Replication-Transciption Switch in Human Mitochondria

Coordinated replication and expression of mitochondrial genome is critical for metabolically active cells during various stages of development. However, it is not known whether replication and transcription can occur simultaneously without interfering with each other and whether mtDNA copy number can be regulated by the transcription machinery. Human mitochondrial RNA polymerase (mtRNAP) is a central enzyme involved in gene expression in mitochondria. It generates genome-size polycistronic transcripts and also makes replication primers at two origins of replication. MtRNAP is distantly related to phage T7 RNAP. While T7 RNAP is optimized to produce large amounts of transcripts to overcompete the bacterial RNAP, mtRNAP must coordinate RNA synthesis with processing and translation. We hypothesized that mtRNAP must be slower than T7 RNAP and measured elongation rates for these RNAPs. We found that mtRNAP is about 20 times slower than T7 RNAP. We also found that mtRNAP is inherently non-processive and cannot synthesize long transcripts. We proposed that low processivity and slow elongation rates of mtRNAP requires assistance of an additional elongation factor. We show that interaction of a recently identified human transcription elongation factor, TEFM, with mtRNAP dramatically increases processivity and elongation rates of the mitochondrial transcription machinery. Importantly, we found that TEFM prevents premature transcription termination and thus generation of replication primers by mtRNAP. Thus, TEFM serves as a component of a molecular switch between replication and transcription, which appear to be mutually exclusive processes in mitochondria. The switch likely allows avoiding the detrimental consequences of head-on collisions between replication and transcription machineries. Regulation of TEFM may explain how mtRNAP transcription rates and, as consequence, respiration and ATP production, can be increased in mitochondria without the need to replicate mtDNA, which has been observed during different developmental processes

A novel intermediate in transcription initiation by human mitochondrial RNA polymerase

The mitochondrial genome is transcribed by a single-subunit T7 phage-like RNA polymerase (mtRNAP),structurally unrelated to cellular RNAPs. In higher eukaryotes, mtRNAP requires two transcription factors for efficient initiation-TFAM, a major nucleoid protein, and TFB2M, a transient component of mtRNAP catalytic site. The mechanisms behind assembly of the mitochondrial transcription machinery and its regulation are poorly understood. We isolated and identified a previously unknown human mitochondrial transcription intermediate-a pre-initiation complex that includes mtRNAP, TFAM and promoter DNA. Using protein-protein cross-linking, we demonstrate that human TFAM binds to the N-terminal domain of mtRNAP, which results in bending of the promoter DNA around mtRNAP. The subsequent recruitment of TFB2M induces promoter melting and formation of an open initiation complex. Our data indicate that the pre-initiation complex is likely to be an important target for transcription regulation and provide basis for further structural, biochemical and biophysical studies of mitochondrial transcription

Stringent Constraint on Galactic Positron Production

The intense 0.511 MeV gamma-ray line emission from the Galactic Center observed by INTEGRAL requires a large annihilation rate of nonrelativistic positrons. If these positrons are injected at even mildly relativistic energies, higher-energy gamma rays will also be produced. We calculate the gamma-ray spectrum due to inflight annihilation and compare to the observed diffuse Galactic gamma-ray data. Even in a simplified but conservative treatment, we find that the positron injection energies must be $\lesssim 3$ MeV, which strongly constrains models for Galactic positron production.Comment: 4 pages, 2 figures; minor revisions, accepted for publication in PR

Locating the VHE source in the Galactic Centre with milli-arcsecond accuracy

Very high-energy gamma-rays (VHE; E>100 GeV) have been detected from the direction of the Galactic Centre up to energies E>10 TeV. Up to now, the origin of this emission is unknown due to the limited positional accuracy of the observing instruments. One of the counterpart candidates is the super-massive black hole (SMBH) Sgr A*. If the VHE emission is produced within ~10^{15} cm ~1000 r_G (r_G=G M/c^2 is the Schwarzschild radius) of the SMBH, a decrease of the VHE photon flux in the energy range 100--300 GeV is expected whenever an early type or giant star approaches the line of sight within ~ milli-arcseconds (mas). The dimming of the flux is due to absorption by pair-production of the VHE photons in the soft photon field of the star, an effect we refer to as pair-production eclipse (PPE). Based upon the currently known orbits of stars in the inner arcsecond of the Galaxy we find that PPEs lead to a systematic dimming in the 100--300 GeV band at the level of a few per cent and lasts for several weeks. Since the PPE affects only a narrow energy band and is well correlated with the passage of the star, it can be clearly discriminated against other systematic or even source-intrinsic effects. While the effect is too small to be observable with the current generation of VHE detectors, upcoming high count-rate experiments like the Cherenkov telescope array (CTA) will be sufficiently sensitive. Measuring the temporal signature of the PPE bears the potential to locate the position and size of the VHE emitting region within the inner 1000 r_G or in the case of a non-detection exclude the immediate environment of the SMBH as the site of gamma-ray production altogether.Comment: 7 pages, published in MNRAS 402, pg. 1342-134

