Assembly in Vitro of the 50 S subunit from Escherichia coli ribosomes: Proteins essential for the first heat-dependent conformational change

An early intermediate during the assembly in vitro of the ribosomal 50 S subunit is the RI 50(1) particle, which undergoes a drastic change in s value to form the RI 50 ∗(1) particle. The formation of this RI 50 ∗(1) particle, which is essential for the assembly of a highly active 50 S particle, was analyzed. Total reconstitution experiments with purified proteins revealed that six ribosomal components (23 S RNA and the proteins L4, L13, L20, L22 and L24) are essential for the RI 50 ∗(1) formation. Protein L3 had a stimulatory effect, but was not essential. With these seven components, a particle with the RI 50 ∗ conformation could be formed. A comparison with assembly in vivo demonstrated that the pathways of protein assembly in vitro and in vivo are very similar at the beginning, but diverge towards the end of the process. Furthermore, the sequence in which the proteins can be split off the 50 S subunit by increasing concentrations of LiCl corresponds, to a first approximation, to the reverse of the order of assembly in vitro.