Small genes/gene-products inEscherichia coliK-12

Forty-two protein spots of observed Mr 6–15 kDa were resolved by two-dimensional gel electrophoresis, stained by Coomassie blue and subjected to Edman microsequencing. All of the proteins could be related back to their encoding open reading frames, thereby vindicating the bioinformatic tools currently utilised in their identification. However, only 14/42 gene-products were expressed as annotated. Translation was confirmed for 14 open reading frames with no attributed function (EcoGene Y-entries), while N-terminal sequence allowed the start codon to be accurately annotated for the genes yjgF, yccU, yqiC, ynfD, and yeeX. The methionine start codon was cleaved in 11 gene-products (AtpE, Hns, RpoZ, RplL, CspC, YccJ, YggX, YjgF, HimA, InfA, RpsQ) and a further five showed loss of a signal peptide (PspE, HdeB, HdeA, YnfD, YkfE). Internal (Tig, AtpA, TufA) and N-terminal fragmentation (CspD, RpsF, AtcU) of much larger proteins was also detected, which may have resulted from physiological or translational processes. Mr and pI isoforms were detected respectively for PtsH and GatB, each being phosphoproteins, as well as RplY which manifested differences with respect to predicted Mr and pI. In addition, YjgF was shown to belong to a small gene family of unknown function with ancient conserved regions across procaryotes and eucaryotes. YgiN was revealed to have a paralogue and orthologues in Bacillus subtilis, Synechocystis sp., Mycobacterium tuberculosis, Neisseria gonorrhoea, and Rhodococcus erythropolis. Orthologues are also reported for YihD, YccU and YeeX. Of the 14 Y-genes, only YkfE possessed no detectable orthologues. These results highlight the need to complement genomic analysis with detailed proteomics in order to gain a better understanding of cellular molecular biology, while the confirmation of the open reading frame start codon using Edman degradation protein microsequencing has yet to be superseded by recent advances in mass spectrometry.