title: Identification of an AU-rich translational enhancer within the Escherichia coli fepB leader RNA.
abstract: The fepB gene encodes a periplasmic binding protein that is essential for the uptake of ferric enterobactin by Escherichia coli. Its transcription is regulated in response to iron levels by the Fur repressor. The fepB transcript includes a 217-nucleotide leader sequence with several features suggestive of posttranscriptional regulation. To investigate the fepB leader for its contribution to fepB expression, defined deletions and substitution mutations in the leader were characterized using fepB-phoA translational fusions. The fepB leader was found to be necessary for maximal fepB expression, primarily due to the influence of an AU-rich translational enhancer (TE) located 5' to the Shine-Dalgarno sequence. Deletions or substitutions within the TE sequence decreased fepB-phoA expression fivefold. RNase protection and in vitro transcription-translation assays demonstrated that the TE augmented translational efficiency, as well as RNA levels. Moreover, primer extension inhibition assays showed that the TE increases ribosome binding. In contrast to the enhancing effect of the TE, the natural fepB GUG start codon decreased ribosome binding and reduced fepB expression 2.5-fold compared with the results obtained with leaders bearing an AUG initiation codon. Thus, the TE-GUG organization in fepB results in an intermediate level of expression compared to the level with AUG, with or without the TE. Furthermore, we found that the TE-GUG sequence is conserved among the eight gram-negative strains examined that have fepB genes, suggesting that this organization may provide a selective advantage.
authors.affiliation: Department of Molecular Microbiology andd Immunology, University of Missouri School of Medicine, Columbia, Missouri 65212, USA. indiah@intra.niddk.nih.gov
authors.forename: India G
authors.initials: IG
authors.lastname: Hook-Barnard
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Brickman
authors.forename: Mark A
authors.initials: MA
authors.lastname: McIntosh
mesh_terms: D001482:Base Composition; D001483:Base Sequence; D018387:Codon, Initiator / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational / Q000235:genetics; D033903:Periplasmic Proteins / Q000235:genetics* / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D020038:RNA, Spliced Leader / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
chemical_list: D018387:Codon, Initiator; D017931:DNA Primers; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D033903:Periplasmic Proteins; D012329:RNA, Bacterial; D020038:RNA, Spliced Leader; C061266:fepB protein, E coli