Biophysical studies of bacterial ribosome assembly

The assembly of the bacterial ribosome involves the association of over 50 proteins to 3 large RNA molecules, and it represents a major metabolic activity for rapidly growing bacteria. The availability of atomic structures of the ribosome and the application of biochemical and biophysical methods have led to rapid progress in understanding the mechanistic details of ribosome assembly. The basic steps required to assemble a ribosome are outlined, and the contributions of mass spectrometry, computational methods, and RNA-folding studies in understanding these steps are detailed. This complex process takes place with both sequential and parallel processing that is coordinated to ensure efficient and complete assembly of ribosomes to meet the demands of cell growth.     

Kinetic studies of the rates and mechanism of assembly of the protein synthesis initiation complex.

Rate constants for a number of the assembly reactions involved in forming Escherichia coli ribosome initiation complexes have been measured. These reactions were monitored in a stopped-flow device in which Rayleigh scattering and fluorescence anisotropy were followed as a function of time. Fluorescence was induced by laser excitation modulated at 50 kHz. Aminoacyl-tRNA, initiation factor 3 (IF3), and 70S ribosomes were labeled with fluorescent probes. The light-scattering and fluorescence data show that the antiassociation model for IF3 function cannot be correct. IF3 can be considered to act as an effector in an allosteric model for ribosome function. Fluorescence anisotropy stopped-flow experiments provided rate constants for the binding of IF3 to both 30S subunits and to the intact 70S ribosome. Aminoacyl-tRNA's and nucleotide triplets appear to bind rapidly to 70S ribosomes and then a slow first-order conformational change occurs.     

Protein-independent folding pathway of the 16S rRNA 5' domain

Evolution of the ribosome from an RNA catalyst suggests that the intrinsic folding pathway of the rRNA dictates the hierarchy of ribosome assembly. To address this possibility, we probed the tertiary folding pathway of the 5' domain of the Escherichia coli 16S rRNA at 20 ms intervals using X-ray-dependent hydroxyl radical footprinting. Comparison with crystallographic structures and footprinting reactions on native 30S ribosomes showed that the RNA formed all of the predicted tertiary interactions in the absence of proteins. In 20 mM MgCl2, many tertiary interactions appeared within 20 ms. By contrast, interactions between H6, H15 and H17 near the spur of the 30S ribosome evolved over several minutes, likely due to mispairing of a central helix junction. The kinetic folding pathway of the RNA corresponded to the expected order of protein binding, suggesting that the RNA folding pathway forms the basis for early steps of ribosome assembly.     

Ribosome biogenesis: of knobs and RNA processing

The synthesis of ribosomes in eukaryotes involves processing of pre-ribosomal RNA (pre-rRNA) and sequential assembly of a large number of ribosomal proteins on the rRNAs. Although we have gained tremendous insights into the processing of pre-rRNA intermediates in the last three decades, little was known about the dynamic nature of ribosome biogenesis. Only recently the development of efficient affinity-purification procedures and mass-spectrometry techniques has allowed the isolation of large pre-ribosomal complexes, which led to the identification of several ribosome assembly intermediates and a large number of novel ribosome assembly factors. In this mini-review, we summarize some of the discoveries that have been made in the field of ribosome biogenesis in the past 30 years and highlight some key aspects about what remains to be learned.     

Ribosome Synthesis in Thermally Shocked Cells of Staphylococcus aureus

Thermally shocked cells of Staphylococcus aureus rapidly synthesized ribonucleic acid (RNA) during the early stages of recovery. During this period, protein synthesis was not observed and occurred only after RNA had reached a maximum level. Even in the absence of coordinated protein synthesis, a large portion of the RNA appeared in newly synthesized ribosomes. Although the 30S subunit was specifically destroyed by the heating process, both ribosomal particles were reassembled during recovery. The addition of chloramphenicol did not inhibit the formation of the ribosomal subunits, nor was the presence of immature chloramphenicol particles detected. Extended recovery with highly prelabeled cells showed that the original ribosomal proteins present before heating are conserved and recycled. Furthermore, the data indicate that the 50S subunit is turned over and used as a source of protein for new ribosome assembly. Kinetic studies of the assembly process by pulse labeling have not revealed the presence of the normally reported precursor particles. Rather, the data suggest that assembly may occur, in this system, in a manner similar to that reported for in vitro assembly of Escherichia coli subunits.     

