Functional annotation of human long noncoding RNAs via molecular phenotyping.

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-to-date lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.

Streptomyces rare codon UUA: from features associated with 2 adpA related locations to candidate phage regulatory translational bypassing.

In Streptomyces species, the cell cycle involves a switch from an early and vegetative state to a later phase where secondary products including antibiotics are synthesized, aerial hyphae form and sporulation occurs. AdpA, which has two domains, activates the expression of numerous genes involved in the switch from the vegetative growth phase. The adpA mRNA of many Streptomyces species has a UUA codon in a linker region between 5' sequence encoding one domain and 3' sequence encoding its other and C-terminal domain. UUA codons are exceptionally rare in Streptomyces, and its functional cognate tRNA is not present in a fully modified and acylated form, in the early and vegetative phase of the cell cycle though it is aminoacylated later. Here, we report candidate recoding signals that may influence decoding of the linker region UUA. Additionally, a short ORF 5' of the main ORF has been identified with a GUG at, or near, its 5' end and an in-frame UUA near its 3' end. The latter is commonly 5 nucleotides 5' of the main ORF start. Ribosome profiling data show translation of that 5' region. Ten years ago, UUA-mediated translational bypassing was proposed as a sensor by a Streptomyces phage of its host's cell cycle stage and an effector of its lytic/lysogeny switch. We provide the first experimental evidence supportive of this proposal.

Unraveling the initial steps of the ignition chemistry of the hypergolic ionic liquid 1-ethyl-3-methylimidazolium cyanoborohydride ([EMIM][CBH]) with nitric acid (HNO3) exploiting chirped pulse triggered droplet merging.

The composition of the products and the mechanistic routes for the reaction of the hypergolic ionic liquid (HIL) 1-ethyl-3-methylimidazolium cyanoborohydride ([EMIM][CBH]) and nitric acid (HNO3) at various concentrations from 10% to 70% were explored using a contactless single droplet merging within an ultrasonic levitation setup in an inert atmosphere of argon to reveal the initial steps that cause hypergolicity. The reactions were initiated through controlled droplet-merging manipulation triggered by a frequency chirp pulse amplitude modulation. Utilizing the high-speed optical and infrared cameras surrounding the levitation process chamber, intriguing visual images were unveiled: (i) extensive gas release and (ii) temperature rises of up to 435 K in the merged droplets. The gas development was validated qualitatively and quantitatively with Fourier Transform Infrared Spectroscopy (FTIR) indicating the major gas-phase products to be hydrogen cyanide (HCN) and nitrous oxide (N2O). The merged droplet was also probed by pulsed Raman spectroscopy which deciphered features for key functional groups of the reaction products and intermediates (-BH, -BH2, -BH3, -NCO); reaction kinetics revealed that the reaction was initiated by the interaction of the [CBH]- anion of the HIL with the oxidizer (HNO3) through proton transfer. Computations indicate the formation of a van-der-Waals complex between the [CBH]- anion and HNO3 initially, followed by proton transfer from the acid to the anion and subsequent extensive isomerization; these rearrangements were found to be essential for the formation of HCN and N2O. The exoergicity observed during the merging process provides a molar enthalpy change up to 10 kJ mol-1 to the system, which could be sufficient for a significant fraction of the reactants of about 11% to overcome the reaction barriers in the individual steps of the computationally determined minimum energy pathways.

Dramatic Conformer-Dependent Reactivity of the Acetaldehyde Oxide Criegee Intermediate with Dimethylamine Via a 1,2-Insertion Mechanism.

The reactivity of carbonyl oxides has previously been shown to exhibit strong conformer and substituent dependencies. Through a combination of synchrotron-multiplexed photoionization mass spectrometry experiments (298 K and 4 Torr) and high-level theory [CCSD(T)-F12/cc-pVTZ-F12//B2PLYP-D3/cc-pVTZ with an added CCSDT(Q) correction], we explore the conformer dependence of the reaction of acetaldehyde oxide (CH3CHOO) with dimethylamine (DMA). The experimental data support the theoretically predicted 1,2-insertion mechanism and the formation of an amine-functionalized hydroperoxide reaction product. Tunable-vacuum ultraviolet photoionization probing of anti- or anti- + syn-CH3CHOO reveals a strong conformer dependence of the title reaction. The rate coefficient of DMA with anti-CH3CHOO is predicted to exceed that for the reaction with syn-CH3CHOO by a factor of ∼34,000, which is attributed to submerged barrier (syn) versus barrierless (anti) mechanisms for energetically downhill reactions.

