Mechanisms of mitochondrial promoter recognition in humans and other mammalian species.

Recognition of mammalian mitochondrial promoters requires the concerted action of mitochondrial RNA polymerase (mtRNAP) and transcription initiation factors TFAM and TFB2M. In this work, we found that transcript slippage results in heterogeneity of the human mitochondrial transcripts in vivo and in vitro. This allowed us to correctly interpret the RNAseq data, identify the bona fide transcription start sites (TSS), and assign mitochondrial promoters for > 50% of mammalian species and some other vertebrates. The divergent structure of the mammalian promoters reveals previously unappreciated aspects of mtDNA evolution. The correct assignment of TSS also enabled us to establish the precise register of the DNA in the initiation complex and permitted investigation of the sequence-specific protein-DNA interactions. We determined the molecular basis of promoter recognition by mtRNAP and TFB2M, which cooperatively recognize bases near TSS in a species-specific manner. Our findings reveal a role of mitochondrial transcription machinery in mitonuclear coevolution and speciation.

Mechanism of transcription initiation and primer generation at the mitochondrial replication origin OriL.

The intricate process of human mtDNA replication requires the coordinated action of both transcription and replication machineries. Transcription and replication events at the lagging strand of mtDNA prompt the formation of a stem-loop structure (OriL) and the synthesis of a ∼25 nt RNA primer by mitochondrial RNA polymerase (mtRNAP). The mechanisms by which mtRNAP recognizes OriL, initiates transcription, and transfers the primer to the replisome are poorly understood. We found that transcription initiation at OriL involves slippage of the nascent transcript. The transcript slippage is essential for initiation complex stability and its ability to translocate the mitochondrial DNA polymerase gamma, PolG, which pre-binds to OriL, downstream of the replication origin thus allowing for the primer synthesis. Our data suggest the primosome assembly at OriL-a complex of mtRNAP and PolG-can efficiently generate the primer, transfer it to the replisome, and protect it from degradation by mitochondrial endonucleases.

The pentatricopeptide repeat protein Rmd9 recognizes the dodecameric element in the 3'-UTRs of yeast mitochondrial mRNAs.

Stabilization of messenger RNA is an important step in posttranscriptional gene regulation. In the nucleus and cytoplasm of eukaryotic cells it is generally achieved by 5' capping and 3' polyadenylation, whereas additional mechanisms exist in bacteria and organelles. The mitochondrial mRNAs in the yeast Saccharomyces cerevisiae comprise a dodecamer sequence element that confers RNA stability and 3'-end processing via an unknown mechanism. Here, we isolated the protein that binds the dodecamer and identified it as Rmd9, a factor that is known to stabilize yeast mitochondrial RNA. We show that Rmd9 associates with mRNA around dodecamer elements in vivo and that recombinant Rmd9 specifically binds the element in vitro. The crystal structure of Rmd9 bound to its dodecamer target reveals that Rmd9 belongs to the family of pentatricopeptide (PPR) proteins and uses a previously unobserved mode of specific RNA recognition. Rmd9 protects RNA from degradation by the mitochondrial 3'-exoribonuclease complex mtEXO in vitro, indicating that recognition and binding of the dodecamer element by Rmd9 confers stability to yeast mitochondrial mRNAs.

Yeast mitochondrial protein Pet111p binds directly to two distinct targets in COX2 mRNA, suggesting a mechanism of translational activation.

The genes in mitochondrial DNA code for essential subunits of the respiratory chain complexes. In yeast, expression of mitochondrial genes is controlled by a group of gene-specific translational activators encoded in the nucleus. These factors appear to be part of a regulatory system that enables concerted expression of the necessary genes from both nuclear and mitochondrial genomes to produce functional respiratory complexes. Many of the translational activators are believed to act on the 5'-untranslated regions of target mRNAs, but the molecular mechanisms involved in this regulation remain obscure. In this study, we used a combination of in vivo and in vitro analyses to characterize the interactions of one of these translational activators, the pentatricopeptide repeat protein Pet111p, with its presumed target, COX2 mRNA, which encodes subunit II of cytochrome c oxidase. Using photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation analysis, we found that Pet111p binds directly and specifically to a 5'-end proximal region of the COX2 transcript. Further, we applied in vitro RNase footprinting and mapped two binding targets of the protein, of which one is located in the 5'-untranslated leader and the other is within the coding sequence. Combined with the available genetic data, these results suggest a plausible mechanism of translational activation, in which binding of Pet111p may prevent inhibitory secondary structures from forming in the translation initiation region, thus rendering the mRNA available for interaction with the ribosome.

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.

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.

