Assembly of the 30S Ribosomal Subunit
GLORIA M. CULVER* AND NARAYANASWAMY KIRTHI
[SECTION EDITOR: MICHAEL O’CONNOR]
Posted January 31, 2008
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA
*Corresponding author. Present address: Department of Biology, University of Rochester, Rochester, NY 14627. Phone: (585) 276-3602, Fax: (585) 275-2070, E-mail:
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Protein synthesis involves nearly a third of the total molecules in a typical bacterial cell. Within the cell, protein synthesis is performed by the ribosomes, and research over several decades has investigated ribosomal formation, structure, and function. Eubacterial ribosomes are composed of approximately 65% RNA and 35% protein, while eukaryotic ribosomes are composed of approximately equal amounts of RNA and protein, and archaeal ribosomes represent a hybrid or composite version of the two. Ribosomes from all kingdoms are composed of two asymmetrical subunits. In bacteria, these are designated the 30S (small) and 50S (large) ribosomal subunits. Prokaryotic ribosomes, in particular, the Escherichia coli ribosome, have been the extensively studied research model. This chapter gives an overview of our current understanding of the assembly of the E. coli 30S ribosomal subunit.
The E. coli 30S subunit contains one rRNA molecule (16S) and 21 ribosomal proteins (r-proteins; S1 to S21). This large macromolecular complex has a molecular mass of approximately 0.85 MDa. The majority of the mass of the 30S subunit is contributed by 16S rRNA, which is 1,542 nucleotides in length. The r-proteins from various organisms have been reasonably conserved throughout evolution. The r-proteins from different bacterial families show significant identity (often greater than 50%) and also show significant homology to their eukaryotic counterparts (33). However, this level of homology pales when compared with that observed for rRNAs. Analyses of sequences of 16S rRNA from many different organisms have revealed that, at the nucleotide level, 16S rRNA is highly conserved. Thus, sequences of 16S rRNA can be used to explore prokaryotic phylogeny (21).
Phylogenetic comparisons, along with structural studies, have been used to determine the secondary structure of 16S rRNA, which contains approximately 45 regular helices connected by irregular single-stranded loops (Fig. 1) (8, 23, 44, 67). This structure can be divided into three major domains: the 5', central, and 3' domains (Fig. 1B) (44). These domains, which are evident in the secondary structure of 16S rRNA, also correspond to specific structural domains, the body (5'), the platform (central), and the head (3' major), that are readily observed in the three-dimensional structure of the 30S subunit (Fig. 1C) (44). The body of the 30S subunit is composed of the 5' domain (helices 1 to 18) and its associated proteins (S4, S5, S8, S12, S16, S17, and S20). The central domain of 16S rRNA (helices 19 to 27) binds five r-proteins, S6, S11, S15, S18, and S21, and forms the platform of the 30S ribosomal subunit. The head of the 30S subunit contains the 3' major domain of 16S rRNA (helices 2 and 28 to 43) and its associated proteins, S2, S3, S7, S9, S10, S13, S14, and S19. The 3' minor domain (helices 44 and 45) stretches across the body of the 30S subunit.
Fragments of 16S rRNA corresponding to the three major domains can independently assemble in vitro with their associated proteins and form discrete ribonucleoprotein complexes (RNPs) (1, 4, 56). Electron microscopy, RNA structure probing, and X-ray crystallography have shown the RNPs to be well structured (1, 2, 3, 4, 56). Additionally, high-resolution structural analyses of the central domain and three associated proteins suggest that the domain structure is similar to that seen within the full 30S subunit (2, 58, 67). All of these results indicate the structural independence of the three domains, and these findings suggest that the assembly of each domain can occur independently and in isolation.
The formation of functional subunits can occur as a self-assembly process in vitro; i.e., all the information required for the formation of active ribosomes resides in the primary sequences of the r-proteins and rRNAs. This property has aided in the dissection of the steps in the assembly pathways. In vitro reconstitution of functional 30S subunits can be carried out by using a mixture of TP30 (total proteins isolated from 30S subunits), individually purified natural (27) or recombinant (15) r-proteins, and natural 16S rRNA. In vitro-transcribed 16S rRNA has also been reconstituted into functional 30S subunits (35). Thus, the above-mentioned systems have allowed detailed analysis of 30S subunit formation.
