Characterization of a trp RNA-binding attenuation protein (TRAP) mutant with tryptophan independent RNA binding activity.
TRAP (trp RNA-binding attenuation protein) is an 11 subunit RNA-binding protein that regulates expression of genes involved in tryptophan metabolism (trp) in Bacillus subtilis in response to changes in intracellular tryptophan concentration. When activated by binding up to 11 tryptophan residues, TRAP binds to the mRNAs of several trp genes and down-regulates their expression. Recently, a TRAP mutant was found that binds RNA in the absence of tryptophan. In this mutant protein, Thr30, which is part of the tryptophan-binding site, is replaced with Val (T30V). We have compared the RNA-binding properties of T30V and wild-type (WT) TRAP, as well as of a series of hetero-11-mers containing mixtures of WT and T30V TRAP subunits. The most significant difference between the interaction of T30V and WT TRAP with RNA is that the affinity of T30V TRAP is more dependent on ionic strength. Analysis of the hetero-11-mers allowed us to examine how subunits interact within an 11-mer with regard to binding to tryptophan or RNA. Our data suggest that individual subunits retain properties similar to those observed when they are in homo-11-mers and that individual G/UAG triplets within the RNA can bind to TRAP differently.
Li
Pan T X
PT
Department of Biological Sciences, State University of New York, Buffalo, NY 14260, USA.
Gollnick
Paul
P
D001412:Bacillus subtilis / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008024:Ligands; D020125:Mutation, Missense; D009994:Osmolar Concentration; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D021122:Protein Subunits / Q000235:genetics; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014364:Tryptophan / Q000031:analogs & derivatives / Q000378:metabolism*
D001426:Bacterial Proteins; D008024:Ligands; C079092:MtrB protein, Bacteria; D021122:Protein Subunits; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D012313:RNA; D014364:Tryptophan