Sequence and transcriptional activity of the Escherichia coli K-12 chromosome region between rrnC and ilvGMEDA

Abstract

We previously identified a protein related to the expression of the ilvGMEDA cluster of Escherichia coli K-12. It was observed that this ilv-related protein was produced at higher levels in UV irradiated cells infected with lambda dilvGMEDA phage with specific ilvG mutations (ValR), compared to phage carrying the wild-type(ValS) ilvG allele. The gene encoding this protein was further localized to a region between rrnC and ilvGMEDA by analyzing restriction fragment subsets in maxicells. We have now determined the nucleotide (nt) sequence of the 3.5-kb segment between rrnC and ilvGMEDA, and two open reading frames (ORFs) are present in the region expected to contain the ilv-related gene. These ORFs predicts Mrs of 18,751 (ORFI) and 20,085 (ORFII) Da, and both ORFs have a strong probability to encode proteins based on codon frequency analysis. Maxicell analysis revealed that a 1319-bp HindIII-SmaI fragment containing ORFI encodes the ilv-related peptide. We deleted a ClaI fragment that removed a portion of ORFI encoding the C-terminal region of the peptide, and maxicell analysis revealed a decrease in the size of the protein produced in accord with the prediction. RNA slot blots and Northern blots were used to characterize transcripts encoding ORFI. A transcript initiated 112 nt from the ilvGp2 promoter, but proceeding in the opposite direction, may encode the ORFI peptide.