Mitochondrial biology. Replication-transcription switch in human mitochondria.

Coordinated replication and expression of the 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 mitochondrial DNA copy number can be regulated by the transcription machinery. We found that interaction of human transcription elongation factor TEFM with mitochondrial RNA polymerase and nascent transcript prevents the generation of replication primers and increases transcription processivity and thereby serves as a molecular switch between replication and transcription, which appear to be mutually exclusive processes in mitochondria. TEFM may allow mitochondria to increase transcription rates and, as a consequence, respiration and adenosine triphosphate production without the need to replicate mitochondrial DNA, as has been observed during spermatogenesis and the early stages of embryogenesis.

The presence of an RNA:DNA hybrid that is prone to slippage promotes termination by T7 RNA polymerase.

Intrinsic termination signals for multisubunit bacterial RNA polymerases (RNAPs) encode a GC-rich stem-loop structure followed by a polyuridine [poly(U)] tract, and it has been proposed that steric clash of the stem-loop with the exit pore of the RNAP imposes a shearing force on the RNA in the downstream RNA:DNA hybrid, resulting in misalignment of the active site. The structurally unrelated T7 RNAP terminates at a similar type of signal (TΦ), suggesting a common mechanism for termination. In the absence of a hairpin (passive conditions), T7 RNAP slips efficiently in both homopolymeric A and U tracts, and we have found that replacement of the U tract in TΦ with a slippage-prone A tract still allows efficient termination. Under passive conditions, incorporation of a single G residue following a poly(U) tract (which is the situation during termination at TΦ) results in a "locked" complex that is unable to extend the transcript. Our results support a model in which transmission of the shearing force generated by steric clash of the hairpin with the exit pore is promoted by the presence of a slippery tracts downstream, resulting in alterations in the active site and the formation of a locked complex that represents an early step in the termination pathway.

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.

Human mitochondrial transcription revisited: only TFAM and TFB2M are required for transcription of the mitochondrial genes in vitro.

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.

Multiple functions of yeast mitochondrial transcription factor Mtf1p during initiation.

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 sigma. Photocross-linking 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.

TFB2 is a transient component of the catalytic site of the human mitochondrial RNA polymerase.

Transcription in human mitochondria is carried out by a single-subunit, T7-like RNA polymerase assisted by several auxiliary factors. We demonstrate that an essential initiation factor, TFB2, forms a network of interactions with DNA near the transcription start site and facilitates promoter melting but may not be essential for promoter recognition. Unexpectedly, catalytic autolabeling reveals that TFB2 interacts with the priming substrate, suggesting that TFB2 acts as a transient component of the catalytic site of the initiation complex. Mapping of TFB2 identifies a region of its N-terminal domain that is involved in simultaneous interactions with the priming substrate and the templating (+1) DNA base. Our data indicate that the transcriptional machinery in human mitochondria has evolved into a system that combines features inherited from self-sufficient, T7-like RNA polymerase and those typically found in systems comprising cellular multi-subunit polymerases, and provide insights into the molecular mechanisms of transcription regulation in mitochondria.

Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification.

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.

Maintenance of RNA-DNA hybrid length in bacterial RNA polymerases.

During transcription elongation the nascent RNA remains base-paired to the template strand of the DNA before it is displaced and the two strands of the DNA reanneal, resulting in the formation of a transcription "bubble" of approximately 10 bp. To examine how the length of the RNA-DNA hybrid is maintained, we assembled transcription elongation complexes on synthetic nucleic acid scaffolds that mimic the situation in which transcript displacement is compromised and the polymerase synthesizes an extended hybrid. We found that in such complexes bacterial RNA polymerase exhibit an intrinsic endonucleolytic cleavage activity that restores the hybrid to its normal length. Mutations in the region of the RNA polymerase near the site of RNA-DNA separation result in altered RNA displacement and translocation functions and as a consequence in different patterns of proofreading activities. Our data corroborate structural findings concerning the elements involved in the maintenance of the length of the RNA-DNA hybrid and suggest interplay between polymerase translocation, DNA strand separation, and intrinsic endonucleolytic activity.

Phosphorylation of connexin43 induced by Src: regulation of gap junctional communication between transformed cells.

Cx43 is a widely expressed gap junction protein that mediates communication between many cell types. In general, tumor cells display less intercellular communication than their nontransformed precursors. The Src tyrosine kinase has been implicated in progression of a wide variety of cancers. Src can phosphorylate Cx43, and this event is associated with the suppression of gap junction communication. However, Src activates multiple signaling pathways that can also affect intercellular communication. For example, serine kinases including PKC and MAPK are downstream effectors of Src that can also phosphorylate Cx43 and disrupt gap junctional communication. In addition, Src can affect the expression of other proteins that may affect intercellular communication. Indeed, disruption of gap junctions by Src appears to be complex. It has become clear that Src can affect Cx43 activity by multiple mechanisms. Here, we review how Src may orchestrate events that regulate intercellular communication mediated by Cx43.

