The assembly of ribosomes

Nomura, Masayasu; Traub, Peter; Guthrie, Christine; Nashimoto, H

doi:10.1002/jcp.1040740428

Cited by 41 publications

(23 citation statements)

References 22 publications

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“…The established existence of protein-protein contacts between S7, S9, and S19 (20) and the proximity of S7 and S9 (22) suggest that the observed second mass center is probably due to the folding of the rRNA stabilized by protein-protein contacts. Evidence for the formation of two mass centers (independent nucleation sites) (28) also arises from reconstitution studies where the 16S rRNA folded into a two-domain structure, one controlled by S4 at the 5' domain and the other controlled by S7 at the 3' domain (28,29).…”

Section: Resultsmentioning

confidence: 99%

Assembly of the Escherichia coli 30S ribosomal subunit reveals protein-dependent folding of the 16S rRNA domains.

Mandiyan

Tumminia

Wall

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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Protein-nucleic acid interactions involved in the assembly process of the Escherichia coli 30S ribosomal subunit were quantitatively analyzed by high-resolution scanning transmission electron microscopy. The in vitro reconstituted ribonucleoprotein (core) particles were characterized by their morphology, mass, and radii of gyration. During the assembly of the 30S subunit, the 16S rRNA underwent significant conformational changes that were governed by the cooperative interactions of the ribosomal proteins. The sequential association of the first 12 proteins with the 16S rRNA resulted in the formation of core particles containing up to three mass centers at distinct stages of the assembly process. These globular mass centers may correspond to the three major domains (5', central, and 3') of the 16S rRNA. Through the subsequent interactions of the late assembly proteins with the 16S rRNA, two of the three domains merge, yielding the basic structural traits ofthe native 30S subunit. The fine morphological features of the native 30S subunit became distinctly resolved only after the addition of the full complement of proteins. The fully reconstituted 30S subunits are active in polyphenylalanine synthesis assays. Visualiztion of the assembly mechanism of the E. coli 30S ribosomal subunit revealed domain-specific folding of the 16S rRNA through the formation bf distinct intermediate core particles hitherto not observed.Ribosomes are ubiquitous macromolecular assemblies of proteins and ribonucleic acids involved in protein biosynthesis. This bipartite cellular organelle is composed of a large and a small subunit. The structure-function relationship of the small (30S) ribosomal subunit ofEscherichia coli has been extensively studied. This subunit is actively involved in the initiation of protein synthesis by its interactions with initiation factors, messenger RNAs, transfer RNAs, and the large (50S) ribosomal subunit (1-4). The 30S subunit possesses characteristic structural features known as the head, body, cleft, and platform (5, 6) and is composed of a single 16S rRNA molecule and 21 different ribosomal proteins (S1-S21). Several ribosomal proteins interact with the 16S rRNA through a sequential and cooperative process as revealed from the in vitro assembly of the 30S subunit (7-9). Results obtained from these studies led to the classification of the proteins as primary binding proteins, which bind directly to the 16S rRNA, and secondary binding proteins, which require the previous association of the primary binding proteins. Apart from being the structural base for protein interactions, the 16S rRNA has an established role in protein biosynthesis (10-14). However, the role of the ribosomal proteins in ribosome structure and function remains unclear.Scanning transmission electron microscopy (STEM) was used to quantitatively evaluate the conformational changes induced in the 16S rRNA molecule by its interactions with the ribosomal proteins under nondenaturing conditions (15). Unlike other physical techniques tha...

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Section: Resultsmentioning

confidence: 99%

Assembly of the Escherichia coli 30S ribosomal subunit reveals protein-dependent folding of the 16S rRNA domains.

Mandiyan

Tumminia

Wall

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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“…Even though the synthesis of all r-proteins analyzed was stimulated at least to some extent (on the average, 1.6-fold stimulation), the degree of the stimulation is substantially less than the degree of stimulation of rRNA synthesis, which was at least twofold. It is known that the assembly of ribosomal subunits in vitro is not completely cooperative; that is, there are more than one independent nucleation site (estimated to be two to three for the 30S assembly [28] and two for the 50S assembly [29]; see discussion in reference 25) (10,27), and stimulation of r-protein synthesis will match the stimulation of rRNA synthesis via translational regulation. It appears that the degree of feedback repression of r-protein synthesis is sufficiently high (i.e., the capacity for derepression is sufficiently high) during normal growth so that cells are able to respond to sudden changes in rRNA synthesis induced by changes in environmental conditions.…”

Section: Resultsmentioning

confidence: 99%

Effects of induction of rRNA overproduction on ribosomal protein synthesis and ribosome subunit assembly in Escherichia coli