Two Cobalt Chelatase Subunits Can Be Generated from a Single chlD Gene via Programed Frameshifting.

Magnesium chelatase chlIDH and cobalt chelatase cobNST enzymes are required for biosynthesis of (bacterio)chlorophyll and cobalamin (vitamin B12), respectively. Each enzyme consists of large, medium, and small subunits. Structural and primary sequence similarities indicate common evolutionary origin of the corresponding subunits. It has been reported earlier that some of vitamin B12 synthesizing organisms utilized unusual cobalt chelatase enzyme consisting of a large cobalt chelatase subunit (cobN) along with a medium (chlD) and a small (chlI) subunits of magnesium chelatase. In attempt to understand the nature of this phenomenon, we analyzed >1,200 diverse genomes of cobalamin and/or chlorophyll producing prokaryotes. We found that, surprisingly, genomes of many cobalamin producers contained cobN and chlD genes only; a small subunit gene was absent. Further on, we have discovered a diverse group of chlD genes with functional programed ribosomal frameshifting signals. Given a high similarity between the small subunit and the N-terminal part of the medium subunit, we proposed that programed translational frameshifting may allow chlD mRNA to produce both subunits. Indeed, in genomes where genes for small subunits were absent, we observed statistically significant enrichment of programed frameshifting signals in chlD genes. Interestingly, the details of the frameshifting mechanisms producing small and medium subunits from a single chlD gene could be prokaryotic taxa specific. All over, this programed frameshifting phenomenon was observed to be highly conserved and present in both bacteria and archaea.

Prediction of lncRNAs and their interactions with nucleic acids: benchmarking bioinformatics tools.

The genomes of mammalian species are pervasively transcribed producing as many noncoding as protein-coding RNAs. There is a growing body of evidence supporting their functional role. Long noncoding RNA (lncRNA) can bind both nucleic acids and proteins through several mechanisms. A reliable computational prediction of the most probable mechanism of lncRNA interaction can facilitate experimental validation of its function. In this study, we benchmarked computational tools capable to discriminate lncRNA from mRNA and predict lncRNA interactions with other nucleic acids. We assessed the performance of 9 tools for distinguishing protein-coding from noncoding RNAs, as well as 19 tools for prediction of RNA-RNA and RNA-DNA interactions. Our conclusions about the considered tools were based on their performances on the entire genome/transcriptome level, as it is the most common task nowadays. We found that FEELnc and CPAT distinguish between coding and noncoding mammalian transcripts in the most accurate manner. ASSA, RIBlast and LASTAL, as well as Triplexator, turned out to be the best predictors of RNA-RNA and RNA-DNA interactions, respectively. We showed that the normalization of the predicted interaction strength to the transcript length and GC content may improve the accuracy of inferring RNA interactions. Yet, all the current tools have difficulties to make accurate predictions of short-trans RNA-RNA interactions-stretches of sparse contacts. All over, there is still room for improvement in each category, especially for predictions of RNA interactions.

Cooling of a Zero-Nuclear-Spin Molecular Ion to a Selected Rotational State.

We demonstrate rotational cooling of the silicon monoxide cation via optical pumping by a spectrally filtered broadband laser. Compared with diatomic hydrides, SiO^{+} is more challenging to cool because of its smaller rotational interval. However, the rotational level spacing and the large dipole moment of SiO^{+} allows for direct manipulation by microwaves, and the absence of hyperfine structure in its dominant isotopologue greatly reduces demands for pure quantum state preparation. These features make ^{28}Si^{16}O^{+} a good candidate for future applications such as quantum information processing. Cooling to the ground rotational state is achieved on a 100 ms timescale and attains a population of 94(3)%, with an equivalent temperature T=0.53(6) K. We also describe a novel spectral-filtering approach to cool into arbitrary rotational states and use it to demonstrate a narrow rotational population distribution (N±1) around a selected state.

