Ahy

Figure 7.9. Scheme of self-assembly (reconstitution) of the 30S ribosomal subunit from 16S ribosomal RNA and 21 proteins: "assembly map" (Modified from S. Mizushima & M. Nomura, Nature 226, 1214-1218, 1970; W.A. Held, B. Ballon, S. Mizushima & M. Nomura, J. Biol. Chem. 249, 3103-3111, 1974). The thick arrows from the RNA to a protein or from one protein to another symbolize the great dependence of the binding of the subsequent partner on the previous one; the thin arrows indicate a weaker dependence. Some weak interactions are omitted for the sake of clarity.

the scheme of Fig. 7.9 are not strictly sequential but appear to proceed concurrently. In other words, the compactization and binding of nine proteins do not greatly depend on each other and may proceed in parallel; in vitro, step II can be accomplished even after step III has been completed.)

Only after the ribonucleoprotein has undergone transition to compact conformation can the last set of proteins, consisting of S3, S10, S14, S21, as well as S2 and S1, be added to the complex (Fig. 7.9 step IV); this step yields the completed biologically active 30S ribosomal subunit. The incorporation of each of these proteins into the complex requires the presence of proteins bound at previous stages, as well as the final overall folding of the 16S RNA. The binding of protein S10 requires the presence of protein S9, the addition of protein S14 depends on protein S19, protein S3 may become incorporated only if proteins S5 and S10 are present, and the binding of protein S21 is stimulated by the presence of protein S11. The binding of protein S2 is affected by protein S3 and probably by the whole local structure of the ribonucleoprotein. The binding of the largest acidic protein, S1, also requires the correct folding of the ribonucleoprotein; however, it is difficult to determine which specific proteins are necessary for its addition.

An analysis of the complete map of 30S ribosomal subunit reconstitution (Fig. 7.9) demonstrates that the assembly of each structural lobe of the particle proceeds on the corresponding domain of 16S RNA more or less independently. Thus, proteins S4, S16, S17, S20, as well as S12, are assembled on the 5'-ter-minal domain (I), forming the subunit body. The middle domain (II) binds proteins S8, S15, S6:S18, as well as S11 and S21, yielding the assembled side bulge of the particle. The 3'-proximal domain (III) with protein S7 incorporates proteins S9, S13, and S19, followed by proteins S10 and S14, and forms the head of the 30S ribosomal subunit. The independence of the assembly of the structural lobes of the 30S subunit has been confirmed in experiments where the isolated RNA fragments representing all three main domains of the 16S rRNA are shown to form compact and specifically shaped ribonucleoprotein particles with corresponding cognate sets of ribosomal proteins (Weitzmann et al., 1993; Samaha et al., 1994; Agalarov et al., 1998). The specific in vitro assembly of the 30S subunit fragments equivalent or similar to the main structural lobes of the integral ribosomal particle supports the idea of a large-block organization of ribos-omal particles in general.

At the same time, interdomain and interlobe interactions should also receive some attention. The most characteristic cases are the addition of protein S5, which depends simultaneously on domains I and II with proteins contained therein; and the attachment of protein S3, which depends on all three domains of RNA and their corresponding proteins (Fig. 7.9). It is likely that protein S5 finds its place somewhere on the boundary between the subunit body and its side bulge, while protein S3 is located at the junction of the head, body, and side bulge of the 30S ribosomal subunit.

A similar analysis of the E. coli 50S ribosomal subunit assembly from 23S RNA, 5S RNA, and 32 proteins revealing the interdependence of protein binding and the sequence of stages can also be conducted on the basis of the experimental data available.

There is every reason to assume that the assembly of ribosomes in vivo proceeds mainly via the route demonstrated in the course of their reconstitution in vitro. In the case of the 50S ribosomal subunit, however, it should be emphasized that the correct reconstitution of the biologically active 50S subunit requires post-transcriptional modifications of the 23S ribosomal RNA, in particular in the region of the pep-tidyl transferase center (Section 9.3) of domain V (m2G 2445, D 2449, Y 2457, Cm 2498, m2A 2503, Y 2504).

0 0

Post a comment