Streptolydigin (Stl) is a potent inhibitor of bacterial RNA polymerases (RNAPs). The 2.4 A resolution structure of the Thermus thermophilus RNAP-Stl complex showed that, in full agreement with the available genetic data, the inhibitor binding site is located 20 A away from the RNAP active site and encompasses the bridge helix and the trigger loop, two elements that are considered to be crucial for RNAP catalytic center function. Structure-based biochemical experiments revealed additional determinants of Stl binding and demonstrated that Stl does not affect NTP substrate binding, DNA translocation, and phosphodiester bond formation. The RNAP-Stl complex structure, its comparison with the closely related substrate bound eukaryotic transcription elongation complexes, and biochemical analysis suggest an inhibitory mechanism in which Stl stabilizes catalytically inactive (preinsertion) substrate bound transcription intermediate, thereby blocking structural isomerization of RNAP to an active configuration. The results provide a basis for a design of new antibiotics utilizing the Stl-like mechanism.

Human mitochondrial transcription is driven by a single subunit RNA polymerase and a set of basal transcription factors. The development of a recombinant in vitro transcription system has allowed for a detailed molecular characterization of the individual components and their contribution to transcription initiation. We found that TFAM and TFB2M act synergistically and increase transcription efficiency 100 -200-fold as compared with RNA polymerase alone. Both the light-strand promoter (LSP) and the HSP1 promoters displayed maximal levels of in vitro transcription when TFAM was present in an amount equimolar to the DNA template. Importantly, we did not detect any significant transcription activity in the presence of the TFB2M paralog, TFB1M, or when templates containing the putative HSP2 promoter were used. These data confirm previous observations that TFB1M does not function as a bona fide transcription factor and raise questions as to whether HSP2 serves as a functional promoter in vivo. In addition, we did not detect transcription stimulation by the ribosomal protein MRPL12. Thus, only two essential initiation factors, TFAM and TFB2M, and two promoters, LSP and HSP1, are required to drive transcription of the mitochondrial genome.Transcription of the human mitochondrial genome is governed by a nuclear-encoded single-subunit RNA polymerase (POLRMT) that is assisted by two transcription initiation factors, TFAM and TFB2M (see Refs. 1 and 2 and references therein). POLRMT possesses promoter recognition functions but depends on TFAM and TFB2M for promoter melting (3). TFAM, a high mobility group class protein, binds to mitochondrial DNA, protects a region 14 -35 bp upstream of the lightstrand promoter (LSP) 4 transcription start site, and assists in assembly of the initiation complex by attracting POLRMT-TFB2M and/or by causing initial melting of the promoter (4). The primary role of TFB2M is to melt the promoter and to stabilize the open promoter complex by simultaneous binding of the priming substrate and the templating DNA base (5). Although the basic requirements for mitochondrial transcription have been established, a number of existing controversial observations preclude a comprehensive view of gene transcription and its regulation in mitochondria. For example, in addition to TFB2M, human mitochondria also contain a homologous factor TFB1M that was reported to stimulate transcription initiation in vitro with 10 -100-fold lower efficiency (6, 7). However, in vivo studies demonstrated that although TFB1M is an essential methyltransferase required to methylate 12 S ribosomal RNA, it plays no role in transcription (8).Another paradox in the field of mitochondrial transcription concerns the existence of an additional promoter in the heavy strand of mtDNA. Transcription initiated at the LSP results in synthesis of a single mRNA that encodes subunit 6 of the NADH dehydrogenase and eight tRNAs (9). The heavy strand of mtDNA encodes 12 polypeptides, two rRNAs, and the rest of the tRNAs; transcription of this strand...

The ECF sigma family was identified 23 years ago as a distinct group of σ70-like factors. ECF sigma factors have since emerged as a major form of bacterial signal transduction that can be grouped into over 50 phylogenetically distinct subfamilies. Advances in our understanding of these sigma factors and the signaling pathways governing their activity have elucidated conserved features as well as aspects that have evolved over time. All ECF sigma factors are predicted to share a common streamlined domain structure and mode of promoter interaction. The activity of most ECF sigma factors is controlled by an anti-sigma factor. The nature of the anti-sigma factor and the activating signaling pathways appear to be conserved within ECF families, while considerable diversity exists between different families.

