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.

Ribosomes containing mutants of L4 ribosomal protein from Thermus thermophilus display multiple defects in ribosomal functions and sensitivity against erythromycin.

Protein L4 from Thermus thermophilus (TthL4) was heterologously overproduced in Escherichia coli cells. To study the implication of the extended loop of TthL4 in the exit-tunnel and peptidyltransferase functions, the highly conserved E56 was replaced by D or Q, while the semiconserved G55 was changed to E or S. Moreover, the sequence -G55E56- was inverted to -E55G56-.

Structural destabilization of the recombinant thermophilic TthL11 ribosomal protein by a single amino acid substitution.

Thermus thermophilus L11 protein has previously been reported to be resistant against tryptic and chymotryptic proteolysis under native conditions. With a single amino acid substitution, namely Trp101Arg, conformational changes were induced that resulted in the exhibition of specific amino acids that served as targets for tryptic and chymotryptic action and rendered the protein highly unstable even during purification. This unexpected process was evidenced by the isolation with size exclusion gel chromatography of the well-structured chymotryptic N-terminal domain in a high amount and its characterization both by Edman degradation and QTOF-EMS spectroscopy.

On the structural and functional importance of the highly conserved Glu56 of Thermus thermophilus L4 ribosomal protein.

The structural and functional importance of the highly conserved amino acid residue glutamic acid 56 (Glu56) of the ribosomal protein L4 from Thermus thermophilus (TthL4) has been investigated by replacing this residue by alanine or glutamine, and by incorporating the resulted mutants into Escherichia coli ribosomes. The catalytic properties of peptidyltransferase estimated for the mutants as well as for the wild-type TthL4 by the puromycin reaction, were quite different. The binding of tRNA to the P and A-site was affected.