Yamagishi

Nomura

1988

J Bacteriol

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Overproduction of rRNA was artificially induced in Escherichia colh cells to test whether the synthesis of ribosomal protein (r-protein) is normally repressed by feedback regulation. When rRNA was overproduced more than twofold from a hybrid plasmid carrying the rrnB operon fused to the A PL promoter (pL-rrnB), synthesis of individual r-proteins increased by an average of about 60%. This demonstrates that the synthesis of r-proteins is repressed under normal conditions. The increase of r-protein production, however, for unknown reasons, was not as great as the increase in rRNA synthesis and resulted in an imbalance between the amounts of rRNA and r-protein synthesis. Therefore, only a small (less than 20%) increase in the synthesis of complete 30S and 50S ribosome subunits was detected, and a considerable fraction of the excess rRNA was degraded. Lack of complete cooperativity in the assembly of ribosome subunits in vivo is discussed as a possible explanation for the absence of a large stimulation of ribosome synthesis observed under these conditions. In addition to the induction of intact rRNA overproduction from the PL-rrnB operon, the effects of unbalanced overproduction of each of the two large rRNAs, 16S rRNA and 23S rRNA, on r-protein synthesis were examined using PL-rrnB derivatives carrying a large deletion in either the 23S rRNA gene or the 16S rRNA gene. Operon-specific derepression of r-protein synthesis was observed, although the degrees of derepression were generally lower than those observed after simultaneous overproduction of both 16S and 23S rRNAs; the pattern of r-protein derepression after 23S or 16S rRNA overproduction correlated with the overproduction of rRNA containing the target site for the operon-specific repressor r-protein. These results are discussed to explain the apparent coupling of the assembly of one ribosomal subunit with that of the other which was observed in earlier studies on conditionally lethal mutants with defects in ribosome assembly.There is now ample evidence that the synthesis of many ribosomal proteins (r-proteins) is 'regulated by a feedback mechanism(s) involving certain key r-proteins as repressors and that such mechanisms are important for the coordination of r-protein synthesis to rRNA synthesis (for reviews, see references 19, 22, 26, 27). Thus, for example, the synthesis of r-proteins in the spc, a, str, Lll, L10, and S20 operons is feedback regulated at a posttranscriptional step by S8 (6, 37), S4 (5, 37), S7 (7), Li (5, 37), L10 (2, 9, 38), and S20 (34), respectively, and the synthesis of r-proteins in the S10 operon is feedback regulated by L4 both at a transcription step (8,20,21) and at a translation step (8,39). However, the question has often'been raised as to the significance of the feedback regulation under conditions of normal growth (e.g., reference 32). We have previously studied this question regarding the a r-protein operon and the Lll r-protein operon. In the former case, we found that a mutational alteration in the translational repressor S4 l...

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“…In particular, cold-sensitive mutants have been a rich source of assembly mutants in bacteria, probably because ribosome assembly has a high activation energy (6,21,44,46,62). Heat-sensitive assembly mutants have been isolated as well (10,40,43,49,55).…”

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confidence: 99%

Assembly of 60S ribosomal subunits is perturbed in temperature-sensitive yeast mutants defective in ribosomal protein L16.

Moritz¹,

Pulaski²,

Woolford³

1991

Mol. Cell. Biol.

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Temperature-sensitive mutants defective in 60S ribosomal subunit protein L16 of Saccharomyces cerevisiae were isolated through hydroxylamine mutagenesis of the RPL16B gene and plasmid shuffling. Two heatsensitive and two cold-sensitive isolates were characterized. The growth of the four mutants is inhibited at their restrictive temperatures. However, many of the cells remain viable if returned to their permissive temperatures. All of the mutants are deficient in 60S ribosomal subunits and therefore accumulate translational preinitiation complexes. Three of the mutants exhibit a shortage of mature 25S rRNA, and one accumulates rRNA precursors. The accumulation of rRNA precursors suggests that ribosome assembly may be slowed in this mutant. These phenotypes lead us to propose that mutants containing the rpll6b alleles are defective for 60S subunit assembly rather than function. In the mutant carrying the rp116b-1 allele, ribosomes initiate translation at the noncanonical codon AUA, at least on the rp1l6b-1 mRNA, bringing to light a possible connection between the rate and the fidelity of translation initiation.Most eukaryotic ribosomes are assembled from approximately 75 different ribosomal proteins (r-proteins) and 4 rRNA molecules in a process involving multiple cellular compartments. Newly synthesized r-proteins make their way from the cytoplasm to the nucleolus by a largely unknown mechanism and there associate with nascent rRNA. By analogy to Escherichia coli, the different r-proteins probably associate with assembling ribosomes in a strict order (reviewed in reference 60). Nearly all of the r-proteins assemble into subunits in the nucleolus. The ribosomes then are exported to the cytoplasm, where a few late-assembling proteins join them (72). The ribosomes then can assume their role as the protein-synthesizing machinery of the cell (reviewed in reference 70).Very little is known about how eukaryotic ribosomes assemble, although maintaining the stoichiometry of ribosomal components appears to be important. In yeasts, r-proteins that are synthesized in excess of their counterparts are rapidly degraded, presumably in the absence of their ability to assemble into ribosomes (14,39,65,71). Similarly, if the synthesis of a single r-protein or rRNA is terminated, most other proteins and RNAs of the same subunit of which that molecule is a part are synthesized and then degraded (1,15,41,42,68,73). In these cases, turnover of ribosomal constituents may result upon disassembly of incorrectly or incompletely assembled subunits.Temperature-sensitive mutants defective for ribosome assembly have also been isolated. In particular, cold-sensitive mutants have been a rich source of assembly mutants in bacteria, probably because ribosome assembly has a high activation energy (6,21,44,46,62). Heat-sensitive assembly mutants have been isolated as well (10,40,43,49,55

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The assembly of ribosomes

Cited by 41 publications

References 22 publications

Assembly of the Escherichia coli 30S ribosomal subunit reveals protein-dependent folding of the 16S rRNA domains.

Assembly of the Escherichia coli 30S ribosomal subunit reveals protein-dependent folding of the 16S rRNA domains.

Effects of induction of rRNA overproduction on ribosomal protein synthesis and ribosome subunit assembly in Escherichia coli

Assembly of 60S ribosomal subunits is perturbed in temperature-sensitive yeast mutants defective in ribosomal protein L16.

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