On the role of protein S4 N-terminal residues 1 through 30 in 30S ribosome function.

30S ribosomal protein S4 contains a single cysteine residue at position 31. We have selectively cleaved the peptide bond adjacent to this residue using the reagent 2-nitro-5-thiocyanobenzoic acid. The two resultant fragments were purified. The smaller S4-fragment (1-30) was found to be incapable of interacting with 16S RNA directly. This fragment also is not incorporated into a particle reconstituted from 16S RNA and 20 purified proteins with S4 missing. In contrast, the large S4-fragment (31-203) appears to be fully functional in ribosome assembly. Replacement of S4 with this fragment in the reconstitution reaction leads to a complete 30S ribosome containing all 30S proteins. This particle has a full capacity to bind poly U but has lost all activity for poly U directed phe-tRNA binding. We therefore propose that the N-terminus of protein S4 is not critical for ribosome assembly but is essential for tRNA binding.     

Understanding ribosome assembly: the structure of in vivo assembled immature 30S subunits revealed by cryo-electron microscopy

Four decades after early in vitro assembly studies demonstrated that ribosome assembly is a controlled process, our understanding of ribosome assembly is still incomplete. Just as structure determination has been so important to understanding ribosome function, so too will it be critical to sorting out the assembly process. Here, we used a viable deletion in the yjeQ gene, a recognized ribosome assembly factor, to isolate and structurally characterize immature 30S subunits assembled in vivo. These small ribosome subunits contained unprocessed 17S rRNA and lacked some late ribosomal proteins. Cryo-electron microscopy reconstructions revealed that the presence of precursor sequences in the rRNA induces a severe distortion in the 3' minor domain of the subunit involved in the decoding of mRNA and interaction with the large ribosome subunit. These findings suggest that rRNA processing events induce key local conformational changes directing the structure toward the mature assembly. We concluded that rRNA processing, folding, and the entry of tertiary r-proteins are interdependent events in the late stages of 30S subunit assembly. In addition, we demonstrate how studies of emerging assembly factors in ribosome biogenesis can help to elucidate the path of subunit assembly in vivo.     

A computational investigation on the connection between dynamics properties of ribosomal proteins and ribosome assembly

Assembly of the ribosome from its protein and RNA constituents has been studied extensively over the past 50 years, and experimental evidence suggests that prokaryotic ribosomal proteins undergo conformational changes during assembly. However, to date, no studies have attempted to elucidate these conformational changes. The present work utilizes computational methods to analyze protein dynamics and to investigate the linkage between dynamics and binding of these proteins during the assembly of the ribosome. Ribosomal proteins are known to be positively charged and we find the percentage of positive residues in r-proteins to be about twice that of the average protein: Lys+Arg is 18.7% for E. coli and 21.2% for T. thermophilus. Also, positive residues constitute a large proportion of RNA contacting residues: 39% for E. coli and 46% for T. thermophilus. This affirms the known importance of charge-charge interactions in the assembly of the ribosome. We studied the dynamics of three primary proteins from E. coli and T. thermophilus 30S subunits that bind early in the assembly (S15, S17, and S20) with atomic molecular dynamic simulations, followed by a study of all r-proteins using elastic network models. Molecular dynamics simulations show that solvent-exposed proteins (S15 and S17) tend to adopt more stable solution conformations than an RNA-embedded protein (S20). We also find protein residues that contact the 16S rRNA are generally more mobile in comparison with the other residues. This is because there is a larger proportion of contacting residues located in flexible loop regions. By the use of elastic network models, which are computationally more efficient, we show that this trend holds for most of the 30S r-proteins.     