Practical Guidance in Genome-Wide RNA:DNA Triple Helix Prediction.

Long noncoding RNAs (lncRNAs) play a key role in many cellular processes including chromatin regulation. To modify chromatin, lncRNAs often interact with DNA in a sequence-specific manner forming RNA:DNA triple helices. Computational tools for triple helix search do not always provide genome-wide predictions of sufficient quality. Here, we used four human lncRNAs (MEG3, DACOR1, TERC and HOTAIR) and their experimentally determined binding regions for evaluating triplex parameters that provide the highest prediction accuracy. Additionally, we combined triplex prediction with the lncRNA secondary structure and demonstrated that considering only single-stranded fragments of lncRNA can further improve DNA-RNA triplexes prediction.

Experimental and computational studies of Criegee intermediate reactions with NH3 and CH3NH2.

Ammonia and amines are emitted into the troposphere by various natural and anthropogenic sources, where they have a significant role in aerosol formation. Here, we explore the significance of their removal by reaction with Criegee intermediates, which are produced in the troposphere by ozonolysis of alkenes. Rate coefficients for the reactions of two representative Criegee intermediates, formaldehyde oxide (CH2OO) and acetone oxide ((CH3)2COO) with NH3 and CH3NH2 were measured using cavity ring-down spectroscopy. Temperature-dependent rate coefficients, k(CH2OO + NH3) = (3.1 ± 0.5) × 10-20T2 exp(1011 ± 48/T) cm3 s-1 and k(CH2OO + CH3NH2) = (5 ± 2) × 10-19T2 exp(1384 ± 96/T) cm3 s-1 were obtained in the 240 to 320 K range. Both the reactions of CH2OO were found to be independent of pressure in the 10 to 100 Torr (N2) range, and average rate coefficients k(CH2OO + NH3) = (8.4 ± 1.2) × 10-14 cm3 s-1 and k(CH2OO + CH3NH2) = (5.6 ± 0.4) × 10-12 cm3 s-1 were deduced at 293 K. An upper limit of ≤2.7 × 10-15 cm3 s-1 was estimated for the rate coefficient of the (CH3)2COO + NH3 reaction. Complementary measurements were performed with mass spectrometry using synchrotron radiation photoionization giving k(CH2OO + CH3NH2) = (4.3 ± 0.5) × 10-12 cm3 s-1 at 298 K and 4 Torr (He). Photoionization mass spectra indicated production of NH2CH2OOH and CH3N(H)CH2OOH functionalized organic hydroperoxide adducts from CH2OO + NH3 and CH2OO + CH3NH2 reactions, respectively. Ab initio calculations performed at the CCSD(T)(F12*)/cc-pVQZ-F12//CCSD(T)(F12*)/cc-pVDZ-F12 level of theory predicted pre-reactive complex formation, consistent with previous studies. Master equation simulations of the experimental data using the ab initio computed structures identified submerged barrier heights of -2.1 ± 0.1 kJ mol-1 and -22.4 ± 0.2 kJ mol-1 for the CH2OO + NH3 and CH2OO + CH3NH2 reactions, respectively. The reactions of NH3 and CH3NH2 with CH2OO are not expected to compete with its removal by reaction with (H2O)2 in the troposphere. Similarly, losses of NH3 and CH3NH2 by reaction with Criegee intermediates will be insignificant compared with reactions with OH radicals.

Sensitive Mass Spectrometer for Time-Resolved Gas-Phase Chemistry Studies at High Pressures.

