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Journal of Bacteriology, October 2001, p. 5918-5926, Vol. 183, No. 20
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.20.5918-5926.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Expression of the Bacillus subtilis trpEDCFBA Operon Is Influenced by Translational Coupling and Rho Termination Factor

Helen Yakhnin, Joshua E. Babiarz,dagger Alexander V. Yakhnin, and Paul Babitzke*

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802

Received 19 March 2001/Accepted 19 July 2001

The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation and translational control mechanisms. Both mechanisms require binding of tryptophan-activated TRAP to 11 (G/U)AG repeats in the trp leader transcript. trpE translational control involves formation of a TRAP-dependent RNA structure that sequesters the trpE Shine-Dalgarno (SD) sequence (the SD blocking hairpin). By comparing expression levels from trpE'-'lacZ translational fusions controlled by the wild-type leader or by a leader that cannot form the SD blocking hairpin, we found that translational control requires a tryptophan concentration higher than that required for transcription attenuation. We also found that inhibition of trpE translation by the SD blocking hairpin does not alter the stability of the downstream message. Since the coding sequences for trpE and trpD overlap by 29 nucleotides, we examined expression levels from trpED'-'lacZ translational fusions to determine if these two genes are translationally coupled. We found that introduction of a UAA stop codon in trpE resulted in a substantial reduction in expression. Since expression was partially restored in the presence of a tRNA suppressor, our results indicate that trpE and trpD are translationally coupled. We determined that the coupling mechanism is TRAP independent and that formation of the SD blocking hairpin regulates trpD translation via translational coupling. We also constructed a rho mutation to investigate the role of Rho-dependent termination in trp operon expression. We found that TRAP-dependent formation of the SD blocking hairpin allows Rho access to the nascent transcript, causing transcriptional polarity.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802. Phone: (814) 865-0002. Fax: (814) 863-7024. E-mail: pxb28{at}psu.edu.

dagger Present address: 6 Center Dr., MSC2790, Building 6B, Room 2B231, NICHD, Bethesda, MD 20892.

Journal of Bacteriology, October 2001, p. 5918-5926, Vol. 183, No. 20
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.20.5918-5926.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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