MOTIVATION: Epistasis, the context-dependence of the contribution of an amino acid substitution to fitness, is common in evolution. To detect epistasis, fitness must be measured for at least four genotypes: the reference genotype, two different single mutants and a double mutant with both of the single mutations. For higher-order epistasis of the order n, fitness has to be measured for all 2n genotypes of an n-dimensional hypercube in genotype space forming a "combinatorially complete dataset". So far, only a handful of such datasets have been produced by manual curation. Concurrently, random mutagenesis experiments have produced measurements of fitness and other phenotypes in a high-throughput manner, potentially containing a number of combinatorially complete datasets. RESULTS: We present an effective recursive algorithm for finding all hypercube structures in random mutagenesis experimental data. To test the algorithm, we applied it to the data from a recent HIS3 protein dataset and found all 199,847,053 unique combinatorially complete genotype combinations of dimensionality ranging from two to twelve. The algorithm may be useful for researchers looking for higher-order epistasis in their high-throughput experimental data. AVAILABILITY: https://github.com/ivankovlab/HypercubeME.git. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Motivation: Computational prediction of the effect of mutations on protein stability is used by researchers in many fields. The utility of the prediction methods is affected by their accuracy and bias. Bias, a systematic shift of the predicted change of stability, has been noted as an issue for several methods, but has not been investigated systematically. Presence of the bias may lead to misleading results especially when exploring the effects of combination of different mutations. Results: Here we use a protocol to measure the bias as a function of the number of introduced mutations. It is based on a self-consistency test of the reciprocity the effect of a mutation. An advantage of the used approach is that it relies solely on crystal structures without experimentally measured stability values. We applied the protocol to four popular algorithms predicting change of protein stability upon mutation, FoldX, Eris, Rosetta and I-Mutant, and found an inherent bias. For one program, FoldX, we manage to substantially reduce the bias using additional relaxation by Modeller. Authors using algorithms for predicting effects of mutations should be aware of the bias described here. Availability and implementation: All calculations were implemented by in-house PERL scripts. Supplementary information: Supplementary data are available at Bioinformatics online. Note: The article 10.1093/bioinformatics/bty348, published alongside this paper, also addresses the problem of biases in protein stability change predictions.

BACKGROUND: Mitochondrial tRNAs have been the subject of study for structural biologists interested in their secondary structure characteristics, evolutionary biologists have researched patterns of compensatory and structural evolution and medical studies have been directed towards understanding the basis of human disease. However, an up to date, manually curated database of mitochondrially encoded tRNAs from higher animals is currently not available. DESCRIPTION: We obtained the complete mitochondrial sequence for 277 tetrapod species from GenBank and re-annotated all of the tRNAs based on a multiple alignment of each tRNA gene and secondary structure prediction made independently for each tRNA. The mitochondrial (mt) tRNA sequences and the secondary structure based multiple alignments are freely available as Supplemental Information online. CONCLUSION: We compiled a manually curated database of mitochondrially encoded tRNAs from tetrapods with completely sequenced genomes. In the course of our work, we reannotated more than 10% of all tetrapod mt-tRNAs and subsequently predicted the secondary structures of 6060 mitochondrial tRNAs. This carefully constructed database can be utilized to enhance our knowledge in several different fields including the evolution of mt-tRNA secondary structure and prediction of pathogenic mt-tRNA mutations. In addition, researchers reporting novel mitochondrial genome sequences should check their tRNA gene annotations against our database to ensure a higher level of fidelity of their annotation.

MOTIVATION: The context of the start codon (typically, AUG) and the features of the 5' Untranslated Regions (5' UTRs) are important for understanding translation regulation in eukaryotic mRNAs and for accurate prediction of the coding region in genomic and cDNA sequences. The presence of AUG triplets in 5' UTRs (upstream AUGs) might effect the initiation rate and, in the context of gene prediction, could reduce the accuracy of the identification of the authentic start. To reveal potential connections between the presence of upstream AUGs and other features of 5' UTRs, such as their length and the start codon context, we undertook a systematic analysis of the available eukaryotic 5' UTR sequences. RESULTS: We show that a large fraction of 5' UTRs in the available cDNA sequences, 15-53% depending on the organism, contain upstream ATGs. A negative correlation was observed between the information content of the translation start signal and the length of the 5' UTR. Similarly, a negative correlation exists between the 'strength' of the start context and the number of upstream ATGs. Typically, cDNAs containing long 5' UTRs with multiple upstream ATGs have a 'weak' start context, and in contrast, cDNAs containing short 5' UTRs without ATGs have 'strong' starts. These counter-intuitive results may be interpreted in terms of upstream AUGs having an important role in the regulation of translation efficiency by ensuring low basal translation level via double negative control and creating the potential for additional regulatory mechanisms. One of such mechanisms, supported by experimental studies of some mRNAs, includes removal of the AUG-containing portion of the 5' UTR by alternative splicing. AVAILABILITY: An ATG_ EVALUATOR program is available upon request or at www.itba.mi.cnr.it/webgene. CONTACT: [email protected], [email protected].