Interaction of the trp RNA-Binding attenuation protein (TRAP) of Bacillus subtilis with RNA: effects of the number of GAG repeats, the nucleotides separating adjacent repeats, and RNA secondary structure.
The 11-subunit trp RNA-binding attenuation protein of Bacillus subtilis, TRAP, regulates transcription and translation by binding to several (G/U)AG repeats present in the trp leader and trpG transcripts. Filter binding assays were used to study interactions between L-tryptophan-activated TRAP and synthetic RNAs. RNAs that contained GAG and/or UAG repeats were tested while the length and sequence of the nucleotides separating adjacent trinucleotide repeats were altered. TRAP-RNA complexes formed with transcripts containing GAG repeats were more stable than those with transcripts containing UAG repeats or alternating GAG and UAG repeats. The stability of TRAP-RNA complexes also increased substantially when the number of GAG repeats was increased from five to six and from six to seven. A gradual increase in complex stability was observed when the number of GAG repeats was increased from 7 to 11. The optimal spacer between adjacent trinucleotide repeats was found to be 2 nucleotides, with A and U residues preferred over G and C residues. TRAP binding was specific for single-stranded RNA; TRAP could not bind to RNA containing GAG repeats base paired in a stable RNA duplex. Overall, our findings suggest that each L-tryptophan-activated TRAP subunit can bind one (G/U)AG repeat and that multiple TRAP subunit-RNA binding site interactions are required for stable TRAP-RNA association.
Babitzke
P
P
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802, USA.
Yealy
J
J
Campanelli
D
D
D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D018911:Trinucleotide Repeats
D001426:Bacterial Proteins; C079092:MtrB protein, Bacteria; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; D014157:Transcription Factors