Accessorizing the human mitochondrial transcription machinery

Abstract

The human genome comprises large chromosomes in the nucleus and mitochondrial DNA (mtDNA) housed in the dynamic mitochondrial network. Human cells contain up to thousands of copies of the double-stranded, circular mtDNA molecule that encodes essential subunits of the oxidative phosphorylation complexes and the rRNAs and tRNAs needed to translate these in the organelle matrix. Transcription of human mtDNA is directed by a single-subunit RNA polymerase, POLRMT, which requires two primary transcription factors, TFB2M (transcription factor B2, mitochondrial) and TFAM (transcription factor A, mitochondrial), to achieve basal regulation of the system. Here, we review recent advances in understanding the structure and function of the primary human transcription machinery and the other factors that facilitate steps in transcription beyond initiation and provide more intricate control over the system.

Figure 1. The human mitochondrial genome

Human mtDNA is depicted with the heavy (H) strand in black and the light (L) strand in gray. The individual rRNAs (purple), mRNAs (green), and tRNAs (blue, letters represent cognate amino acids) are indicated with H-strand genes labeled outside and L-strand genes labeled inside the circle. H-strand transcription is initiated from two promoter sites, HSP1 and HSP2. HSP1 transcripts are terminated at the 22-bp termination sequence (TERM) within the tRNA-Leu(UUR) gene, where MTERF1 binds. HSP2 transcripts generate near full-length polycistronic transcripts that are then processed into the individual RNAs. L-strand transcription is initiated from a single promoter site, LSP, which also generates near full-length polycistronic messages that are processed. The primary replication origins of both the H- and L-strands are indicated (O_H and O_L, respectively). The D-loop control region, the major non-coding segment of human mtDNA, is shown expanded above. Indicated are HSP1, HSP2, and LSP, the TFAM binding sites at LSP and HSP1 (arrows indicate relative binding orientation of TFAM) within the inter-promoter region, conserved sequence blocks (CSBs I, II, III) important for proper RNA primer formation involved in H-strand replication, and the H-strand origin of replication (O_H). mtDNA: mitochondrial DNA, HSP: heavy-strand promoter, LSP: light-strand promoter, MTERF1: mitochondrial termination factor 1, TFAM: transcription factor A mitochondrial.

Figure 2. TFAM differentially regulates mtDNA promoters and packages mtDNA

TFAM is a multi-functional protein. (A) TFAM activation of transcription at the LSP is shown. TFAM, via its C-terminal tail that interacts with TFB2M (green) and unique DNA-bending capacity, promotes high levels of specific initiation by POLRMT (blue) and TFB2M. (B) TFAM can also inhibit transcription at HSP2 in vitro at concentrations that activate LSP and HSP1. It is postulated that this is due to a unique binding mode at this site that competitively inhibits promoter binding by POLRMT and TFB2M. C) In addition to its role in transcription, TFAM also packages mtDNA to facilitate nucleoid formation. This is accomplished by its ability to bind many sites on mtDNA in a more or less nonspecific manner and bend the DNA, albeit to a lesser degree than promoter DNA. TFAM is phosphorylated at sites within the HMG-box domains (denoted as round circles with a “P”), which reduces DNA binding and promotes its degradation by Lon protease. This may allow dynamic remodeling of nucleoids to achieve specific outcomes (e.g. to relieve inhibition of HSP2 transcription). TFAM: transcription factor A mitochondrial, TFB2M: transcription factor B2 mitochondrial, POLRMT: mitochondrial RNA polymerase, LSP: light-strand promoter, HSP: heavy-strand promoter, mtDNA: mitochondrial DNA, HMG box: high-mobility-group box.

Figure 3. Mitochondrial transcription accessory factors

A portion of the human mtDNA with salient cis-acting transcriptional regulatory elements (LSP, HSP1, HSP2, and TERM) is shown at the top. Accessory factors discussed in the main text that have been implicated in human mitochondrial transcriptional regulation are depicted as colored ovals, with stimulatory (arrows) or inhibitory (“T”) functions indicated. Known interactions with POLRMT are shown for MRPL12, LRPPRC and TEFM. That it is currently unknown if or how MTERF1-3 interact with the primary mitochondrial transcription machinery (POLRMT, TFB2M and TFAM) is indicated by the question mark. As indicated, MTERF1, 2 and 3 have been shown to interact in a complex. MTERF1 is shown bound to the TERM site, where it facilitates transcription termination. mtDNA: mitochondrial DNA, LSP: light-strand promoter, HSP: heavy-strand promoter, TERM: termination sequence, POLRMT: mitochondrial RNA polymerase, TFB2M: transcription factor B2 mitochondrial, TFAM: transcription factor A mitochondrial, MTERF: mitochondrial termination factor, LRPPRC: leucine-rich pentatricopeptide repeat containing, MRPL12: mitochondrial ribosomal protein L12, TEFM: transcription elongation factor mitochondrial.