NusA-stimulated RNA polymerase pausing and termination participates in the Bacillus subtilis trp operon attenuation mechanism in vitro

被引:81
作者
Yakhnin, AV [1 ]
Babitzke, P [1 ]
机构
[1] Penn State Univ, Dept Biochem & Mol Biol, University Pk, PA 16802 USA
关键词
D O I
10.1073/pnas.162373299
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation and translation control mechanisms. Both mechanisms require the binding of tryptophan-activated TRAP to the 11 (G/U)AG-repeat segment in the trp leader transcript. To promote termination, TRAP must bind to the nascent RNA before the antiterminator structure forms. Because only 20 nucleotides separate the TRAP-binding site from the 3' end of the antiterminator, TRAP has a short time frame to control this regulatory decision. Synchronization of factor binding and/or RNA folding with the RNA polymerase position is a major challenge in all attenuation mechanisms. Because RNA polymerase pausing allows this synchronization in many attenuation mechanisms, we performed experiments in vitro to determine whether pausing participates in the B. subtilis trp attenuation mechanism. We identified two NusA-stimulated pause sites in the trp leader region. Formation of pause hairpins participates in pausing at both positions. The first pause occurred at the nucleotide just preceding the critical overlap between the alternative antiterminator and terminator structures. TRAP binding to transcripts containing preexisting pause complexes releases RNA polymerase, suggesting that pausing provides additional time for TRAP to bind and promote termination. The second pause is downstream from the trp leader termination point, raising the possibility that this pause event participates in the trpE translation control mechanism. NusA also increases the efficiency of termination in the trp leader region and shifts termination one nucleotide upstream. Finally, NusA-stimulated termination is cooperative, suggesting that binding of multiple NusA molecules influences termination.
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页码:11067 / 11072
页数:6
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