We report a new experimental apparatus for direct time-resolved probing of high-pressure gas-phase chemical reactions by photoionization mass spectrometry. The apparatus uses a laser photolysis flow reactor, capable of operating at P = 0.3-100 bar and T = 300-1000 K. We initiate reactions in homogeneous gas mixtures by the photolysis of an appropriate radical precursor using laser pulses at repetition rates of 1-10 Hz. The reacting mixture is continuously sampled into a vacuum chamber, ionized by vacuum-ultraviolet (VUV) photons from laboratory-based discharge lamps or from a synchrotron beamline, and analyzed by a custom mass spectrometer. Soft near-threshold ionization by tunable synchrotron radiation enables spectroscopic quantification of many key intermediates and products of chemical reactions. A novel ionization scheme in the high-density region of the sample jet expansion increases the experimental sensitivity 100-fold, compared with the existing instruments, without compromising mass resolution. A 40 kHz pulsed reflectron time-of-flight mass spectrometer achieves simultaneous detection of all ionized species with 25 µs time resolution. We demonstrate the capability of this apparatus by probing the ethyl radical oxidation reaction using very dilute (<1012 molecules·cm-3) ethyl concentrations at pressures up to 25 bar.

To Boldly Look Where No One Has Looked Before: Identifying the Primary Photoproducts of Acetylacetone.

We investigate the gas-phase photochemistry of the enolone tautomer of acetylacetone (pentane-2,4-dione) following S2(ππ*) ← S0 excitation at λ = 266 and 248 nm, using three complementary time-resolved spectroscopic methods. Contrary to earlier reports, which claimed to study one-photon excitation of acetylacetone and found OH and CH3 as the only important gas-phase products, we detect 15 unique primary photoproducts and demonstrate that five of them, including OH and CH3, arise solely by multiphoton excitation. We assign the one-photon products to six photochemical channels and show that the most significant pathway is phototautomerization to the diketone form, which is likely an intermediate in several of the other product channels. Furthermore, we measure the equilibrium constant of the tautomerization of the enolone to diketone on S0 from 320 to 600 K and extract Δ H = 4.1 ± 0.3 kcal·mol-1 and Δ S = 6.8 ± 0.5 cal·mol-1·K-1 using a van't Hoff analysis. We correct the C-OH bond dissociation energy in acetylacetone, previously determined as 90 kcal·mol-1 by theory and experiment, to a new value of 121.7 kcal·mol-1. Our experiments and electronic structure calculations provide evidence that some of the product channels, including phototautomerization, occur on S0, while others likely occur on excited triplet surfaces. Although the large oscillator strength of the S2 ← S0 transition results from the (ππ*) excitation of the C═C-C═O backbone, similar to conjugated polyenes, the participation of triplets in the dissociation pathways of acetylacetone appears to have more in common with ketone photochemistry.

Influence of the Ether Functional Group on Ketohydroperoxide Formation in Cyclic Hydrocarbons: Tetrahydropyran and Cyclohexane.

Photolytically initiated oxidation experiments were conducted on cyclohexane and tetrahydropyran using multiplexed photoionization mass spectrometry to assess the impact of the ether functional group in the latter species on reaction mechanisms relevant to autoignition. Pseudo-first-order conditions, with [O2]0:[R•]0 > 2000, were used to ensure that R• + O2 → products were the dominant reactions. Quasi-continuous, tunable vacuum ultraviolet light from a synchrotron was employed over the range 8.0-11.0 eV to measure photoionization spectra of the products at two pressures (10 and 1520 Torr) and three temperatures (500, 600, and 700 K). Photoionization spectra of ketohydroperoxides were measured in both species and were qualitatively identical, within the limit of experimental noise, to those of analogous species formed in n-butane oxidation. However, differences were noted in the temperature dependence of ketohydroperoxide formation between the two species. Whereas the yield from cyclohexane is evident up to 700 K, ketohydroperoxides in tetrahydropyran were not detected above 650 K. The difference indicates that reaction mechanisms change due to the ether group, likely affecting the requisite •QOOH + O2 addition step. Branching fractions of nine species from tetrahydropyran were quantified with the objective of determining the role of ring-opening reactions in diminishing ketohydroperoxide. The results indicate that products formed from unimolecular decomposition of R• and •QOOH radicals via concerted C-C and C-O ß-scission are pronounced in tetrahydropyran and are insignificant in cyclohexane oxidation. The main conclusion drawn is that, under the conditions herein, ring-opening pathways reduce the already low steady-state concentration of •QOOH, which in the case of tetrahydropyran prevents •QOOH + O2 reactions necessary for ketohydroperoxide formation. Carbon balance calculations reveal that products from ring opening of both R• and •QOOH, at 700 K, account for >70% at 10 Torr and >55% at 1520 Torr. Three pathways are confirmed to contribute to the depletion of •QOOH in tetrahydropyran including (i) γ-•QOOH → pentanedial + •OH, (ii) γ-•QOOH → vinyl formate + ethene + •OH, and (iii) γ-•QOOH → 3-butenal + formaldehyde + •OH. Analogous mechanisms in cyclohexane oxidation leading to similar intermediates are compared and, on the basis of mass spectral results, confirm that no such ring-opening reactions occur. The implication from the comparison to cyclohexane is that the ether group in tetrahydropyran increases the propensity for ring-opening reactions and inhibits the formation of ketohydroperoxide isomers that precede chain-branching. On the contrary, the absence of such reactions in cyclohexane enables ketohydroperoxide formation up to 700 K and perhaps higher temperature.

