Experimental RNomics in Aquifex aeolicus: identification of small non-coding RNAs and the putative 6S RNA homolog

By an experimental RNomics approach, we have generated a cDNA library from small RNAs expressed from the genome of the hyperthermophilic bacterium Aquifex aeolicus. The library included RNAs that were antisense to mRNAs and tRNAs as well as RNAs encoded in intergenic regions. Substantial steady-state levels in A.aeolicus cells were confirmed for several of the cloned RNAs by northern blot analysis. The most abundant intergenic RNA of the library was identified as the 6S RNA homolog of A.aeolicus. Although shorter in size (150 nt) than its γ-proteobacterial homologs (∼185 nt), it is predicted to have the most stable structure among known 6S RNAs. As in the γ-proteobacteria, the A.aeolicus 6S RNA gene (ssrS) is located immediately upstream of the ygfA gene encoding a widely conserved 5-formyltetrahydrofolate cyclo-ligase. We identifed novel 6S RNA candidates within the γ-proteobacteria but were unable to identify reasonable 6S RNA candidates in other bacterial branches, utilizing mfold analyses of the region immediately upstream of ygfA combined with 6S RNA blastn searches. By RACE experiments, we mapped the major transcription initiation site of A.aeolicus 6S RNA primary transcripts, located within the pheT gene preceding ygfA, as well as three processing sites

Atomic mutagenesis of stop codon nucleotides reveals the chemical prerequisites for release factor-mediated peptide release.

Termination of protein synthesis is triggered by the recognition of a stop codon at the ribosomal A site and is mediated by class I release factors (RFs). Whereas in bacteria, RF1 and RF2 promote termination at UAA/UAG and UAA/UGA stop codons, respectively, eukaryotes only depend on one RF (eRF1) to initiate peptide release at all three stop codons. Based on several structural as well as biochemical studies, interactions between mRNA, tRNA, and rRNA have been proposed to be required for stop codon recognition. In this study, the influence of these interactions was investigated by using chemically modified stop codons. Single functional groups within stop codon nucleotides were substituted to weaken or completely eliminate specific interactions between the respective mRNA and RFs. Our findings provide detailed insight into the recognition mode of bacterial and eukaryotic RFs, thereby revealing the chemical groups of nucleotides that define the identity of stop codons and provide the means to discriminate against noncognate stop codons or UGG sense codons

Small ncRNA transcriptome analysis from Aspergillus fumigatus suggests a novel mechanism for regulation of protein synthesis

Small non-protein-coding RNAs (ncRNAs) have systematically been studied in various model organisms from Escherichia coli to Homo sapiens. Here, we analyse the small ncRNA transcriptome from the pathogenic filamentous fungus Aspergillus fumigatus. To that aim, we experimentally screened for ncRNAs, expressed under various growth conditions or during specific developmental stages, by generating a specialized cDNA library from size-selected small RNA species. Our screen revealed 30 novel ncRNA candidates from known ncRNA classes such as small nuclear RNAs (snRNAs) and C/D box-type small nucleolar RNAs (C/D box snoRNAs). Additionally, several candidates for H/ACA box snoRNAs could be predicted by a bioinformatical screen. We also identified 15 candidates for ncRNAs, which could not be assigned to any known ncRNA class. Some of these ncRNA species are developmentally regulated implying a possible novel function in A. fumigatus development. Surprisingly, in addition to full-length tRNAs, we also identified 5′- or 3′-halves of tRNAs, only, which are likely generated by tRNA cleavage within the anti-codon loop. We show that conidiation induces tRNA cleavage resulting in tRNA depletion within conidia. Since conidia represent the resting state of A. fumigatus we propose that conidial tRNA depletion might be a novel mechanism to down-regulate protein synthesis in a filamentous fungus

Chemical engineering of the peptidyl transferase center reveals an important role of the 2′-hydroxyl group of A2451

The main enzymatic reaction of the large ribosomal subunit is peptide bond formation. Ribosome crystallography showed that A2451 of 23S rRNA makes the closest approach to the attacking amino group of aminoacyl-tRNA. Mutations of A2451 had relatively small effects on transpeptidation and failed to unequivocally identify the crucial functional group(s). Here, we employed an in vitro reconstitution system for chemical engineering the peptidyl transferase center by introducing non-natural nucleosides at position A2451. This allowed us to investigate the peptidyl transfer reaction performed by a ribosome that contained a modified nucleoside at the active site. The main finding is that ribosomes carrying a 2′-deoxyribose at A2451 showed a compromised peptidyl transferase activity. In variance, adenine base modifications and even the removal of the entire nucleobase at A2451 had only little impact on peptide bond formation, as long as the 2′-hydroxyl was present. This implicates a functional or structural role of the 2′-hydroxyl group at A2451 for transpeptidation

