Alanine-scanning mutagenesis of Bacillus subtilis trp RNA-binding attenuation protein (TRAP) reveals residues involved in tryptophan binding and RNA binding.
In Bacillus subtilis, expression of the trp genes is negatively regulated by an RNA binding protein called TRAP (trp RNA-binding Attenuation Protein), which is activated to bind RNA by binding l-tryptophan. TRAP contains 11 identical subunits assembled in a symmetric ring. We have used alanine-scanning mutagenesis to analyze the functions of surface amino acid residues of TRAP. The in vivo regulatory activity of each mutant TRAP was analyzed in a B. subtilis reporter strain containing a trpE'-'lacZ fusion. Mutant TRAP proteins with defective in vivo regulatory activities were characterized in vitro by measuring their tryptophan binding and RNA binding activities. Most of the mutant proteins with altered tryptophan binding, either affinity or cooperativity, contained substituted residues located on two loops formed by residues 25 to 33 and residues 49 to 52, as well as on the beta-strand and beta-turn contiguous with these loops. Substitution of three residues (Lys37, Lys56 and Arg58) with alanine resulted in significant decreases in the RNA binding activity of TRAP without altering tryptophan binding. Structural analysis shows that these three residues are directly aligned on the outer edge of TRAP. Further mutagenic analysis of these three residues revealed that only lysine or arginine residues at positions 37 or 58 allow proper TRAP function, whereas at position 56, only lysine is functional. Residue Asn20 is the only other residue in TRAP that is located on the line formed by residues 37, 56 and 58, and virtually any amino acid residue is functional at position 20. We propose that RNA wraps around TRAP by interacting with residues Lys37, Lys56 and Arg58.
Yang
M
M
Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY 14260, USA.
Chen
X p
Xp
Militello
K
K
Hoffman
R
R
Fernandez
B
B
Baumann
C
C
Gollnick
P
P
D000409:Alanine; D001216:Asparagine; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D001665:Binding Sites; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014364:Tryptophan / Q000378:metabolism*
D001426:Bacterial Proteins; C079092:MtrB protein, Bacteria; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D012313:RNA; D001216:Asparagine; D014364:Tryptophan; D000409:Alanine