19] Preparation of functional ribosomal complexes and effect of buffer conditions on tRNA positions observed by cryoelectron microscopy

Abstract

Publisher Summary This chapter discusses the isolation of the ribosomes and the preparation of functional complexes and provides an overview of the possibilities for analyzing ribosomal complexes. It summarizes and discusses the results of recent cryoelectron microscopy studies that reflect the effect of buffer conditions. Studies have established that the ribosome has three transfer RNA (tRNA) binding sites, but 3-D cryo-electron microscopy (EM) has revealed five different tRNA positions on the ribosome, classified as A, P, P/E, E, and E2. The occupancy of some of these positions strongly depends on the buffer conditions used and the charge state of the tRNA. In the presence of the polyamine buffer, mimicking the in vivo conditions, only occupancy of A, P, and E sites are observed in complexes of the initiating and elongating ribosomes. The procedure described in the chapter for the small-scale isolation of tightly coupled ribosomes yields highly active and intact ribosomes, an important prerequisite for the preparation of functional complexes. The chapter describes the isolation of ribosomal subunits that can be used to prepare reassociated ribosomes. Reassociated ribosomes show a more efficient tRNA binding as compared to tightly coupled ribosomes, because the saturation of tRNA binding is reached at molar ratios slightly above stoichiometric ones. This can be attributed to at least two factors: (1) a selective pressure for active particles in the reassociation step and (2) the loss of residual amounts of tRNAs and of mitochondrial RNA (mRNA) fragments.