Products of Criegee intermediate reactions with NO2: experimental measurements and tropospheric implications.

The reactions of Criegee intermediates with NO2 have been proposed as a potentially significant source of the important nighttime oxidant NO3, particularly in urban environments where concentrations of ozone, alkenes and NOx are high. However, previous efforts to characterize the yield of NO3 from these reactions have been inconclusive, with many studies failing to detect NO3. In the present work, the reactions of formaldehyde oxide (CH2OO) and acetaldehyde oxide (CH3CHOO) with NO2 are revisited to further explore the product formation over a pressure range of 4-40 Torr. NO3 is not observed; however, temporally resolved and [NO2]-dependent signal is observed at the mass of the Criegee-NO2 adduct for both formaldehyde- and acetaldehyde-oxide systems, and the structure of this adduct is explored through ab initio calculations. The atmospheric implications of the title reaction are investigated through global modelling.

Time-resolved measurements of product formation in the low-temperature (550-675 K) oxidation of neopentane: a probe to investigate chain-branching mechanism.

Product formation, in particular ketohydroperoxide formation and decomposition, were investigated in time-resolved, Cl-atom initiated neopentane oxidation experiments in the temperature range 550-675 K using a photoionization time-of-flight mass spectrometer. Ionization light was provided either by Advanced Light Source tunable synchrotron radiation or ∼10.2 eV fixed energy radiation from a H2-discharge lamp. Experiments were performed both at 1-2 atm pressure using a high-pressure reactor and also at ∼9 Torr pressure employing a low-pressure reactor for comparison. Because of the highly symmetric structure of neopentane, ketohydroperoxide signal can be attributed to a 3-hydroperoxy-2,2-dimethylpropanal isomer, i.e. from a γ-ketohydroperoxide (γ-KHP). The photoionization spectra of the γ-KHP measured at low- and high pressures and varying oxygen concentrations agree well with each other, further supporting they originate from the single isomer. Measurements performed in this work also suggest that the "Korcek" mechanism may play an important role in the decomposition of 3-hydroperoxy-2,2-dimethylpropanal, especially at lower temperatures. However, at higher temperatures where γ-KHP decomposition to hydroxyl radical and oxy-radical dominates, oxidation of the oxy-radical yields a new important channel leading to acetone, carbon monoxide, and OH radical. Starting from the initial neopentyl + O2 reaction, this channel releases altogether three OH radicals. A strongly temperature-dependent reaction product is observed at m/z = 100, likely attributable to 2,2-dimethylpropanedial.

Pressure-Dependent Competition among Reaction Pathways from First- and Second-O2 Additions in the Low-Temperature Oxidation of Tetrahydrofuran.

We report a combined experimental and quantum chemistry study of the initial reactions in low-temperature oxidation of tetrahydrofuran (THF). Using synchrotron-based time-resolved VUV photoionization mass spectrometry, we probe numerous transient intermediates and products at P = 10-2000 Torr and T = 400-700 K. A key reaction sequence, revealed by our experiments, is the conversion of THF-yl peroxy to hydroperoxy-THF-yl radicals (QOOH), followed by a second O2 addition and subsequent decomposition to dihydrofuranyl hydroperoxide + HO2 or to γ-butyrolactone hydroperoxide + OH. The competition between these two pathways affects the degree of radical chain-branching and is likely of central importance in modeling the autoignition of THF. We interpret our data with the aid of quantum chemical calculations of the THF-yl + O2 and QOOH + O2 potential energy surfaces. On the basis of our results, we propose a simplified THF oxidation mechanism below 700 K, which involves the competition among unimolecular decomposition and oxidation pathways of QOOH.