The transition to an elongation complex by T7 RNA polymerase is a multistep process.

During the transition from an initiation complex to an elongation complex (EC), T7 RNA polymerase undergoes major conformational changes that involve reorientation of a "core" subdomain as a rigid body and extensive refolding of other elements in the 266 residue N-terminal domain. The pathway and timing of these events is poorly understood. To examine this, we introduced proline residues into regions of the N-terminal domain that become alpha-helical during the reorganization and changed the charge of a key residue that interacts with the RNA:DNA hybrid 5 bp upstream of the active site in the EC but not in the initiation complex. These alterations resulted in a diminished ability to make products >5-7 nt and/or a slow transition through this point. The results indicate that the transition to an EC is a multistep process and that the movement of the core subdomain and reorganization of certain elements in the N-terminal domain commence prior to promoter release (at 8-9 nt).

Multisubunit RNA polymerases melt only a single DNA base pair downstream of the active site.

To extend the nascent transcript, RNA polymerases must melt the DNA duplex downstream from the active site to expose the next acceptor base for substrate binding and incorporation. A number of mechanisms have been proposed to account for the manner in which the correct substrate is selected, and these differ in their predictions as to how far the downstream DNA is melted. Using fluorescence quenching experiments, we provide evidence that cellular RNA polymerases from bacteria and yeast melt only one DNA base pair downstream from the active site. These data argue against a model in which multiple NTPs are lined up downstream of the active site.

Template misalignment in multisubunit RNA polymerases and transcription fidelity.

Recent work showed that the single-subunit T7 RNA polymerase (RNAP) can generate misincorporation errors by a mechanism that involves misalignment of the DNA template strand. Here, we show that the same mechanism can produce errors during transcription by the multisubunit yeast RNAP II and bacterial RNAPs. Fluorescence spectroscopy reveals a reorganization of the template strand during this process, and molecular modeling suggests an open space above the polymerase active site that could accommodate a misaligned base. Substrate competition assays indicate that template misalignment, not misincorporation, is the preferred mechanism for substitution errors by cellular RNAPs. Misalignment could account for data previously taken as evidence for additional NTP binding sites downstream of the active site. Analysis of the effects of different template topologies on misincorporation indicates that the duplex DNA immediately downstream of the active site plays an important role in transcription fidelity.

A mechanism of nucleotide misincorporation during transcription due to template-strand misalignment.

Transcription errors by T7 RNA polymerase (RNAP) may occur as the result of a mechanism in which the template base two positions downstream of the 3' end of the RNA (the TSn+1 base) is utilized during two consecutive nucleotide-addition cycles. In the first cycle, misalignment of the template strand leads to incorporation of a nucleotide that is complementary to the TSn+1 base. In the second cycle, the template is realigned and the mismatched primer is efficiently extended, resulting in a substitution error. Proper organization of the transcription bubble is required for maintaining the correct register of the DNA template, as the presence of a complementary nontemplate strand opposite the TSn+1 base suppresses template misalignment. Our findings for T7 RNAP are in contrast to related DNA polymerases of the Pol I type, which fail to extend mismatches efficiently and generate predominantly deletion errors as a result of template-strand misalignment.

Elongation complexes of Thermus thermophilus RNA polymerase that possess distinct translocation conformations.

We have characterized elongation complexes (ECs) of RNA polymerase from the extremely thermophilic bacterium, Thermus thermophilus. We found that complexes assembled on nucleic acid scaffolds are transcriptionally competent at high temperature (50-80 degrees C) and, depending upon the organization of the scaffold, possess distinct translocation conformations. ECs assembled on scaffolds with a 9 bp RNA:DNA hybrid are highly stable, resistant to pyrophosphorolysis, and are in the posttranslocated state. ECs with an RNA:DNA hybrid longer or shorter than 9 bp appear to be in a pretranslocated state, as evidenced by their sensitivity to pyrophosphorolysis, GreA-induced cleavage, and exonuclease footprinting. Both pretranslocated (8 bp RNA:DNA hybrid) and posttranslocated (9 bp RNA:DNA hybrid) complexes were crystallized in distinct crystal forms, supporting the homogeneity of the conformational states in these complexes. Crystals of a posttranslocated complex were used to collect diffraction data at atomic resolution.