Positron annihilation as a cosmic-ray probe

Recently, the gamma-ray telescopes AGILE and Fermi observed several middle-aged supernova remnants (SNRs) interacting with molecular clouds. A plausible emission mechanism of the gamma rays is the decay of neutral pions produced by cosmic ray (CR) nuclei (hadronic processes). However, observations do not rule out contributions from bremsstrahlung emission due to CR electrons. TeV gamma-ray telescopes also observed many SNRs and discovered many unidentified sources. It is still unclear whether the TeV gamma-ray emission is produced via leptonic processes or hadronic processes. In this Letter, we propose that annihilation emission of secondary positrons produced by CR nuclei is a diagnostic tool of the hadronic processes. We investigate MeV emissions from secondary positrons and electrons produced by CR protons in molecular clouds. The annihilation emission of the secondary positrons from SNRs can be robustly estimated from the observed gamma-ray flux. The expected flux of the annihilation line from SNRs observed by AGILE and Fermi is sufficient for the future Advanced Compton Telescope to detect. Moreover, synchrotron emission from secondary positrons and electrons and bremsstrahlung emission from CR protons can be also observed by the future X-ray telescope NuSTAR and ASTRO-H.Comment: 6 pages, 4 figures, accepted for publication in the MNRAS letter

Non-thermal Processes in Black-Hole-Jet Magnetospheres

The environs of supermassive black holes are among the universe's most extreme phenomena. Understanding the physical processes occurring in the vicinity of black holes may provide the key to answer a number of fundamental astrophysical questions including the detectability of strong gravity effects, the formation and propagation of relativistic jets, the origin of the highest energy gamma-rays and cosmic-rays, and the nature and evolution of the central engine in Active Galactic Nuclei (AGN). As a step towards this direction, this paper reviews some of the progress achieved in the field based on observations in the very high energy domain. It particularly focuses on non-thermal particle acceleration and emission processes that may occur in the rotating magnetospheres originating from accreting, supermassive black hole systems. Topics covered include direct electric field acceleration in the black hole's magnetosphere, ultra-high energy cosmic ray production, Blandford-Znajek mechanism, centrifugal acceleration and magnetic reconnection, along with the relevant efficiency constraints imposed by interactions with matter, radiation and fields. By way of application, a detailed discussion of well-known sources (Sgr A*; Cen A; M87; NGC1399) is presented.Comment: invited review for International Journal of Modern Physics D, 49 pages, 15 figures; minor typos corrected to match published versio

Mechanism of Transcription Anti-termination in Human Mitochondria.

In human mitochondria, transcription termination events at a G-quadruplex region near the replication origin are thought to drive replication of mtDNA by generation of an RNA primer. This process is suppressed by a key regulator of mtDNA-the transcription factor TEFM. We determined the structure of an anti-termination complex in which TEFM is bound to transcribing mtRNAP. The structure reveals interactions of the dimeric pseudonuclease core of TEFM with mobile structural elements in mtRNAP and the nucleic acid components of the elongation complex (EC). Binding of TEFM to the DNA forms a downstream sliding clamp, providing high processivity to the EC. TEFM also binds near the RNA exit channel to prevent formation of the RNA G-quadruplex structure required for termination and thus synthesis of the replication primer. Our data provide insights into target specificity of TEFM and mechanisms by which it regulates the switch between transcription and replication of mtDNA

The Star-Forming Galaxy Contribution to the Cosmic MeV and GeV Gamma-Ray Background

While star-forming galaxies could be major contributors to the cosmic GeV $\gamma$-ray background, they are expected to be MeV-dim because of the "pion bump" falling off below ~100 MeV. However, there are very few observations of galaxies in the MeV range, and other emission processes could be present. We investigate the MeV background from star-forming galaxies by running one-zone models of cosmic ray populations, including Inverse Compton and bremsstrahlung, as well as nuclear lines (including $^{26}$Al), emission from core-collapse supernovae, and positron annihilation emission, in addition to the pionic emission. We use the Milky Way and M82 as templates of normal and starburst galaxies, and compare our models to radio and GeV--TeV $\gamma$-ray data. We find that (1) higher gas densities in high-z normal galaxies lead to a strong pion bump, (2) starbursts may have significant MeV emission if their magnetic field strengths are low, and (3) cascades can contribute to the MeV emission of starbursts if they emit mainly hadronic $\gamma$-rays. Our fiducial model predicts that most of the unresolved GeV background is from star-forming galaxies, but this prediction is uncertain by an order of magnitude. About ~2% of the claimed 1 MeV background is diffuse emission from star-forming galaxies; we place a firm upper limit of <~10% based on the spectral shape of the background. The star-formation contribution is constrained to be small, because its spectrum is peaked, while the observed background is steeply falling with energy through the MeV-GeV range.Comment: Published in ApJ, 27 pages, emulateapj format. Readers may be interested in the concurrent paper by Chakraborty and Fields (arXiv:1206.0770), a calculation of the Inverse Compton background from star-forming galaxie

A model for transcription initiation in human mitochondria.

Regulation of transcription of mtDNA is thought to be crucial for maintenance of redox potential and vitality of the cell but is poorly understood at the molecular level. In this study we mapped the binding sites of the core transcription initiation factors TFAM and TFB2M on human mitochondrial RNA polymerase, and interactions of the latter with promoter DNA. This allowed us to construct a detailed structural model, which displays a remarkable level of interaction between the components of the initiation complex (IC). The architecture of the mitochondrial IC suggests mechanisms of promoter binding and recognition that are distinct from the mechanisms found in RNAPs operating in all domains of life, and illuminates strategies of transcription regulation developed at the very early stages of evolution of gene expression