Ribosome assembly in HeLa cells: labeling pattern of ribosomal proteins by two-dimensional resolution.

A high resolution, two-dimensional gel electrophoresis of the proteins from HeLa cell large ribosomal subunits and their nucleolar precursor particles is described. There are 40 major spots in the mature particles and about 65 in the precursors. Proteins in the precursor particles include 30 spots which are similar to those in mature large subunits, and at least 33 major spots which are restricted to the precursor stage. Labeling patterns of ribosomes showed a limited number of proteins associated with mature large subunits that incorporate radioactive amino acids more rapidly, indicating those proteins that are recycled in the cytoplasm. Among the proteins associated with pre-ribosomal particles, those that are similar to the proteins of mature ribosomes labeled more rapidly than the precursor-specific nucleolar proteins. The latter are apparently reutilized for ribosome assembly in the nucleolus. Thus, in addition to resolution of the proteins only transiently associated with precursor particles, results indicate the differences in their labeling properties, consistent with their behaviour during ribosome assembly in HeLa cells.     

Constructing ribosomes along the Danube

The EMBO Conference on Ribosome Synthesis held last summer explored the latest breakthroughs in ribosome assembly and how it affects disease. Both of these topics have recently seen important advances that enlighten how almost 200 proteins cooperate to produce a ribosome and how the cell responds to a malfunction in this process.Medieval Regensburg, Germany played host from 26 to 30 August 2009 to more than 230 participants in the eighth triennial international meeting on ‘Ribosome Synthesis,” organized by Herbert Tschochner, Gernot Längst, Philipp Milkereit, David Tollervey and John Woolford. This meeting marked a clear step forward for two main reasons: a substantial advance in understanding the incredibly complex process of eukaryotic ribosome assembly, and an increasing appreciation of the fact that aberrant ribosome synthesis has a critical role in human diseases such as anaemia and cancer.     

Powering through ribosome assembly

Ribosome assembly is required for cell growth in all organisms. Classic in vitro work in bacteria has led to a detailed understanding of the biophysical, thermodynamic, and structural basis for the ordered and correct assembly of ribosomal proteins on ribosomal RNA. Furthermore, it has enabled reconstitution of active subunits from ribosomal RNA and proteins in vitro. Nevertheless, recent work has shown that eukaryotic ribosome assembly requires a large macromolecular machinery in vivo. Many of these assembly factors such as ATPases, GTPases, and kinases hydrolyze nucleotide triphosphates. Because these enzymes are likely regulatory proteins, much work to date has focused on understanding their role in the assembly process. Here, we review these factors, as well as other sources of energy, and their roles in the ribosome assembly process. In addition, we propose roles of energy-releasing enzymes in the assembly process, to explain why energy is used for a process that occurs largely spontaneously in bacteria. Finally, we use literature data to suggest testable models for how these enzymes could be used as targets for regulation of ribosome assembly.     

Quantitative Proteomic Analysis of Ribosome Assembly and Turnover In Vivo

Although high-resolution structures of the ribosome have been solved in a series of functional states, relatively little is known about how the ribosome assembles, particularly in vivo. Here, a general method is presented for studying the dynamics of ribosome assembly and ribosomal assembly intermediates. Since significant quantities of assembly intermediates are not present under normal growth conditions, the antibiotic neomycin is used to perturb wild-type Escherichia coli. Treatment of E. coli with the antibiotic neomycin results in the accumulation of a continuum of assembly intermediates for both the 30S and 50S subunits. The protein composition and the protein stoichiometry of these intermediates were determined by quantitative mass spectrometry using purified unlabeled and ¹⁵N-labeled wild-type ribosomes as external standards. The intermediates throughout the continuum are heterogeneous and are largely depleted of late-binding proteins. Pulse-labeling with ¹⁵N-labeled medium time-stamps the ribosomal proteins based on their time of synthesis. The assembly intermediates contain both newly synthesized proteins and proteins that originated in previously synthesized intact subunits. This observation requires either a significant amount of ribosome degradation or the exchange or reuse of ribosomal proteins. These specific methods can be applied to any system where ribosomal assembly intermediates accumulate, including strains with deletions or mutations of assembly factors. This general approach can be applied to study the dynamics of assembly and turnover of other macromolecular complexes that can be isolated from cells.     