Optimizing hot electron harvesting at planar metal–semiconductor interfaces with titanium oxynitride thin films

Understanding metal-semiconductor interfaces is critical to the advancement of photocatalysis and sub-bandgap solar energy harvesting where electrons in the metal can be excited by sub-bandgap photons and extracted into the semiconductor. In this work, we compare the electron extraction efficiency across Au/TiO2 and titanium oxynitride (TiON)/TiO2-x interfaces, where in the latter case the spontaneously forming oxide layer (TiO2-x) creates a metal-semiconductor contact. Time-resolved pump-probe spectroscopy is used to study the electron recombination rates in both cases. Unlike the nanosecond recombination lifetimes in Au/TiO2, we find a bottleneck in the electron relaxation in the TiON system, which we explain using a trap-mediated recombination model. Using this model, we investigate the tunability of the relaxation dynamics with oxygen content in the parent film. The optimized film (TiO0.5N0.5) exhibits the highest carrier extraction efficiency (NFC ≈ 2.8 × 1019 m-3), slowest trapping, and an appreciable hot electron population reaching the surface oxide (NHE ≈ 1.6 × 1018 m-3). Our results demonstrate the productive role oxygen can play in enhancing electron harvesting and prolonging electron lifetimes, providing an optimized metal-semiconductor interface using only the native oxide of titanium oxynitride

k-Space Hyperspectral Imaging by a Birefringent Common-Path Interferometer

Fourier-plane microscopy is a powerful tool for measuring the angular optical response of a plethora of materials and photonic devices. Among them, optical microcavities feature distinctive energy-momentum dispersions, crucial for a broad range of fundamental studies and applications. However, measuring the whole momentum space (k-space) with sufficient spectral resolution using standard spectroscopic techniques is challenging, requiring long and alignment-sensitive scans. Here, we introduce a k-space hyperspectral microscope, which uses a common-path birefringent interferometer to image photoluminescent organic microcavities, obtaining an angle- and wavelength-resolved view of the samples in only one measurement. The exceptional combination of angular and spectral resolution of our technique allows us to reconstruct a three-dimensional (3D) map of the cavity dispersion in the energy-momentum space, revealing the polarization-dependent behavior of the resonant cavity modes. Furthermore, we apply our technique for the characterization of a dielectric nanodisk metasurface, evidencing the angular and spectral behavior of its anapole mode. This approach is able to provide a complete optical characterization for materials and devices with nontrivial angle-/wavelength-dependent properties, fundamental for future developments in the fields of topological photonics and optical metamaterials

Seed-based IntaRNA prediction combined with GFP-reporter system identifies mRNA targets of the small RNA Yfr1

Motivation: Prochlorococcus possesses the smallest genome of all sequenced photoautotrophs. Although the number of regulatory proteins in the genome is very small, the relative number of small regulatory RNAs is comparable with that of other bacteria. The compact genome size of Prochlorococcus offers an ideal system to search for targets of small RNAs (sRNAs) and to refine existing target prediction algorithms

R2R - software to speed the depiction of aesthetic consensus RNA secondary structures

<p>Abstract</p> <p>Background</p> <p>With continuing identification of novel structured noncoding RNAs, there is an increasing need to create schematic diagrams showing the consensus features of these molecules. RNA structural diagrams are typically made either with general-purpose drawing programs like Adobe Illustrator, or with automated or interactive programs specific to RNA. Unfortunately, the use of applications like Illustrator is extremely time consuming, while existing RNA-specific programs produce figures that are useful, but usually not of the same aesthetic quality as those produced at great cost in Illustrator. Additionally, most existing RNA-specific applications are designed for drawing single RNA molecules, not consensus diagrams.</p> <p>Results</p> <p>We created R2R, a computer program that facilitates the generation of aesthetic and readable drawings of RNA consensus diagrams in a fraction of the time required with general-purpose drawing programs. Since the inference of a consensus RNA structure typically requires a multiple-sequence alignment, the R2R user annotates the alignment with commands directing the layout and annotation of the RNA. R2R creates SVG or PDF output that can be imported into Adobe Illustrator, Inkscape or CorelDRAW. R2R can be used to create consensus sequence and secondary structure models for novel RNA structures or to revise models when new representatives for known RNA classes become available. Although R2R does not currently have a graphical user interface, it has proven useful in our efforts to create 100 schematic models of distinct noncoding RNA classes.</p> <p>Conclusions</p> <p>R2R makes it possible to obtain high-quality drawings of the consensus sequence and structural models of many diverse RNA structures with a more practical amount of effort. R2R software is available at <url>http://breaker.research.yale.edu/R2R</url> and as an Additional file.</p