Transcription of the yeast mitochondrial genome is carried out by an RNA polymerase (Rpo41p) that is related to single subunit bacteriophage RNA polymerases but requires an additional factor (Mtf1p) for initiation. In this work we show that Mtf1p is involved in multiple roles during initiation including discrimination of upstream base pairs in the promoter, initial melting of three to four base pairs around the site of transcript initiation, and suppression of nonspecific initiation. It, thus, appears that Mtf1p is functionally analogous to initiation factors of multisubunit RNA polymerases, such as . Photocrosslinking experiments reveal close proximity between Mtf1p and the promoter DNA and show that the C-terminal domain makes contacts with the template strand in the vicinity of the start site. Interestingly, Mtf1p is related to a class of RNA methyltransferases, suggesting an early evolutionary link between RNA synthesis and processing.Energy production in most eukaryotic cells depends largely upon the function of mitochondria, and a number of human diseases have been attributed to disruption of the activity of these organelles. Whereas most proteins that are essential for mitochondrial processes are encoded by nuclear genes and imported into the organelle, a subset of critical proteins is encoded by the mitochondrial genome (1). Expression of the nuclear and mitochondrial genes must, therefore, be coordinately regulated to ensure proper mitochondrial function. However, little is known about how this regulation occurs.Mitochondrial genomes are transcribed by RNA polymerases (RNAPs) 3 that are related to the single subunit RNAPs encoded by T7-like phages (2-4). Unlike T7 RNAP, however, mitochondrial RNAPs require additional factors for transcription initiation. In the yeast Saccharomyces cerevisiae the catalytic subunit of mitochondrial RNAP (Rpo41p) requires a 40-kDa protein, Mtf1p, for efficient initiation. Earlier work suggested that Mtf1p might be a functional analog of the bacterial initiation factor (5-7). For example, as is the case for , Mtf1p is required for promoter binding (8) and the formation of an open complex (9) but is lost from the complex shortly after initiation (10). However, subsequent results revealed that Mtf1p is structurally unrelated to factors (11) and that Rpo41p can initiate transcription on a pre-melted promoter in the absence of Mtf1p (12). The latter observation led to a revised view that promoter specificity determinants are intrinsic to Rpo41p and that the role of Mtf1p is limited to melting of the promoter and/or to stabilization of an open promoter complex (12).The manner in which the Rpo41p⅐Mtf1p complex recognizes the promoter and initiates transcription has not yet been determined. Because of conservation among phage and mitochondrial RNAPs (see Fig. 1), it might be anticipated that a similar mechanism of promoter recognition and melting would be employed during the formation of an open promoter complex by mitochondrial RNAP. Indeed, it has recently been shown that, lik...

The abundance of mitochondrial (mt) transcripts varies under different conditions, and is thought to depend upon rates of transcription initiation, transcription termination/attenuation and RNA processing/degradation. The requirement to maintain the balance between RNA synthesis and processing may involve coordination between these processes; however, little is known about factors that regulate the activity of mtRNA polymerase (mtRNAP). Recent attempts to identify mtRNAP–protein interactions in yeast by means of a generalized tandem affinity purification (TAP) protocol were not successful, most likely because they involved a C-terminal mtRNAP–TAP fusion (which is incompatible with mtRNAP function) and because of the use of whole-cell solubilization protocols that did not preserve the integrity of mt protein complexes. Based upon the structure of T7 RNAP (to which mtRNAPs show high sequence similarity), we identified positions in yeast mtRNAP that allow insertion of a small affinity tag, confirmed the mature N-terminus, constructed a functional N-terminal TAP–mtRNAP fusion, pulled down associated proteins, and identified them by LC–MS–MS. Among the proteins found in the pull-down were a DEAD-box protein (Mss116p) and an RNA-binding protein (Pet127p). Previous genetic experiments suggested a role for these proteins in linking transcription and RNA degradation, in that a defect in the mt degradadosome could be suppressed by overexpression of either of these proteins or, independently, by mutations in either mtRNAP or its initiation factor Mtf1p. Further, we found that Mss116p inhibits transcription by mtRNAP in vitro in a steady-state reaction. Our results support the hypothesis that Mss116p and Pet127p are involved in modulation of mtRNAP activity. Copyright © 2009 John Wiley & Sons, Ltd.