Gateway Role for rRNA Precursors in Ribosome Assembly

In Escherichia coli, rRNAs are initially transcribed with precursor sequences, which are subsequently removed through processing reactions. To investigate the role of precursor sequences, we analyzed ribosome assembly in strains containing mutations in the processing RNases. We observed that defects in 23S rRNA processing resulted in an accumulation of ribosomal subunits and caused a significant delay in ribosome assembly. These observations suggest that precursor residues in 23S rRNA control ribosome assembly and could be serving a regulatory role to couple ribosome assembly to rRNA processing. The possible mechanisms through which rRNA processing and ribosome assembly could be linked are discussed.     

Limited proteolysis analysis of the ribosome is affected by subunit association

Our understanding of the structural organization of ribosome assembly intermediates, in particular those intermediates that result from misfolding leading to their eventual degradation within the cell, is limited because of the lack of methods available to characterize assembly intermediate structures. Because conventional structural approaches, such as NMR, X‐ray crystallography, and cryo‐EM, are not ideally suited to characterize the structural organization of these flexible and sometimes heterogeneous assembly intermediates, we have set out to develop an approach combining limited proteolysis with matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) that might be applicable to ribonucleoprotein complexes as large as the ribosome. This study focuses on the limited proteolysis behavior of appropriately assembled ribosome subunits. Isolated subunits were analyzed using limited proteolysis and MALDI‐MS and the results were compared with previous data obtained from 70S ribosomes. Generally, ribosomal proteins were found to be more stable in 70S ribosomes than in their isolated subunits, consistent with a reduction in conformational flexibility on subunit assembly. This approach demonstrates that limited proteolysis combined with MALDI‐MS can reveal structural changes to ribosomes on subunit assembly or disassembly, and provides the appropriate benchmark data from 30S, 50S, and 70S proteins to enable studies of ribosome assembly intermediates. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 410–422, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Dependency map of proteins in the small ribosomal subunit

The assembly of the ribosome has recently become an interesting target for antibiotics in several bacteria. In this work, we extended an analytical procedure to determine native state fluctuations and contact breaking to investigate the protein stability dependence in the 30S small ribosomal subunit of Thermus thermophilus. We determined the causal influence of the presence and absence of proteins in the 30S complex on the binding free energies of other proteins. The predicted dependencies are in overall agreement with the experimentally determined assembly map for another organism, Escherichia coli. We found that the causal influences result from two distinct mechanisms: one is pure internal energy change, the other originates from the entropy change. We discuss the implications on how to target the ribosomal assembly most effectively by suggesting six proteins as targets for mutations or other hindering of their binding. Our results show that by blocking one out of this set of proteins, the association of other proteins is eventually reduced, thus reducing the translation efficiency even more. We could additionally determine the binding dependency of THX—a peptide not present in the ribosome of E. coli—and suggest its assembly path.     

Circularly permuted GTPase YqeH binds 30S ribosomal subunit: Implications for its role in ribosome assembly

YqeH, a circularly permuted GTPase, is conserved among bacteria and eukaryotes including humans. It was shown to be essential for the assembly of small ribosomal (30S) subunit in bacteria. However, whether YqeH interacts with 30S ribosome and how it may participate in 30S assembly are not known. Here, using co-sedimentation experiments, we report that YqeH co-associates with 30S ribosome in the GTP-bound form. In order to probe whether YqeH functions as RNA chaperone in 30S assembly, we assayed for strand dissociation and annealing activity. While YqeH does not exhibit these activities, it binds a non-specific single and double-stranded RNA, which unlike the 30S binding is independent of GTP/GDP binding and does not affect intrinsic GTP hydrolysis rates. Further, S5, a ribosomal protein which participates during the initial stages of 30S assembly, was found to promote GTP hydrolysis and RNA binding activities of YqeH.     