The 30S subunit assembly map was worked out by the study of assembly dependencies and codependencies for the r-proteins (22, 27, 40) (Fig. 2). This map reveals that the 30S subunit assembles in an ordered and cooperative manner in vitro. One set of proteins, the primary binding proteins (S4, S7, S8, S15, S17, and S20), bind directly and independently to 16S rRNA. The secondary binding proteins (S5, S6, S9, S11, S12, S13, S16, S18, and S19) require at least one of the primary proteins to be bound to 16S rRNA prior to their association. The assembly of the third class of proteins, the tertiary binding proteins (S2, S3, S10, S14, and S21), requires that at least one protein from each of the previous sets be bound to the developing RNP (Fig. 2A). The association of these three sets of r-proteins with 16S rRNA allows intact, functional 30S subunits to be formed in vitro. Interestingly, many of the proteins that are linked in the assembly map are neighbors within the fully formed 30S subunit (Fig. 2B) (11, 58, 67, 69).
In vitro reconstitution of 30S subunits can be described as a stepwise process (64) (Fig. 3). At low temperatures (0 to 15°C), the primary and secondary proteins bind to 16S rRNA and form a distinct particle, the reconstitution intermediate (RI) (Fig. 3A). This RNP has a sedimentation coefficient of 21S and is unable to bind tertiary proteins. Upon heating (42°C), the RI undergoes conformational changes to form activated RI (RI*), which has a sedimentation coefficient of 26S (Fig. 3B). Tertiary proteins bind to RI* to give rise to functional 30S subunits (Fig. 3C) (65). The transition from RI to RI* is the rate-limiting step in the in vitro assembly of 30S subunits, and this conformational change represents a high activation barrier for the assembly process (64). As similar intermediates have been observed in vivo (see references 24, 25, and 42 for examples; also see "In Vivo Assembly of the E. coli 30S Subunit," below), studies of these steps and intermediates reveal important aspects of RNA folding and RNA-protein interactions involved in the assembly of the 30S ribosomal subunit.
Chemical probing and primer extension analysis have been used extensively to monitor changes in the reactivities of nucleotides in 16S rRNA during the in vitro reconstitution of 30S subunits. This approach has allowed the study of the binding of each r-protein to 16S rRNA. Additionally, this approach has been exploited to monitor 16S rRNA folding during different stages of 30S subunit assembly.
The temperature dependence of in vitro 30S subunit reconstitution can be used to retard the assembly process, and this step enables the monitoring of r-protein association with 16S rRNA as assembly proceeds. Thus, changes in the assembly state can be monitored by temperature manipulations (50). The observed changes led to the classification of the proteins into four kinetic classes: early, mid, mid-to-late, and late (14, 50). Generally, the primary and secondary proteins which bind to the 5' and central domains of 16S rRNA make up the early-assembly class, while S7, another primary binding protein which initiates the assembly of the 3' domain, is positioned in the midassembly class (Fig. 4). These kinetic assembly data demonstrate that the assembly of the 5' and central domains begins prior to the assembly of the 3' domain (Fig. 4). This finding suggests that the assembly occurs in the 5'-to-3' orientation, which further implies that the assembly may occur cotranscriptionally in vivo.
The temperature-dependent nature of in vitro 30S subunit assembly has also allowed the analysis of discrete 16S rRNA folding events during assembly. Structural differences between the 30S ribosomal subunit assembly intermediates RI and RI* have been characterized by chemical modification and primer extension analyses (30). Approximately 7% of the 16S rRNA nucleotides show altered reactivities in RI* compared to those in the RI, many of these alterations corresponding to functional sites. Also, these alterations are found throughout the subunit, but the majority are localized to the head of the 30S subunit, which is composed of the 3' domain of 16S rRNA (30). These results suggest that the formation of functional sites during assembly may be concerted and that assembly may occur in a polar manner.
Interestingly, temperature-dependent conformational changes can also be observed in significantly smaller ribosomal RNPs. The association of the r-protein S4 with 16S rRNA can occur at low temperatures; however, the complex undergoes temperature-dependent rearrangement (51). These results suggest that the temperature dependence of assembly can occur in discrete steps, which may begin to explain the hierarchical and sequential binding of r-proteins to 16S rRNA during 30S subunit assembly.
