The 16S rRNA binding site of Thermus thermophilus ribosomal protein S15: comparison with Escherichia coli S15, minimum site and structure.
Binding of Escherichia coli and Thermus thermophilus ribosomal proteins S15 to a 16S ribosomal RNA fragment from T. thermophilus (nt 559-753) has been investigated in detail by extensive deletion analysis, filter-binding assays, gel mobility shift, structure probing, footprinting with chemical, enzymatic, and hydroxyl radical probes. Both S15 proteins recognize two distinct sites. The first one maps in the bottom of helix 638-655/717-734 (H22) and in the three-way junction between helix 560-570/737-747 (H20), helix 571-600/606-634 (H21), and H22. The second is located in a conserved purine-rich region in the center of H22. The first site provides a higher contribution to the free energy of binding than the second one, and both are required for efficient binding. A short RNA fragment of 56 nt containing these elements binds S15 with high affinity. The structure of the rRNA is constrained by the three-way junction and requires both magnesium and S15 to be stabilized. A 3D model, derived by computer modeling with the use of experimental data, suggests that the bound form adopts a Y-shaped conformation, with a quasi-coaxial stacking of H22 on H20, and H21 forming an acute angle with H22. In this model, S15 binds to the shallow groove of the RNA on the exterior side of the Y-shaped structure, making contact with the two sites, which are separated by one helix turn.
Serganov
A A
AA
Institute of Protein Research, Pushchino, Moscow, Russia.
Masquida
B
B
Westhof
E
E
Cachia
C
C
Portier
C
C
Garber
M
M
Ehresmann
B
B
Ehresmann
C
C
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003198:Computer Simulation; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D013045:Species Specificity; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000235:genetics / Q000378:metabolism*
D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012269:Ribosomal Proteins; C036004:ribosomal protein S15; D008274:Magnesium