A Combined Experimental and Theoretical Study of the Reaction OH + 2-Butene in the 400-800 K Temperature Range.

We report a combined experimental and theoretical study of the OH + cis-2-butene and OH + trans-2-butene reactions at combustion-relevant conditions: pressures of 1-20 bar and temperatures of 400-800 K. We probe the OH radical time histories by laser-induced fluorescence and analyze these experimental measurements with aid from time-dependent master-equation calculations. Importantly, our investigation covers a temperature range where experimental data on OH + alkene chemistry in general are lacking, and interpretation of such data is challenging due to the complexity of the competing reaction pathways. Guided by theory, we unravel this complex behavior and determine the temperature- and pressure-dependent rate coefficients for the three most important OH + 2-butene reaction channels at our conditions: H abstraction, OH addition to the double bond, and back-dissociation of the OH-butene adduct.

New Insights into Low-Temperature Oxidation of Propane from Synchrotron Photoionization Mass Spectrometry and Multiscale Informatics Modeling.

Low-temperature propane oxidation was studied at P = 4 Torr and T = 530, 600, and 670 K by time-resolved multiplexed photoionization mass spectrometry (MPIMS), which probes the reactants, intermediates, and products with isomeric selectivity using tunable synchrotron vacuum UV ionizing radiation. The oxidation is initiated by pulsed laser photolysis of oxalyl chloride, (COCl)2, at 248 nm, which rapidly generates a ∼1:1 mixture of 1-propyl (n-propyl) and 2-propyl (i-propyl) radicals via the fast Cl + propane reaction. At all three temperatures, the major stable product species is propene, formed in the propyl + O2 reactions by direct HO2 elimination from both n- and i-propyl peroxy radicals. The experimentally derived propene yields relative to the initial concentration of Cl atoms are (20 ± 4)% at 530 K, (55 ± 11)% at 600 K, and (86 ± 17)% at 670 K at a reaction time of 20 ms. The lower yield of propene at low temperature reflects substantial formation of propyl peroxy radicals, which do not completely decompose on the experimental time scale. In addition, C3H6O isomers methyloxirane, oxetane, acetone, and propanal are detected as minor products. Our measured yields of oxetane and methyloxirane, which are coproducts of OH radicals, suggest a revision of the OH formation pathways in models of low-temperature propane oxidation. The experimental results are modeled and interpreted using a multiscale informatics approach, presented in detail in a separate publication (Burke, M. P.; Goldsmith, C. F.; Klippenstein, S. J.; Welz, O.; Huang H.; Antonov I. O.; Savee J. D.; Osborn D. L.; Zádor, J.; Taatjes, C. A.; Sheps, L. Multiscale Informatics for Low-Temperature Propane Oxidation: Further Complexities in Studies of Complex Reactions. J. Phys. Chem A. 2015, DOI: 10.1021/acs.jpca.5b01003). The model predicts the time profiles and yields of the experimentally observed primary products well, and shows satisfactory agreement for products formed mostly via secondary radical-radical reactions.

Multiscale Informatics for Low-Temperature Propane Oxidation: Further Complexities in Studies of Complex Reactions.