Recent advances have allowed some kinetic aspects of in vitro 30S subunit assembly to be revealed. A novel approach using differential labeling and mass spectrometry analysis has allowed the time course of r-protein binding to be deconvoluted in real time (61a). The r-proteins bind to 16S rRNA at different rates, and there is good correlation between the assembly rates and the map. Many different rate-limiting transitions were observed in this study. This finding is in contrast to the single rate-limiting step reported previously (29), and further work will be needed to determine the kinetics of ribosome biogenesis in vivo.
The potential roles for r-proteins in 30S subunit assembly were determined by omitting single proteins in reconstitution experiments (46). The RNPs resulting from single protein omissions were examined in terms of their composition and function to determine the roles of the absent proteins. The particles resulting from omitting any of the proteins S1, S2, S6, S12, S13, S18, S20, and S21 had the same sedimentation coefficients as the native 30S subunits, but the functional capacities of some of the particles were considerably reduced. The omission of other r-proteins led to the formation of particles with sedimentation coefficients ranging between 22S and 28S. The formation of discrete intermediate particles in most omission experiments suggests that 30S subunit assembly proceeds unimpeded in those assembly branches which do not require the omitted protein. These results may support the existence of more than one pathway for the formation of pre-30S and 30S subunits.
Roles for r-proteins were also examined by the addition of specific subsets of r-proteins to 16S rRNA in reconstitution reactions. Experiments to determine the r-proteins that bind to 16S rRNA without cooperation showed only S4 and S7 among the six primary binding proteins to be true assembly initiator proteins (47). The S6, S8, S15, S16, and S20 proteins were found to be dependent on S4, while S3, S8, S9, and S19 were found to be dependent on S7. These results are generally in good agreement with the connections revealed in the assembly map (Fig. 2). Also, S8 and S5 were suggested to have a role in the interconnection of the two assembly branches formed by S4 and S7 (47).
Nomura et al. (46) showed that the omission of S14 results in discrete particles with a sedimentation coefficient of 28S and with negligible activity. Similar results were seen with the omission of S3; the resulting particles had a sedimentation coefficient of 28S and were also functionally defective. The demonstration that the removal of S3 and S14 from an assembled particle still leaves an active subunit confirmed that these r-proteins are not absolutely required for function (52). However, reconstitution using protein mixes depleted of S3 and S14 did not yield functional 30S subunits. This result indicated that S3 and S14 may be essential during the assembly of functional 30S subunits but may not be required for function postassembly.
S16 is the only protein that is known to have an effect on the rate of ribosome assembly (28). Reconstitution reactions performed in the absence of S16 yielded inactive particles with a sedimentation coefficient of 27S. However, over time, these 27S particles were converted into active 30S subunits. This finding suggests that S16 plays a role in the efficient assembly of 30S subunits but may not be formally required for functional 30S subunit formation (28).
To date, only a few definite roles for the r-proteins have been demonstrated. One possible explanation for the lack of obvious assembly roles for r-proteins is redundancy of function. Given the importance of ribosome biogenesis to cell viability, building redundancy into the assembly mechanism via overlapping roles for the r-proteins would be of clear biological advantage.
Recent developments in understanding the structure of the 30S subunit have led to speculation about roles for some of the r-proteins in assembly. The crystal structures of the 30S subunit (58, 67) and the 70S ribosome (69) reveal details of the r-protein and rRNA interactions. This new information has enabled the evaluation of various hypotheses regarding r-protein binding and 30S subunit assembly. The 30S subunit structure confirms that each of the primary binding proteins organizes distinct regions of the 30S subunit. S8 and S15 organize the central domain, S7 assembles the lower part of the 3' major domain, and S4, S17, and S20 organize different parts of the 5' and central domains (9). The crystal structure of the 30S subunit reveals that the primary binding proteins are typically globular proteins and often bind to multistem junctions. Other, later-binding r-proteins like S9 and S12 have long, extended tails which are found encased in rRNA (9). It has been postulated that the assembly of the tails must proceed first, followed by the assembly of other r-proteins in their vicinity (9). This organization would allow the tails to be integrated readily into the functional 30S subunit structure and may facilitate tighter packing of negatively charged rRNA elements during the assembly process.
