the Rpo41 plays a role in transcription related functions has not been determined. Deletion of 1-200 amino acids (aa) causes temperature sensitive petite phenotype in S. cerevisiae and results in mitochondrial genome instability, whereas deletions beyond 200 aa result in the RPO41 null phenotype (9). Subsequently, it was shown that the region 118 -208 NTD of the Rpo41 harbors a binding site for the Nam1p protein, which is involved in ...

Pentatricopeptide motifs in the N-terminal extension domain of yeast mitochondrial RNA polymerase Rpo41p are not essential for its function

“…The observed phenotypes, together with previously reported data obtained using large deletions in the N-terminal extension in vivo [33,44,45] and in vitro [46], also support the conclusion that this region of the protein is not involved in the main enzymatic activity.…”

“…Subsequent studies suggested that this domain couples transcription to RNA processing and translation through interactions with the Nam1 and Sls1 proteins [34,44,45]. Deletion of the first 270 amino acids of the Rpo41p NTE domain does not affect the catalytic activity of the polymerase in vitro, in fact enhancing the productive/abortive ratio of RNA synthesis, whereas a 380 aa deletion decreases initiation from duplex, but not premelted promoters [46]. …”

Conservation of Promoter Melting Mechanisms in Divergent Regions of the Single-Subunit RNA Polymerases

“…5B, lanes 6–10). The MTF1 dependent decrease in runoff transcription on pre-melted promoters has been observed previously and attributed to inhibition of promoter escape due to a too strong interaction with the melted promoter and MTF1(3, 14). It is relieved by deletions in the MtRNAP N-terminal domain that weaken the interaction with MTF1(3, 14).…”

Yeast mitochondrial RNA polymerase primes mitochondrial DNA polymerase at origins of replication and promoter sequences

“…Oris, 14S, and COX II nucleotide sequences from the −20 to the +40 position (+1 indicated the first transcribed ribonucleotide) were cloned into puC19 and used as templates (Table 1S). Transcription at 14S and COX II promoters displays a well studied abortive transcription pattern from 2 to 10 nts Paratkar et al, 2011;Velazquez et al, 2012) and a run-off transcript of 65 and 66 nts respectively (Fig. 3, lanes 1 and 2).…”