The function of RH22, a DEAD RNA helicase, in the biogenesis of the 50S ribosomal subunits of Arabidopsis chloroplasts

The chloroplast ribosome is a large and dynamic ribonucleoprotein machine that is composed of the 30S and 50S subunits. Although the components of the chloroplast ribosome have been identified in the last decade, the molecular mechanisms driving chloroplast ribosome biogenesis remain largely elusive. Here, we show that RNA helicase 22 (RH22), a putative DEAD RNA helicase, is involved in chloroplast ribosome assembly in Arabidopsis (Arabidopsis thaliana). A loss of RH22 was lethal, whereas a knockdown of RH22 expression resulted in virescent seedlings with clear defects in chloroplast ribosomal RNA (rRNA) accumulation. The precursors of 23S and 4.5S, but not 16S, rRNA accumulated in rh22 mutants. Further analysis showed that RH22 was associated with the precursors of 50S ribosomal subunits. These results suggest that RH22 may function in the assembly of 50S ribosomal subunits in chloroplasts. In addition, RH22 interacted with the 50S ribosomal protein RPL24 through yeast two-hybrid and pull-down assays, and it was also bound to a small 23S rRNA fragment encompassing RPL24-binding sites. This action of RH22 may be similar to, but distinct from, that of SrmB, a DEAD RNA helicase that is involved in the ribosomal assembly in Escherichia coli, which suggests that DEAD RNA helicases and rRNA structures may have coevolved with respect to ribosomal assembly and function.     

The essential GTPase YqeH is required for proper ribosome assembly in Bacillus subtilis

Recent work with bacteria and eukaryotes has shown that GTPases play important roles in ribosome assembly. Here we show that the essential GTPase YqeH is required for proper 70S ribosome formation and 30S subunit assembly/stability in Bacillus subtilis.     

Studies on the kinetic sequence of in vitro ribosome assembly using cibacron blue F3GA as a general assembly inhibitor.

We have found that all E. coli ribosomal proteins strongly bind to an agarose affinity column derivatized with the dye Cibacron Blue F3GA. We have also shown that the capacity to bind the dye is lost when the proteins are organized within the structure of the ribosome or are members of pre-formed protein-RNA complexes. We conclude that the binding of ribosomal proteins to this dye involves specific protein-RNA recognition sites. These observations led us to discover that Cibacron Blue can be used to inhibit in vitro ribosome assembly at any stage of the assembly process. This has allowed us to determine a kinetic order of ribosome assembly.     

Global ribosome motions revealed with elastic network model

The motions of large systems such as the ribosome are not fully accessible with conventional molecular simulations. A coarse-grained, less-than-atomic-detail model such as the anisotropic network model (ANM) is a convenient informative tool to study the cooperative motions of the ribosome. The motions of the small 30S subunit, the larger 50S subunit, and the entire 70S assembly of the two subunits have been analyzed using ANM. The lowest frequency collective modes predicted by ANM show that the 50S subunit and 30S subunit are strongly anti-correlated in the motion of the 70S assembly. A ratchet-like motion is observed that corresponds well to the experimentally reported ratchet motion. Other slow modes are also examined because of their potential links to the translocation steps in the ribosome. We identify several modes that may facilitate the E-tRNA exiting from the assembly. The A-site t-RNA and P-site t-RNA are found to be strongly coupled and positively correlated in these slow modes, suggesting that the translocations of these two t-RNAs occur simultaneously, while the motions of the E-site t-RNA are less correlated, and thus less likely to occur simultaneously. Overall the t-RNAs exhibit relatively large deformations. Animations of these slow modes of motion can be viewed at.