The crystal structure of the 30S subunit also allows speculation on the nature of the RI particle. The RI particle in E. coli lacks the tertiary binding proteins S2, S3, S10, S14, and S21. When compared with the structure of the 30S subunit in the absence of the contacts with the tertiary proteins (all except S21, which is absent in Thermus thermophilus), the RI seems to lack most contacts between the head and body, except for those created by S5. Interestingly, S5 is known to be present in substoichiometric amounts on the RI (42). Also, S5 is one of the very few r-proteins that have contact with more than one domain of 16S rRNA in the 30S subunit. The crystal structure of the 30S subunit reveals contacts between S5 and all three major domains. Also, the localization of four of the five tertiary binding proteins in the head indicates that in the RI the head is partially folded. During the conversion of the RI into RI*, there may be a change in the internal conformation of the head, enabling the remaining r-proteins to bind following the transition (9).
The recent crystal structures and the assembly map that came about from many decades of experiments are in strong agreement. However, the exact sequence of events and the landscape of explored conformations during 30S subunit assembly that result in the assembly of functional 30S subunits need to be further characterized.
The assembly of functional ribosomal subunits requires sequential binding events and conformational changes. In vitro perturbation of these events can be revealed in the temperature-dependent nature of assembly. In vivo mutations that alter the ribosome biogenesis but are not lethal have been found to result generally in cold sensitivity. This finding supports the important role of dynamics during in vivo 30S subunit assembly (see references 5, 17, 24, and 42 for examples).
Some of the first mutations found to alter ribosome biosynthesis were isolated more than 30 years ago (24). Studies identified mutations that resulted in the production of 21S particles, with r-protein compositions and sedimentation coefficients similar to those of the in vitro RI particle. 21S-like particles are also observed in vivo in dnaK mutants (5). The appearance of a 21S intermediate in vivo strongly suggests that the paths of in vitro and in vivo 30S subunit assembly are related.
Although the similarities between in vitro and in vivo assembly processes have been noted, it is known that the in vivo assembly is more complicated. One obvious complication is the involvement of the precursor 16S rRNA in vivo. The 21S particles accumulated in vivo contain precursor 16S rRNA and not mature 16S rRNA (42). Precursor 16S rRNA isolated from in vivo-formed 21S particles is undermodified and has extra nucleotides at both the 5' (leader) and 3' (trailer) ends (36). Studies of ribosomes assembled from rRNA operons with mutations in their leader sequences revealed that the folding and structure of 30S subunits are greatly influenced by the highly retained conserved leader sequences (6, 49, 62). Although in vivo ribosome biosynthesis appears to be more complicated than the in vitro process, it occurs very rapidly compared to in vitro 30S subunit reconstitution (29).
Similarities between in vivo and in vitro E. coli 30S subunit assembly processes have validated the use of the in vitro work model system. The nonphysiological requirements of the in vitro reconstitution reaction suggest that extraribosomal or nonribosomal factors may actively participate in ribosome assembly in vivo. Such factors would be notably absent in standard in vitro 30S subunit reconstitution reactions. To date, a few cellular nonribosomal proteins, such as RbfA (17), Era (31), and DnaK (5, 37), have been suggested to aid in the assembly process.
Given the cold-sensitive phenotypes associated with mutations affecting 30S subunit biogenesis, it is not surprising that cold shock proteins have been implicated in this process. RbfA (ribosomal binding factor A) was identified as a suppressor of a cold-sensitive mutation in 16S rRNA (17). Both the original 16S rRNA mutation (16) and the deletion of the RbfA gene (17) result in defects in 30S subunit assembly. RbfA has been implicated in 16S rRNA maturation during cold stress (16, 68) and has been characterized as a cold shock protein (32). RimM is another protein shown to have a role in ribosome assembly; it has been shown to be essential for the efficient processing of pre-16S rRNA (10). Another cold shock protein, CspA, may aid in 30S subunit assembly (32, 63). CspA is a canonical DEAD box protein and can act as an ATP-dependent RNA helicase (32, 63). While results are suggestive of the importance of these proteins in 30S subunit biogenesis, no precise roles for these proteins have been identified.