The present paper describes further development of the multiscale informatics approach to kinetic model formulation of Burke et al. (Burke, M. P.; Klippenstein, S. J.; Harding, L. B. Proc. Combust. Inst. 2013, 34, 547-555) that directly incorporates elementary kinetic theories as a means to provide reliable, physics-based extrapolation of kinetic models to unexplored conditions. Here, we extend and generalize the multiscale informatics strategy to treat systems of considerable complexity-involving multiwell reactions, potentially missing reactions, nonstatistical product branching ratios, and non-Boltzmann (i.e., nonthermal) reactant distributions. The methodology is demonstrated here for a subsystem of low-temperature propane oxidation, as a representative system for low-temperature fuel oxidation. A multiscale model is assembled and informed by a wide variety of targets that include ab initio calculations of molecular properties, rate constant measurements of isolated reactions, and complex systems measurements. Active model parameters are chosen to accommodate both "parametric" and "structural" uncertainties. Theoretical parameters (e.g., barrier heights) are included as active model parameters to account for parametric uncertainties in the theoretical treatment; experimental parameters (e.g., initial temperatures) are included to account for parametric uncertainties in the physical models of the experiments. RMG software is used to assess potential structural uncertainties due to missing reactions. Additionally, branching ratios among product channels are included as active model parameters to account for structural uncertainties related to difficulties in modeling sequences of multiple chemically activated steps. The approach is demonstrated here for interpreting time-resolved measurements of OH, HO2, n-propyl, i-propyl, propene, oxetane, and methyloxirane from photolysis-initiated low-temperature oxidation of propane at pressures from 4 to 60 Torr and temperatures from 300 to 700 K. In particular, the multiscale informed model provides a consistent quantitative explanation of both ab initio calculations and time-resolved species measurements. The present results show that interpretations of OH measurements are significantly more complicated than previously thought-in addition to barrier heights for key transition states considered previously, OH profiles also depend on additional theoretical parameters for R + O2 reactions, secondary reactions, QOOH + O2 reactions, and treatment of non-Boltzmann reaction sequences. Extraction of physically rigorous information from those measurements may require more sophisticated treatment of all of those model aspects, as well as additional experimental data under more conditions, to discriminate among possible interpretations and ensure model reliability.

GeneTack database: genes with frameshifts in prokaryotic genomes and eukaryotic mRNA sequences.

Database annotations of prokaryotic genomes and eukaryotic mRNA sequences pay relatively low attention to frame transitions that disrupt protein-coding genes. Frame transitions (frameshifts) could be caused by sequencing errors or indel mutations inside protein-coding regions. Other observed frameshifts are related to recoding events (that evolved to control expression of some genes). Earlier, we have developed an algorithm and software program GeneTack for ab initio frameshift finding in intronless genes. Here, we describe a database (freely available at http://topaz.gatech.edu/GeneTack/db.html) containing genes with frameshifts (fs-genes) predicted by GeneTack. The database includes 206 991 fs-genes from 1106 complete prokaryotic genomes and 45 295 frameshifts predicted in mRNA sequences from 100 eukaryotic genomes. The whole set of fs-genes was grouped into clusters based on sequence similarity between fs-proteins (conceptually translated fs-genes), conservation of the frameshift position and frameshift direction (-1, +1). The fs-genes can be retrieved by similarity search to a given query sequence via a web interface, by fs-gene cluster browsing, etc. Clusters of fs-genes are characterized with respect to their likely origin, such as pseudogenization, phase variation, etc. The largest clusters contain fs-genes with programed frameshifts (related to recoding events).

Identification of the nature of reading frame transitions observed in prokaryotic genomes.

Our goal was to identify evolutionary conserved frame transitions in protein coding regions and to uncover an underlying functional role of these structural aberrations. We used the ab initio frameshift prediction program, GeneTack, to detect reading frame transitions in 206 991 genes (fs-genes) from 1106 complete prokaryotic genomes. We grouped 102 731 fs-genes into 19 430 clusters based on sequence similarity between protein products (fs-proteins) as well as conservation of predicted position of the frameshift and its direction. We identified 4010 pseudogene clusters and 146 clusters of fs-genes apparently using recoding (local deviation from using standard genetic code) due to possessing specific sequence motifs near frameshift positions. Particularly interesting was finding of a novel type of organization of the dnaX gene, where recoding is required for synthesis of the longer subunit, τ. We selected 20 clusters of predicted recoding candidates and designed a series of genetic constructs with a reporter gene or affinity tag whose expression would require a frameshift event. Expression of the constructs in Escherichia coli demonstrated enrichment of the set of candidates with sequences that trigger genuine programmed ribosomal frameshifting; we have experimentally confirmed four new families of programmed frameshifts.