Water vapour in the atmosphere of a transiting extrasolar planet

Water is predicted to be among, if not the most abundant molecular species after hydrogen in the atmospheres of close-in extrasolar giant planets (hot-Jupiters) Several attempts have been made to detect water on an exoplanet, but have failed to find compelling evidence for it or led to claims that should be taken with caution. Here we report an analysis of recent observations of the hot-Jupiter HD189733b taken during the transit, where the planet passed in front of its parent star. We find that absorption by water vapour is the most likely cause of the wavelength-dependent variations in the effective radius of the planet at the infrared wavelengths 3.6, 5.8 and 8 microns. The larger effective radius observed at visible wavelengths may be due to either star variability or the presence of clouds/hazes. We explain the most recent thermal infrared observations of the planet during secondary transit behind the star, reporting a non-detection of water on HD189733b, as being a consequence of the nearly isothermal vertical profile of the planet.s atmosphere. Our results show that water is detectable on extrasolar planets using the primary transit technique and that the infrared should be a better wavelength region than the visible, for such searches

Dissection of Pol II Trigger Loop Function and Pol II Activity–Dependent Control of Start Site Selection In Vivo

Structural and biochemical studies have revealed the importance of a conserved, mobile domain of RNA Polymerase II (Pol II), the Trigger Loop (TL), in substrate selection and catalysis. The relative contributions of different residues within the TL to Pol II function and how Pol II activity defects correlate with gene expression alteration in vivo are unknown. Using Saccharomyces cerevisiae Pol II as a model, we uncover complex genetic relationships between mutated TL residues by combinatorial analysis of multiply substituted TL variants. We show that in vitro biochemical activity is highly predictive of in vivo transcription phenotypes, suggesting direct relationships between phenotypes and Pol II activity. Interestingly, while multiple TL residues function together to promote proper transcription, individual residues can be separated into distinct functional classes likely relevant to the TL mechanism. In vivo, Pol II activity defects disrupt regulation of the GTP-sensitive IMD2 gene, explaining sensitivities to GTP-production inhibitors, but contrasting with commonly cited models for this sensitivity in the literature. Our data provide support for an existing model whereby Pol II transcriptional activity provides a proxy for direct sensing of NTP levels in vivo leading to IMD2 activation. Finally, we connect Pol II activity to transcription start site selection in vivo, implicating the Pol II active site and transcription itself as a driver for start site scanning, contravening current models for this process

Structure of human mitochondrial RNA polymerase

Transcription of the mitochondrial genome is performed by a single-subunit RNA polymerase (mtRNAP) that is distantly related to the RNAP of bacteriophage T7, the pol I family of DNA polymerases, and single-subunit RNAPs from chloroplasts1, 2, 3, 4. Whereas T7 RNAP can initiate transcription by itself, mtRNAP requires the factors TFAM and TFB2M for binding and melting promoter DNA5, 6, 7. TFAM is an abundant protein that binds and bends promoter DNA 15–40 base pairs upstream of the transcription start site, and stimulates the recruitment of mtRNAP and TFB2M to the promoter8, 9. TFB2M assists mtRNAP in promoter melting and reaches the active site of mtRNAP to interact with the first base pair of the RNA–DNA hybrid10. Here we report the X-ray structure of human mtRNAP at 2.5 Å resolution, which reveals a T7-like catalytic carboxy-terminal domain, an amino-terminal domain that remotely resembles the T7 promoter-binding domain, a novel pentatricopeptide repeat domain, and a flexible N-terminal extension. The pentatricopeptide repeat domain sequesters an AT-rich recognition loop, which binds promoter DNA in T7 RNAP, probably explaining the need for TFAM during promoter binding. Consistent with this, substitution of a conserved arginine residue in the AT-rich recognition loop, or release of this loop by deletion of the N-terminal part of mtRNAP, had no effect on transcription. The fingers domain and the intercalating hairpin, which melts DNA in phage RNAPs, are repositioned, explaining the need for TFB2M during promoter melting. Our results provide a new venue for the mechanistic analysis of mitochondrial transcription. They also indicate how an early phage-like mtRNAP lost functions in promoter binding and melting, which were provided by initiation factors in trans during evolution, to enable mitochondrial gene regulation and the adaptation of mitochondrial function to changes in the environment

Structural basis of mitochondrial transcription initiation.

Transcription in human mitochondria is driven by a single-subunit, factor-dependent RNA polymerase (mtRNAP). Despite its critical role in both expression and replication of the mitochondrial genome, transcription initiation by mtRNAP remains poorly understood. Here, we report crystal structures of human mitochondrial transcription initiation complexes assembled on both light and heavy strand promoters. The structures reveal how transcription factors TFAM and TFB2M assist mtRNAP to achieve promoter-dependent initiation. TFAM tethers the N-terminal region of mtRNAP to recruit the polymerase to the promoter whereas TFB2M induces structural changes in mtRNAP to enable promoter opening and trapping of the DNA non-template strand. Structural comparisons demonstrate that the initiation mechanism in mitochondria is distinct from that in the well-studied nuclear, bacterial, or bacteriophage transcription systems but that similarities are found on the topological and conceptual level. These results provide a framework for studying the regulation of gene expression and DNA replication in mitochondria

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 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

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

Probing conformational changes in T7 RNA polymerase during initiation and termination by using engineered disulfide linkages

During the transition from an initiation complex to an elongation complex (EC), the single-subunit bacteriophage T7 RNA polymerase (RNAP) undergoes dramatic conformational changes. To explore the significance of these changes, we constructed mutant RNAPs that are able to form disulfide bonds that limit the mobility of elements that are involved in the transition (or its reversal) and examined the effects of the crosslinks on initiation and termination. A crosslink that is specific to the initiation complex conformation blocks transcription at 5–6 nt, presumably by preventing isomerization to an EC. A crosslink that is specific to the EC conformation has relatively little effect on elongation or on termination at a class I terminator (Tφ), which involves the formation of a stable stem–loop structure in the RNA. Crosslinked ECs also pause and resume transcription normally at a class II pause site (concatamer junction) but are deficient in termination at a class II terminator (PTH, which is found in human preparathyroid hormone gene), both of which involve a specific recognition sequence. The crosslinked amino acids in the EC lie close to the upstream end of the RNA–DNA hybrid and may prevent a movement of the polymerase that would assist in displacing or releasing RNA from a relatively unstable DNA–RNA hybrid in the paused PTH complex