title: Interaction analysis between tmRNA and SmpB from Thermus thermophilus.
abstract: Small protein B, SmpB, is a tmRNA-specific binding protein essential for trans-translation. We examined the interaction between SmpB and tmRNA from Thermus thermophilus, using biochemical and NMR methods. Chemical footprinting analyses using full-length tmRNA demonstrated that the sites protected upon SmpB binding are located exclusively in the tRNA-like domain (TLD) of tmRNA. To clarify the SmpB binding sites, we constructed several segments derived from TLD. Optical biosensor interaction analyses and melting profile analyses with mutational studies showed that SmpB efficiently binds to only a 30-nt segment that forms a stem and loop, with the 5' and 3' extensions composed of the D-loop and variable-loop analogues. The conserved sequences, 16UCGA and 319GAC, in the extensions are responsible for the SmpB binding. These results agree with the those visualized by the cocrystal structure of TLD and SmpB from Aquifex aeolicus. In addition, NMR chemical shift mapping analyses, using the 30-nt segment and (15)N-labeled SmpB, revealed the characteristic RNA binding mode. The hydrogen bond pattern around beta2 changes, with the Gly in beta2, which acts as a hinge, showing the largest chemical shift change. It appears that SmpB undergoes structural changes indicating an induced fit upon binding to the specific region of TLD.
authors.affiliation: Department of Industrial Chemistry, Faculty of Engineering, Chiba Institute of Technology, Chiba 275-0016.
authors.forename: Nobukazu
authors.initials: N
authors.lastname: Nameki
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Someya
authors.forename: Satoshi
authors.initials: S
authors.lastname: Okano
authors.forename: Reiko
authors.initials: R
authors.lastname: Suemasa
authors.forename: Michiko
authors.initials: M
authors.lastname: Kimoto
authors.forename: Kyoko
authors.initials: K
authors.lastname: Hanawa-Suetsugu
authors.forename: Takaho
authors.initials: T
authors.lastname: Terada
authors.forename: Mikako
authors.initials: M
authors.lastname: Shirouzu
authors.forename: Ichiro
authors.initials: I
authors.lastname: Hirao
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takaku
authors.forename: Hyouta
authors.initials: H
authors.lastname: Himeno
authors.forename: Akira
authors.initials: A
authors.lastname: Muto
authors.forename: Seiki
authors.initials: S
authors.lastname: Kuramitsu
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.forename: Gota
authors.initials: G
authors.lastname: Kawai
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D016963:Thermus thermophilus / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; C430328:small protein B; C096222:tmRNA