Cloning and sequence analysis of the Escherichia coli 4.5 S RNA gene

Abstract

The structure of the Escherichia coli gene coding for the metabolically stable 4.5 S RNA has been determined by cloning and DNA sequence analysis. Results from Southern hybridization assays carried out prior to cloning show the 4.5 S DNA to be limited to a single locus in the E. coli K12 genome. A 5.4 X 10(3) base DNA fragment containing the 4.5 S DNA was cloned into plasmid pBR322 for restriction, hybridization and sequence analyses. Cells harboring the cloned gene overproduce the 4.5 S RNA by 15-fold under normal culturing conditions; however, no effect on growth rate is observed. DNA sequencing revealed only one copy of the 4.5 S RNA gene, with a deduced RNA sequence both longer at 114 bases and slightly different from the RNA sequence reported earlier. A promoter structure immediately preceding the structural gene shows good agreement with the prokaryotic consensus sequence at both the -35 and -10 regions. In addition, a G + C-rich sequence between the Pribnow box and the start of transcription agrees well with an apparent consensus sequence found for other stable RNA genes also under stringent control. No clearly recognizable termination signal was found immediately downstream from the 3' terminus of the 4.5 S DNA, although structural elements with that potential appear to occur. A potential coding sequence for a protein occurs about 100 bases downstream from the 4.5 S DNA, suggesting the possibility of a dual function 4.5 S RNA-mRNA transcript.