Biochemical evidence for the heptameric complex L10(L12)6 in the Thermus thermophilus ribosome: in vitro analysis of its molecular assembly and functional properties.

The stalk protein L12 is the only multiple component in 50S ribosomal subunit. In Escherichia coli, two L12 dimers bind to the C-terminal domain of L10 to form a pentameric complex, L10[(L12)(2)](2), while the recent X-ray crystallographic study and tandem MS analyses revealed the presence of a heptameric complex, L10[(L12)(2)](3), in some thermophilic bacteria. We here characterized the complex of Thermus thermophilus (Tt-) L10 and Tt-L12 stalk proteins by biochemical approaches using C-terminally truncated variants of Tt-L10. The C-terminal 44-residues removal (Delta44) resulted in complete loss of interactions with Tt-L12. Quantitative analysis of Tt-L12 assembled onto E. coli 50S core particles, together with Tt-L10 variants, indicated that the wild-type, Delta13 and Delta23 variants bound three, two and one Tt-L12 dimers, respectively. The hybrid ribosomes that contained the T. thermophilus proteins were highly accessible to E. coli elongation factors. The progressive removal of Tt-L12 dimers caused a stepwise reduction of ribosomal activities, which suggested that each individual stalk dimer contributed to ribosomal function. Interestingly, the hybrid ribosomes showed higher EF-G-dependent GTPase activity than E. coli ribosomes, even when two or one Tt-L12 dimer. This result seems to be due to a structural characteristic of Tt-L12 dimer.