Kinetic and thermodynamic parameters of the assembly in vitro of the large subunit from Escherichia coli ribosomes

Abstract

When the 23s and 5s R N A and the total proteins from the large subunit of E. coli ribosomes are subjected to the two-step reconstitution procedure (4 m M Mg2+, 44 OC, -20 m M Mg2+, 50 "C), fully active subunits are formed. Here we show that both the formation of the reconstitution intermediate RI50*( 1) which is the essential product of the first incubation and the formation of reconstituted 50s subunits from RISo(2) particles during the second incubation can be treated as a first-order reaction. That is, alteration of the concentration of the reactants little influenced the kinetic parameters of the first step and did not change those of the second step. Therefore, the rate-limiting steps of the second incubation and most probably also that of the first one are unimolecular reactions. The temperature dependence of the rate constants of both partial reactions was determined and used to calculate the Arrhenius activation energy. When the R N A was isolated by I n the course of total reconstitution of the small subunit from E. coli ribosomes, the reconstitution intermediate R130 is formed which undergoes a conformational change to the RI30* particle. In spite of 22 reactants (21 proteins and the 1 6 s RNA), the kinetics of 30s formation are those of a firstorder reaction; Le., the rate-limiting step of the reconstitution process is the conformational change. The Arrhenius activation energy was determined to be about 160 kJ/mol(38 kcal/mol) (Traub and Nomura, 1969).Reconstitution of the large subunit from E. coli ribosomes requires a two-step incubation (Nierhaus and Dohme, 1974). The essential product of the first incubation is the RI50*( 1) particle, which is formed from RI50( 1) by a conformational change. The RI50( 1) particle contains 2 3 s and 5s R N A and about 20 tightly bound proteins. The R150*( 1) particle can add the remaining proteins in both the first and the second incubation step, thereby forming the RISo(2) intermediate, whereas the conformational change from the RIS0(2) particle to the 50s subunit only proceeds during the second step (Dohme and Nierhaus, 1976).In this paper, we describe the determination of the apparent reaction order and the molecularity of the rate-limiting step in both incubations. Furthermore, the Arrhenius activation energy is calculated from the dependence of the rate constants on temperature for both steps using R N A prepared in two different ways. Finally, we show that the temperature of the first step influences both the initial rate and the extent of R15,,*( 1 ) formation. Experimental ProcedureThe 3 0 s and 50s ribosomal subunits were isolated from E. coli K12, strain A19, as described (Schreiner and Nierhaus, 1973) with the modification of Dohme and Nierhaus (1976). 0006-2960/78/0417-3505$01 .OO/Othe phenol method, we obtained a value of about 290 kJ/mol (70 kcal/mol) for the first step and 225 kJ/mol (54 kcal/mol) for the second one. However, when the R N A was prepared by an acetic acid method, a lower value of 225 kJ/mol(54 kcal/ mol) for the first step was found...

Protein L16 induces a conformational change when incorporated into an L16‐deficient core derived from Escherichia coli ribosomes

“…However, the temperature optimum (40°C) observed in the L16-dependent reaction (rigA) is different from that of the second incubation (50°C, [16]). Furthermore, the activation energy of the latter reaction (225 kJ/mol, [15]) considerably exceeds that of the L16-induced effect (160 kJ/mol). Therefore, the L16.dependent reaction might be part of, but is not identical with, the conformational change occurring during the second incubation of the total reconstitution procedure for the large ribosomal subunit.…”

“…Therefore, it is likely that L16 plays an important role for the conformational change of the last assembly step (R/so (2) ~ 50 S, [9,15]). However, the temperature optimum (40°C) observed in the L16-dependent reaction (rigA) is different from that of the second incubation (50°C, [16]).…”

Escherichia coli rimM and yjeQ null strains accumulate immature 30S subunits of similar structure and protein complement

“…Early in vitro and in vivo experiments were able to identify discrete assembly intermediates of the 30S and 50S subunits (Hayes and Hayes 1971;Lindahl 1975;Sieber and Nierhaus 1978;Nierhaus 1980). These early studies seeded the hypothesis that assembly proceeds through a single pathway characterized by the existence of rate limiting intermediates.…”

Assembly map of the large subunit (50S) of Escherichia coli ribosomes.

“…Similarly, the latter two particles (RI50 [2] and SOS) differ only in their conformation, since the RI50 [2] particle consists ofthe full complement of50S components but is totally inactive. The two conformational changes (RI50 [1] --+RI* [1] and RI50 [2] --50S) require different ionic conditions and temperatures; a two-step incubation is therefore needed for the reconstitution of E. coli SOS subunits starting from totally separated rRNA and protein fractions (3,4).…”