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Journal of Bacteriology, June 2007, p. 4028-4037, Vol. 189, No. 11
0021-9193/07/$08.00+0     doi:10.1128/JB.01924-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of an AU-rich Translational Enhancer within the Escherichia coli fepB Leader RNA{triangledown}

India G. Hook-Barnard,* Timothy J. Brickman,{dagger} and Mark A. McIntosh

Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, Missouri 65212

Received 20 December 2006/ Accepted 22 March 2007

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.

* Corresponding author. Present address: National Institutes of Health, Bldg. 8, Room 2A-24, Bethesda, MD 20892-0830. Phone: (301) 435-3374. Fax: (301) 402-0053. E-mail: indiah{at}intra.niddk.nih.gov

{triangledown} Published ahead of print on 30 March 2007.

{dagger} Present address: Department of Microbiology, University of Minnesota, MMC 196 Mayo, 420 Delaware St. SE, Minneapolis, MN 55455-0312.

Journal of Bacteriology, June 2007, p. 4028-4037, Vol. 189, No. 11
0021-9193/07/$08.00+0     doi:10.1128/JB.01924-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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