Small genes gene-products in Escherichia coli K-12

Forty-two protein spots of observed M-r 6-15 kDa were resolved by two-dimen sional gel electrophoresis, stained by Coomassie blue and subjected to Edma n microsequencing. All of the proteins could be related back to their encod ing open reading Frames: thereby vindicating the bioinformatic tools curren tly utilised in their identification. However; only 14/42 gene-products wer e expressed as annotated. Translation was confirmed for 14 open reading fra mes with no attributed Function (EcoGene Y-entries), while N-terminal seque nce 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 ge ne-products (AtpE, Hns, RpoZ, RplL, CspC, YccJ, YggX, YjgF, HimA, InfA, Rps Q) and a further five showed loss of a signal peptide (PspE, HdeB, HdeA, Yn fD, YkfE). Internal (Tig, AtpA, TufA) and N-terminal fragmentation (CspD, R psF, AtcU) of much larger proteins was also detected, which may have result ed from physiological or translational processes, M-r and pI isoforms were detected respectively for PtsH and GatB, each being phosphoproteins, as wel l as RplY which manifested differences with respect to predicted M-r and pI . In addition, YjgF was shown to belong to a small gene family of unknown f unction with ancient conserved regions across procaryotes and eucaryote. Yg iN was revealed to have a paralogue and orthologues in Bacillus subtilis, S ynechocystis sp., Mycobacterium tuberculosis, Neisseria gonorrhoea, and Rho dococcus erythropolis. Orthologues are also reported for YihD, YccU and Yee X. Of the 14 Y-genes, only YkfE possessed no detectable orthologues. These results highlight the need to complement genomic analysis with detailed pro teomics in order to gain a better understanding of cellular molecular biolo gy, while the confirmation of the open reading frame start codon using Edma n degradation protein microsequencing has yet to be superseded by recent ad vances in mass spectrometry. (C) 1998 Published by Elsevier Science B.V. Al l rights reserved.