Interaction analysis between tmRNA and SmpB from Thermus thermophilus.
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.
Nameki
Nobukazu
N
Department of Industrial Chemistry, Faculty of Engineering, Chiba Institute of Technology, Chiba 275-0016.
Someya
Tatsuhiko
T
Okano
Satoshi
S
Suemasa
Reiko
R
Kimoto
Michiko
M
Hanawa-Suetsugu
Kyoko
K
Terada
Takaho
T
Shirouzu
Mikako
M
Hirao
Ichiro
I
Takaku
Hiroshi
H
Himeno
Hyouta
H
Muto
Akira
A
Kuramitsu
Seiki
S
Yokoyama
Shigeyuki
S
Kawai
Gota
G
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*
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; C430328:small protein B; C096222:tmRNA