Mechanism of transcription initiation by the yeast mitochondrial RNA polymerase

Abstract

Mitochondria are the major supplier of cellular energy in the form of ATP. Defects in normal ATP production due to dysfunctions in mitochondrial gene expression are responsible for many mitochondrial and aging related disorders. Mitochondria carry their own DNA genome which is transcribed by relatively simple transcriptional machinery consisting of the mitochondrial RNAP (mtRNAP) and one or more transcription factors. The mtRNAPs are remarkably similar in sequence and structure to single-subunit bacteriophage T7 RNAP but they require accessory transcription factors for promoter-specific initiation. Comparison of the mechanisms of T7 RNAP and mtRNAP provides a framework to better understand how mtRNAP and the transcription factors work together to facilitate promoter selection, DNA melting, initiating nucleotide binding, and promoter clearance. This review focuses primarily on the mechanistic characterization of transcription initiation by the yeast Saccharomyces cerevisiae mtRNAP (Rpo41) and its transcription factor (Mtf1) drawing insights from the homologous T7 and the human mitochondrial transcription systems. We discuss regulatory mechanisms of mitochondrial transcription and the idea that the mtRNAP acts as the in vivo ATP "sensor" to regulate gene expression. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

Figure 1. Comparison of single-subunit RNAP structures from phage T7 and mitochondria

(A) The crystal structure of T7 RNAP-promoter initiation complex (PDB 1QLN). (B) Modeled structure of yeast mt Rpo41 (PDB Rpo41_IC) with DNA from 1QLN [36]. (C) Crystal structure of human POLRMT without DNA (PDB 3SPA). The conserved C-terminal domains are shown in grey and the N-terminal domains in cyan, NT: non-template strand, and T: template strand. The promoter binding elements: AT-rich recognition loop (brown) and intercalating hairpin (yellow) in the N-terminal domain, and promoter specificity loop (purple) in the C-terminal domain are highlighted. Parts of the missing intercalating hairpin (aa 592-602) and specificity loop (aa 1086-1105) in human POLRMT are constructed based on structural alignment with T7 RNAP using PyMol. (D) Sequence alignment generated from structural homology of Rpo41 with T7 RNAP [36] and POLRMT with T7 RNAP [38]. Secondary-structure elements predicted using PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred) are depicted above the sequences as brown cylinders for α-helices, blue arrows for β-strands, and black lines for loops. Identical residues are denoted in pink, and the conserved ones are marked with an asterisk. Mutations in the specificity loop of Ymt Rpo41 which affect selective promoter utilization are underlined [37, 42].

Figure 2. Mtf1 with the bent/open promoter DNA in the open complex

Crystal structure of Mtf1 (grey, PDB 1I4W) positioned next to a model of bent/open promoter DNA in the open complex. Positions of Mtf1-DNA crosslinks (blue spheres) and Rpo41-DNA (yellow spheres) are shown [36]. The size of the spheres corresponds to the crosslinking efficiency. The C-terminal tail (dotted grey line) of Mtf1 is not present in the crystal structure and placed near the melted template strand in green [60]. Amino acids of Mtf1 known to result in loss of Rpo41 interactions are shown: Cluster A mutants Y42C, H44P, L53H (pink); cluster B mutants V135A, I154T, K157E (red); and cluster C mutants I221K, D225G, S218R [50, 83]. The amino acids in blue, R178, K179, H187, and R189, form a basic cleft at the two-domain interface and when mutated cause loss of activity on duplex but not premelted promoter .

Figure 3. Kinetic mechanism of promoter binding, bending, and melting

First column: Rpo41 (pink) binds the promoter DNA (green) and induces modest DNA bending. The single-molecule FRET time course (bottom panels) shows an example of the infrequent event of Rpo41-induced promoter DNA bending/opening. Second column: Rpo41-Mtf1 binds the promoter DNA more tightly and induces a sharp bend in the DNA that leads to a bent/open DNA with a much longer lifetime. The single-molecule time course shows an example of a more stable bent/open state in the presence of Mtf1. The time trace in the third column shows that the initiating ATP (red star) further stabilizes the bent/open state of the promoter bound to Rpo41-Mtf1. Third column, top panel, shows Rpo41-Mtf1 binding to the non-promoter DNA without bending it [75, 81].