Chris M. Brown

The identity of the base following the stop codon determines the efficiency of in vivo translational termination in Escherichia coli.

A statistical analysis of > 2000 Escherichia coli genes suggested that the base following the translational stop codon might be an important feature of the signal for termination. The strengths of each of 12 possible ‘four base stop signals’ (UAAN, UGAN and UAGN) were tested in an in vivo termination assay that measured termination efficiency by its direct competition with frameshifting. Termination efficiencies varied significantly depending on both the stop codon and the fourth base, ranging from 80 (UAAU) to 7% (UGAC). For both the UAAN and UGAN series, the fourth base hierarchy was U > G > A approximately C. UAG stop codons, which are used rarely in E. coli, showed efficiencies comparable with UAAN and UGAN, but differed in that the hierarchy of the fourth base was G > U approximately A > C. The rate of release factor selection varied 30‐fold at UGAN stop signals, and 10‐fold for both the UAAN and UAGN series; it correlated well with the frequency with which the different UAAN and UGAN signals are found at natural termination sites. The results suggest that the identity of the base following the stop codon determines the efficiency of translational termination in E. coli. They also provide a rationale for the use of the strong UAAU signal in highly expressed genes and for the occurrence of the weaker UGAC signal at several recording sites.

Visualization of RNA-protein interactions in living cells: FMRP and IMP1 interact on mRNAs

Protein expression depends significantly on the stability, translation efficiency and localization of mRNA. These qualities are largely dictated by the RNA‐binding proteins associated with an mRNA. Here, we report a method to visualize and localize RNA–protein interactions in living mammalian cells. Using this method, we found that the fragile X mental retardation protein (FMRP) isoform 18 and the human zipcode‐binding protein 1 ortholog IMP1, an RNA transport factor, were present on common mRNAs. These interactions occurred predominantly in the cytoplasm, in granular structures. In addition, FMRP and IMP1 interacted independently of RNA. Tethering of FMRP to an mRNA caused IMP1 to be recruited to the same mRNA and resulted in granule formation. The intimate association of FMRP and IMP1 suggests a link between mRNA transport and translational repression in mammalian cells.

CRISPRTarget: Bioinformatic prediction and analysis of crRNA targets

The bacterial and archaeal CRISPR/Cas adaptive immune system targets specific protospacer nucleotide sequences in invading organisms. This requires base pairing between processed CRISPR RNA and the target protospacer. For type I and II CRISPR/Cas systems, protospacer adjacent motifs (PAM) are essential for target recognition, and for type III, mismatches in the flanking sequences are important in the antiviral response. In this study, we examine the properties of each class of CRISPR. We use this information to provide a tool (CRISPRTarget) that predicts the most likely targets of CRISPR RNAs (http://bioanalysis.otago.ac.nz/CRISPRTarget). This can be used to discover targets in newly sequenced genomic or metagenomic data. To test its utility, we discover features and targets of well-characterized Streptococcus thermophilus and Sulfolobus solfataricus type II and III CRISPR/Cas systems. Finally, in Pectobacterium species, we identify new CRISPR targets and propose a model of temperate phage exposure and subsequent inhibition by the type I CRISPR/Cas systems.

Global or local? Predicting secondary structure and accessibility in mRNAs

Determining the structural properties of mRNA is key to understanding vital post-transcriptional processes. As experimental data on mRNA structure are scarce, accurate structure prediction is required to characterize RNA regulatory mechanisms. Although various structure prediction approaches are available, it is often unclear which to choose and how to set their parameters. Furthermore, no standard measure to compare predictions of local structure exists. We assessed the performance of different methods using two types of data: transcriptome-wide enzymatic probing information and a large, curated set of cis-regulatory elements. To compare the approaches, we introduced structure accuracy, a measure that is applicable to both global and local methods. Our results showed that local folding was more accurate than the classic global approach. We investigated how the locality parameters, maximum base pair span and window size, influenced the prediction performance. A span of 150 provided a reasonable balance between maximizing the number of accurately predicted base pairs, while minimizing effects of incorrect long-range predictions. We characterized the error at artificial sequence ends, which we reduced by setting the window size sufficiently greater than the maximum span. Our method, LocalFold, diminished all border effects and produced the most robust performance.

Detecting overlapping coding sequences in virus genomes

BackgroundDetecting new coding sequences (CDSs) in viral genomes can be difficult for several reasons. The typically compact genomes often contain a number of overlapping coding and non-coding functional elements, which can result in unusual patterns of codon usage; conservation between related sequences can be difficult to interpret – especially within overlapping genes; and viruses often employ non-canonical translational mechanisms – e.g. frameshifting, stop codon read-through, leaky-scanning and internal ribosome entry sites – which can conceal potentially coding open reading frames (ORFs).ResultsIn a previous paper we introduced a new statistic – MLOGD (Maximum Likelihood Overlapping Gene Detector) – for detecting and analysing overlapping CDSs. Here we present (a) an improved MLOGD statistic, (b) a greatly extended suite of software using MLOGD, (c) a database of results for 640 virus sequence alignments, and (d) a web-interface to the software and database. Tests show that, from an alignment with just 20 mutations, MLOGD can discriminate non-overlapping CDSs from non-coding ORFs with a typical accuracy of up to 98%, and can detect CDSs overlapping known CDSs with a typical accuracy of 90%. In addition, the software produces a variety of statistics and graphics, useful for analysing an input multiple sequence alignment.ConclusionMLOGD is an easy-to-use tool for virus genome annotation, detecting new CDSs – in particular overlapping or short CDSs – and for analysing overlapping CDSs following frameshift sites. The software, web-server, database and supplementary material are available at http://guinevere.otago.ac.nz/mlogd.html.

