trp RNA-binding attenuation protein-5' stem-loop RNA interaction is required for proper transcription attenuation control of the Bacillus subtilis trpEDCFBA operon.
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.
Du
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Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Yakhnin
A V
AV
Dharmaraj
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S
Babitzke
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P
D020121:5' Untranslated Regions / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D008957:Models, Genetic; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D009876:Operon / Q000235:genetics*; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D013728:Terminator Regions, Genetic / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics*; D014364:Tryptophan / Q000235:genetics* / Q000502:physiology
D020121:5' Untranslated Regions; D001426:Bacterial Proteins; C079092:MtrB protein, Bacteria; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D014364:Tryptophan