TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, is a multisubunit complex that appears to recognize G/UAG repeats in the trpEDCFBA and trpG transcripts.
A filter binding assay was developed to study interactions between purified TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, and trp specific transcripts. TRAP formed stable complexes with trpEDCFBA leader RNA; binding was L-tryptophan-dependent and was complete within 60 s. TRAP binds to a segment of the trp leader transcript that includes part of an RNA antiterminator structure. Binding to this segment allows formation of an RNA terminator structure, thereby promoting transcription termination. Using several trpEDCFBA leader deletion transcripts, we identified several closely spaced trinucleotide repeats (seven GAG and four UAG repeats) in the trp leader transcript that appeared to be required for TRAP binding. We also showed that TRAP binds to a segment of the trpG transcript that includes the trpG ribosome binding site; the nucleotide sequence of this segment contains several appropriately spaced trinucleotide repeats (seven GAG, one UAG, and one AAG). TRAP binding to the trpG transcript would block translation initiation. RNA footprint analysis confirmed interaction between TRAP and the trinucleotide repeats in the various transcripts. TRAP, in the presence or absence of L-tryptophan, appears to consist of 11 or 12 identical 8-kDa subunits. Our findings suggest that each tryptophan-activated TRAP subunit can bind one G/UAG repeat in a target transcript. Multiple protein-RNA interactions are required for stable association.
Babitzke
P
P
Department of Biological Sciences, Stanford University, California 94305.
Stults
J T
JT
Shire
S J
SJ
Yanofsky
C
C
D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins; D001483:Base Sequence; D003432:Cross-Linking Reagents; D005798:Genes, Bacterial; D007700:Kinetics; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009876:Operon; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D013728:Terminator Regions, Genetic; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; C079092:MtrB protein, Bacteria; D012329:RNA, Bacterial; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014157:Transcription Factors