Transterm: a database to aid the analysis of regulatory sequences in mRNAs

Messenger RNAs, in addition to coding for proteins, may contain regulatory elements that affect how the protein is translated. These include protein and microRNA-binding sites. Transterm (http://mRNA.otago.ac.nz/Transterm.html) is a database of regions and elements that affect translation with two major unique components. The first is integrated results of analysis of general features that affect translation (initiation, elongation, termination) for species or strains in Genbank, processed through a standard pipeline. The second is curated descriptions of experimentally determined regulatory elements that function as translational control elements in mRNAs. Transterm focuses on protein binding sites, particularly those in 3′-untranslated regions (3′-UTR). For this release the interface has been extensively updated based on user feedback. The data is now accessible by strain rather than species, for example there are 10 Escherichia coli strains (genomes) analysed separately. In addition to providing a repository of data, the database also provides tools for users to query their own mRNA sequences. Users can search sequences for Transterm or user defined regulatory elements, including protein or miRNA targets. Transterm also provides a central core of links to related resources for complementary analyses.

The translational termination signal database

The Translational Termination Database (TransTerm) consists of the immediate context sequences around the natural termination codons from 45 organisms, and summary tables. The influence of termination codon context on their effectivness as stop signals has been widely documented. The SPECIES--TRI.DAT table shows trinucleotide stop codon usage in each organism and for comparison the occurrence of these sequences in the noncoding region. The SPECIES--TETRA.DAT table contains is a similar table of tetranucleotide stop signal usage. The database is available from EMBL.

The Genomes of Three Uneven Siblings: Footprints of the Lifestyles of Three Trichoderma Species

SUMMARY The genus Trichoderma contains fungi with high relevance for humans, with applications in enzyme production for plant cell wall degradation and use in biocontrol. Here, we provide a broad, comprehensive overview of the genomic content of these species for “hot topic” research aspects, including CAZymes, transport, transcription factors, and development, along with a detailed analysis and annotation of less-studied topics, such as signal transduction, genome integrity, chromatin, photobiology, or lipid, sulfur, and nitrogen metabolism in T. reesei, T. atroviride, and T. virens, and we open up new perspectives to those topics discussed previously. In total, we covered more than 2,000 of the predicted 9,000 to 11,000 genes of each Trichoderma species discussed, which is >20% of the respective gene content. Additionally, we considered available transcriptome data for the annotated genes. Highlights of our analyses include overall carbohydrate cleavage preferences due to the different genomic contents and regulation of the respective genes. We found light regulation of many sulfur metabolic genes. Additionally, a new Golgi 1,2-mannosidase likely involved in N-linked glycosylation was detected, as were indications for the ability of Trichoderma spp. to generate hybrid galactose-containing N-linked glycans. The genomic inventory of effector proteins revealed numerous compounds unique to Trichoderma, and these warrant further investigation. We found interesting expansions in the Trichoderma genus in several signaling pathways, such as G-protein-coupled receptors, RAS GTPases, and casein kinases. A particularly interesting feature absolutely unique to T. atroviride is the duplication of the alternative sulfur amino acid synthesis pathway.

Detecting overlapping coding sequences with pairwise alignments

MOTIVATION Overlapping gene coding sequences (CDSs) are particularly common in viruses but also occur in more complex genomes. Detecting such genes with conventional gene-finding algorithms can be difficult for several reasons. If an overlapping CDS is on the same read-strand as a known CDS, then there may not be a distinct promoter or mRNA. Furthermore, the constraints imposed by double-coding can result in atypical codon biases. However, these same constraints lead to particular mutation patterns that may be detectable in sequence alignments. RESULTS In this paper, we investigate several statistics for detecting double-coding sequences with pairwise alignments--including a new maximum-likelihood method. We also develop a model for double-coding sequence evolution. Using simulated sequences generated with the model, we characterize the distribution of each statistic as a function of sequence composition, length, divergence time and double-coding frame. Using these results, we develop several algorithms for detecting overlapping CDSs. The algorithms were tested on known overlapping CDSs and other overlapping open reading frames (ORFs) in the hepatitis B virus (HBV), Escherichia coli and Salmonella typhimurium genomes. The algorithms should prove useful for detecting novel overlapping genes--especially short coding ORFs in viruses. AVAILABILITY Programs may be obtained from the authors. SUPPLEMENTARY INFORMATION http://biochem.otago.ac.nz/double.html.

Interstitial modification of palladium nanoparticles with boron atoms as a green catalyst for selective hydrogenation

Lindlar catalysts comprising of palladium/calcium carbonate modified with lead acetate and quinoline are widely employed industrially for the partial hydrogenation of alkynes. However, their use is restricted, particularly for food, cosmetic and drug manufacture, due to the extremely toxic nature of lead, and the risk of its leaching from catalyst surface. In addition, the catalysts also exhibit poor selectivities in a number of cases. Here we report that a non-surface modification of palladium gives rise to the formation of an ultra-selective nanocatalyst. Boron atoms are found to take residence in palladium interstitial lattice sites with good chemical and thermal stability. This is favoured due to a strong host-guest electronic interaction when supported palladium nanoparticles are treated with a borane tetrahydrofuran solution. The adsorptive properties of palladium are modified by the subsurface boron atoms and display ultra-selectivity in a number of challenging alkyne hydrogenation reactions, which outclass the performance of Lindlar catalysts.