The contribution of the zinc-finger motif to the function of Thermus thermophilus ribosomal protein S14.
In the crystal structure of the 30S ribosomal subunit from Thermus thermophilus, cysteine 24 of ribosomal protein S14 (TthS14) occupies the first position in a CXXC-X12-CXXC motif that coordinates a zinc ion. The structural and functional importance of cysteine 24, which is widely conserved from bacteria to humans, was studied by its replacement with serine and by incorporating the resulting mutant into Escherichia coli ribosomes. The capability of such modified ribosomes in binding tRNA at the P and A-sites was equal to that obtained with ribosomes incorporating wild-type TthS14. In fact, both chimeric ribosomal species exhibited 20% lower tRNA affinity compared with native E. coli ribosomes. In addition, replacement of the native E. coli S14 by wild-type, and particularly by mutant TthS14, resulted in reduced capability of the 30S subunit for association with 50S subunits. Nevertheless, ribosomes from transformed cells sedimented normally and had a full complement of proteins. Unexpectedly, the peptidyl transferase activity in the chimeric ribosomes bearing mutant TthS14 was much lower than that measured in ribosomes incorporating wild-type TthS14. The catalytic center of the ribosome is located within the 50S subunit and, therefore, it is unlikely to be directly affected by changes in the structure of S14. More probably, the perturbing effects of S14 mutation on the catalytic center seem to be propagated by adjacent intersubunit bridges or the P-site tRNA molecule, resulting in weak donor-substrate reactivity. This hypothesis was verified by molecular dynamics simulation analysis.
Xaplanteri
Maria A
MA
Laboratory of Biochemistry, School of Medicine, University of Patras, 26500 Patras, Greece.
Papadopoulos
Georgios
G
Leontiadou
Fotini
F
Choli-Papadopoulou
Theodora
T
Kalpaxis
Dimitrios L
DL
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003198:Computer Simulation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D016963:Thermus thermophilus / Q000737:chemistry*; D016335:Zinc Fingers
D001426:Bacterial Proteins; D021122:Protein Subunits; D012269:Ribosomal Proteins