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Journal of Bacteriology, April 2000, p. 1819-1827, Vol. 182, No. 7
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

trp RNA-Binding Attenuation Protein-5' Stem-Loop RNA Interaction Is Required for Proper Transcription Attenuation Control of the Bacillus subtilis trpEDCFBA Operon

Hansen Du,dagger Alexander V. Yakhnin, Subramanian Dharmaraj,Dagger and Paul Babitzke*

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

Received 25 October 1999/Accepted 10 January 2000

The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by a novel transcription attenuation mechanism. Tryptophan-activated TRAP binds to the nascent trp leader transcript by interacting with 11 (G/U)AG repeats, 6 of which are present in an antiterminator structure. TRAP binding to these repeats prevents formation of the antiterminator, thereby promoting formation of an overlapping intrinsic terminator. A third stem-loop structure that forms at the extreme 5' end of the trp leader transcript also plays a role in the transcription attenuation mechanism. The 5' stem-loop increases the affinity of TRAP for trp leader RNA. Results from RNA structure mapping experiments demonstrate that the 5' stem-loop consists of a 3-bp lower stem, a 5-by-2 asymmetric internal loop, a 6-bp upper stem, and a hexaloop at the apex of the structure. Footprinting results indicate that TRAP interacts with the 5' stem-loop and that this interaction differs depending on the number of downstream (G/U)AG repeats present in the transcript. Expression studies with trpE'-'lacZ translational fusions demonstrate that TRAP-5' stem-loop interaction is required for proper regulation of the trp operon. 3' RNA boundary experiments indicate that the 5' structure reduces the number of (G/U)AG repeats required for stable TRAP-trp leader RNA association. Thus, TRAP-5' stem-loop interaction may increase the likelihood that TRAP will bind to the (G/U)AG repeats in time to block antiterminator formation.

* 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: Department of Biology, MS008, Brandeis University, Waltham, MA 02454.

Dagger Present address: Ambion, Inc., Austin, TX 78744.

Journal of Bacteriology, April 2000, p. 1819-1827, Vol. 182, No. 7
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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