Creating hetero-11-mers composed of wild-type and mutant subunits to study RNA binding to TRAP.
TRAP (trp RNA-binding attenuation protein) is an RNA-binding protein that regulates expression of the tryptophan biosynthetic genes in Bacillus subtilis by binding to RNA targets that contain multiple GAG and UAG repeats. TRAP is composed of 11 identical subunits arranged symmetrically in a ring. The secondary structure of the protein consists entirely of antiparallel beta-sheets, beta-turns, and loops. We show here that the TRAP 11-mer can be reversibly denatured into unfolded monomers by guanidine hydrochloride. Removing the denaturant allows the protein to spontaneously renature into fully functional 11-mers. Based on this finding, we developed a subunit mixing method to hybridize wild-type and mutant subunits into heteromeric 11-mers by denaturation followed by subunit mixing renaturation. This method allows the study of subunit cooperativity in protein-ligand interaction such as RNA binding. Our data further support and extend the previously proposed two-step model for RNA binding to TRAP by showing that the initiation of binding requires at least one fully active subunit in the protein combined with one fully functional repeat in the RNA. The initiation complex tethers the RNA on the protein, thus allowing cooperative interaction with the remainder of the repeats.
Li
Pan T X
PT
Department of Biological Sciences, State University of New York, Buffalo, New York 14260, USA.
Scott
David J
DJ
Gollnick
Paul
P
D001412:Bacillus subtilis / Q000378:metabolism; D001426:Bacterial Proteins; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry*; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry; D014461:Ultracentrifugation
D001426:Bacterial Proteins; C079092:MtrB protein, Bacteria; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D012313:RNA; D014364:Tryptophan; D019791:Guanidine