Identification of prokaryotic small proteins using a comparative genomic approach

Motivation: Accurate prediction of genes encoding small proteins (on the order of 50 amino acids or less) remains an elusive open problem in bioinformatics. Some of the best methods for gene prediction use either sequence composition analysis or sequence similarity to a known protein coding sequence. These methods often fail for small proteins, however, either due to a lack of experimentally verified small protein coding genes or due to the limited statistical significance of statistics on small sequences. Our approach is based upon the hypothesis that true small proteins will be under selective pressure for encoding the particular amino acid sequence, for ease of translation by the ribosome and for structural stability. This stability can be achieved either independently or as part of a larger protein complex. Given this assumption, it follows that small proteins should display conserved local protein structure properties much like larger proteins. Our method incorporates neural-net predictions for three local structure alphabets within a comparative genomic approach using a genomic alignment of 22 closely related bacteria genomes to generate predictions for whether or not a given open reading frame (ORF) encodes for a small protein. Results: We have applied this method to the complete genome for Escherichia coli strain K12 and looked at how well our method performed on a set of 60 experimentally verified small proteins from this organism. Out of a total of 11 407 possible ORFs, we found that 6 of the top 10 and 27 of the top 100 predictions belonged to the set of 60 experimentally verified small proteins. We found 35 of all the true small proteins within the top 200 predictions. We compared our method to Glimmer, using a default Glimmer protocol and a modified small ORF Glimmer protocol with a lower minimum size cutoff. The default Glimmer protocol identified 16 of the true small proteins (all in the top 200 predictions), but failed to predict on 34 due to size cutoffs. The small ORF Glimmer protocol made predictions for all the experimentally verified small proteins but only contained 9 of the 60 true small proteins within the top 200 predictions.