Heat shock proteins (Hsp) have also been shown to play a role in ribosome assembly (5, 20, 37, 57). In the thermophilic archaeon Sulfolobus solfataricus, the Hsp60 homolog has been shown to play a role in 16S rRNA processing (55). The purified DnaK chaperone system (including DnaK, DnaJ, GrpE, and ATP) can facilitate the in vitro reconstitution of functional 30S subunits under otherwise nonpermissive conditions (37). This finding is in good agreement with findings regarding phenotypes associated with dnaK mutations. DnaK may not be an absolute requirement for ribosome biogenesis, as the deletion of dnaK is not lethal (25). Nevertheless, the DnaK chaperone system may accelerate the late steps of ribosome biogenesis, especially at high temperatures in vivo (25). Another class of heat shock proteins, GroEL and GroES, have a role in ribosome assembly when there is altered expression of both DnaK and DnaJ (20). Era, a GTP binding protein, is suggested to have a role in 16S rRNA maturation and ribosome assembly (31). Again, the results are suggestive of the critical roles of these proteins in ribosome assembly, but mechanistic details are greatly lacking.
It has long been known that many antibiotics exert their action by binding to ribosomes and interfering with protein biosynthesis. Most of these antibiotics act directly on the ribosome at their functional sites by analogy with enzyme inhibitors (66). Interestingly, recent studies have also suggested a role for certain antibiotics in stalling 30S subunit assembly.
Paramomycin and neomycin are aminoglycosides that bind to the 30S subunit and cause misreading and mistranslation of mRNA (41, 53, 59). Recently, it has been shown that the presence of paramomycin and neomycin in growth medium results in a reduction in the amount of 30S subunits produced. The accumulation of 21S particles under these conditions was observed as well (38). At higher concentrations of these antibiotics, decreased 50S subunit levels were observed, suggesting that stalling 30S ribosomal assembly has a downstream effect on 50S subunit formation. Therefore, the bactericidal effect of these antibiotics may be twofold, with defects in translational accuracy and the stalling of ribosome assembly both contributing to cell death (38, 39).
A number of antibiotics that bind to the 50S subunit, like erythromycin, are known to have an effect on 50S subunit assembly (13). For most of these drugs, the inhibition of assembly is equivalent to the inhibition of translation. This result suggests that some of these antibiotics bind to their known functional sites on the 50S ribosomal subunits as these subunits are emerging during ribosome assembly.
As discussed for E. coli 30S subunits, E. coli 50S subunits can also be reconstituted in vitro (18, 43, 45). However, the reconstitution of 50S subunits requires more steps and manipulations than 30S subunit reconstitution (19, 60). This observation, along with the increased number of components present in the 50S subunit compared to the 30S subunit, suggests that the assembly of the 50S subunit is more complicated than that of the 30S subunit. Assembly map features for 50S subunit assembly have been worked out (54, 61), although not at the level of detail as those for 30S subunit assembly (7, 26, 34, 48). The crystal structure of the 50S subunit also supports a more complicated assembly process. While 16S rRNA folds into discrete domains in the structure of the 30S subunit, the six domains of 23S rRNA (and 5S rRNA) fold to form a single domain in the 50S subunit (7, 26, 34). Thus, understanding the assembly of the 50S subunit is a formidable process, and 50S subunit assembly has not been characterized as well as 30S subunit assembly.
A variety of factors appear to influence 50S subunit assembly. As for 30S subunit assembly, little detail is available to delineate the specific roles of these factors. There are both genetic and biochemical links between the 30S and 50S subunit biogenesis processes. These observations serve to underscore that the in vivo biosynthesis of ribosomes is a highly complicated and regulated process. Comprehensive understanding of 30S and 50S subunit assembly and also the interdependence of the assembly processes of the two subunits await the elucidation of further details.
While the many advances, discussed above, in the research on 30S subunit assembly have greatly increased our understanding of this process, many questions still remain. The next frontier may in fact be expanding the view of 30S subunit assembly to look beyond the final components and begin to identify new intermediates and additional factors that facilitate or attenuate ribosomal subunit assembly. One major area that will yield important insights is the integration of the roles and timing of rRNA and r-protein modification as these details relate to ribosome assembly. Additionally, very little is known about the timing of pre-rRNA processing events as regards r-protein binding, 16S rRNA domain folding, and the action of extraribosomal assembly factors. All of these questions get at the question of how ribosomal subunits actually assemble in the cell. Perhaps the goal should be to move from thinking of 30S subunit assembly to 30S subunit biogenesis.
We thank Kristi Holmes, Laura Dutca, Nathan Napper, Darrin Lemmer, and Daniel Dickinson for comments on the review. We also thank Kristi Holmes for her help with the figures.
Preparation of the review was supported in part by a grant from the National Institutes of Health (R01GM62432) to G.M.C.
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Module2.5.3
