Evidence that the three-site model for the ribosomal elongation cycle is also valid in the archaebacterium Halobacterium halobium

Summary

The properties of the ribosomal elongation cycle were analyzed in the extreme halophilic archaebacterium Halobacterium halobium under conditions optimal for poly(U)-dependent poly(Phe) synthesis (60 mM Mg²⁺, 6.4 M monovalent cations). For tRNA binding studies the standard nitrocellulose-filter technique could not be used, since due to the high salt concentration the nucleic acids were retained quantitatively on the filters. Instead, an ultracentrifugation technique was applied, which revealed in saturation experiments that the following numbers of tRNA molecules could be bound per poly(U) programmed ribosome: (a) three deacylated tRNA^Phe molecules, or (b) two Phe-tRNA^Phe molecules in the A and P sites, or (c) one AcPhe-tRNA^Phe molecule in either the A or P site (exclusion principle). In the absence of poly(U), AcPhe-tRNA^Phe and deacylated tRNA^Phe bound exclusively to the P site, whereas a low but significant binding of Phe-tRNA^Phe to the A site was observed. Functional analyses were performed with ribosomes carrying a deacylated [¹⁴C]tRNA in the P site and a peptidyl-tRNA in the A site. The results demonstrate that the translocation reaction is not accompanied by a release of deacylated tRNA, but that the deacylated [¹⁴C]tRNA is released in the subsequent round of elongation.

These data are qualitatively identical to those recently reported with E. coli ribosomes, although here different methods under extreme conditions had to be applied. This convincingly indicates the general validity of the three-site model for the description of the ribosomal elongation cycle in eu- and archaebacteria.