The nature of the TRAP-Anti-TRAP complex.
Tryptophan biosynthesis is subject to exquisite control in species of Bacillus and has become one of the best-studied model systems in gene regulation. The protein TRAP (trp RNA-binding attenuation protein) predominantly forms a ring-shaped 11-mer, which binds cognate RNA in the presence of tryptophan to suppress expression of the trp operon. TRAP is itself regulated by the protein Anti-TRAP, which binds to TRAP and prevents RNA binding. To date, the nature of this interaction has proved elusive. Here, we describe mass spectrometry and analytical centrifugation studies of the complex, and 2 crystal structures of the TRAP-Anti-TRAP complex. These crystal structures, both refined to 3.2-A resolution, show that Anti-TRAP binds to TRAP as a trimer, sterically blocking RNA binding. Mass spectrometry shows that 11-mer TRAP may bind up to 5 AT trimers, and an artificial 12-mer TRAP may bind 6. Both forms of TRAP make the same interactions with Anti-TRAP. Crystallization of wild-type TRAP with Anti-TRAP selectively pulls the 12-mer TRAP form out of solution, so the crystal structure of wild-type TRAP-Anti-TRAP complex reflects a minor species from a mixed population.
Watanabe
Masahiro
M
Yokohama City University, Tsurumi, Suehiro 1-7-29, Yokohama 230-0045, Japan.
Heddle
Jonathan G
JG
Kikuchi
Kenichi
K
Unzai
Satoru
S
Akashi
Satoko
S
Park
Sam-Yong
SY
Tame
Jeremy R H
JR
D001407:Bacillus / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D001665:Binding Sites; D018360:Crystallography, X-Ray / Q000379:methods; D015964:Gene Expression Regulation, Bacterial; D006860:Hydrogen Bonding; D013058:Mass Spectrometry; D008954:Models, Biological; D008958:Models, Molecular; D008968:Molecular Conformation; D011485:Protein Binding; D011487:Protein Conformation; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D012997:Solvents / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014157:Transcription Factors / Q000737:chemistry / Q000502:physiology*; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; C079092:MtrB protein, Bacteria; D016601:RNA-Binding Proteins; D012997:Solvents; D014157:Transcription Factors; D014364:Tryptophan