Interdependency and Redundancy Add Complexity and Resilience to Biogenesis of Bacterial Ribosomes.

The pace and efficiency of ribosomal subunit production directly impact the fitness of bacteria. Biogenesis demands more than just the union of ribosomal components, including RNA and proteins, to form this functional ribonucleoprotein particle. Extra-ribosomal protein factors play a fundamental role in the efficiency and efficacy of ribosomal subunit biogenesis. A paucity of data on intermediate steps, multiple and overlapping pathways, and the puzzling number of functions that extra-ribosomal proteins appear to play in vivo make unraveling the formation of this macromolecular assemblage difficult. In this review, we outline with examples the multinodal landscape of factor-assisted mechanisms that influence ribosome synthesis in bacteria. We discuss in detail late-stage events that mediate correct ribosome formation and the transition to translation initiation and thereby ensure high-fidelity protein synthesis.

Uncovering a delicate balance between endonuclease RNase III and ribosomal protein S15 in E. coli ribosome assembly.

The bacterial ribosomal protein S15 is located in the platform, a functional region of the 30S ribosomal subunit. While S15 is critical for in vitro formation of E. coli small subunits (SSUs), it is dispensable for in vivo biogenesis and growth. In this work, a novel synergistic interaction between rpsO, the gene that encodes S15, and rnc (the gene that encodes RNase III), was uncovered in E. coli. RNase III catalyzes processing of precursor ribosomal RNA (rRNA) transcripts and thus is involved in functional ribosome subunit maturation. Strains lacking S15 (ΔrpsO), RNase III (Δrnc) or both genes were examined to understand the relationship between these two factors and the impact of this double deletion on rRNA processing and SSU maturation. The double deletion of rpsO and rnc partially alleviates the observed cold sensitivity of ΔrpsO alone. A novel 16S rRNA precursor (17S∗ rRNA) that is detected in free 30S subunits of Δrnc is incorporated in 70S-like ribosomes in the double deletion. The stable accumulation of 17S∗ rRNA suggests that timing of processing events is closely coupled with SSU formation events in vivo. The double deletion has a suppressive effect on the cell elongation phenotype of ΔrpsO. The alteration of the phenotypes associated with S15 loss, due to the absence of RNase III, indicates that pre-rRNA processing and improvement of growth, relative to that observed for ΔrpsO, are connected. The characterization of the functional link between the two factors illustrates that there are redundancies and compensatory pathways for SSU maturation.

Characterization of 16S rRNA Processing with Pre-30S Subunit Assembly Intermediates from E. coli.

Ribosomal RNA (rRNA) is a major component of ribosomes and is fundamental to the process of translation. In bacteria, 16S rRNA is a component of the small ribosomal subunit and plays a critical role in mRNA decoding. rRNA maturation entails the removal of intervening spacer sequences contained within the pre-rRNA transcript by nucleolytic enzymes. Enzymatic activities involved in maturation of the 5'-end of 16S rRNA have been identified, but those involved in 3'-end maturation of 16S rRNA are more enigmatic. Here, we investigate molecular details of 16S rRNA maturation using purified in vivo-formed small subunit (SSU) assembly intermediates (pre-SSUs) from wild-type Escherichia coli that contain precursor 16S rRNA (17S rRNA). Upon incubation of pre-SSUs with E. coli S100 cell extracts or purified enzymes implicated in 16S rRNA processing, the 17S rRNA is processed into additional intermediates and mature 16S rRNA. These results illustrate that exonucleases RNase R, RNase II, PNPase, and RNase PH can process the 3'-end of pre-SSUs in vitro. However, the endonuclease YbeY did not exhibit nucleolytic activity with pre-SSUs under these conditions. Furthermore, these data demonstrate that multiple pathways facilitate 16S rRNA maturation with pre-SSUs in vitro, with the dominant pathways entailing complete processing of the 5'-end of 17S rRNA prior to 3'-end maturation or partial processing of the 5'-end with concomitant processing of the 3'-end. These results reveal the multifaceted nature of SSU biogenesis and suggest that E. coli may be able to escape inactivation of any one enzyme by using an existing complementary pathway.

Analysis of r-protein and RNA conformation of 30S subunit intermediates in bacteria.

The ribosome is a large macromolecular complex that must be assembled efficiently and accurately for the viability of all organisms. In bacteria, this process must be robust and tunable to support life in diverse conditions from the ice of arctic glaciers to thermal hot springs. Assembly of the Small ribosomal SUbunit (SSU) of Escherichia coli has been extensively studied and is highly temperature-dependent. However, a lack of data on SSU assembly for other bacteria is problematic given the importance of the ribosome in bacterial physiology. To broaden the understanding of how optimal growth temperature may affect SSU assembly, in vitro SSU assembly of two thermophilic bacteria, Geobacillus kaustophilus and Thermus thermophilus, was compared with that of E. coli. Using these phylogenetically, morphologically, and environmentally diverse bacteria, we show that SSU assembly is highly temperature-dependent and efficient SSU assembly occurs at different temperatures for each organism. Surprisingly, the assembly landscape is characterized by at least two distinct intermediate populations in the organisms tested. This novel, second intermediate, is formed in the presence of the full complement of r-proteins, unlike the previously observed RI* particle formed in the absence of late-binding r-proteins in E. coli. This work reveals multiple distinct intermediate populations are present during SSU assembly in vitro for several bacteria, yielding insights into RNP formation and possible antimicrobial development toward this common SSU target.

Multiple in vivo pathways for Escherichia coli small ribosomal subunit assembly occur on one pre-rRNA.

Processing of transcribed precursor ribosomal RNA (pre-rRNA) to a mature state is a conserved aspect of ribosome biogenesis in vivo. We developed an affinity-purification system to isolate and analyze in vivo-formed pre-rRNA-containing ribonucleoprotein (RNP) particles (rRNPs) from wild-type E. coli. We observed that the first processing intermediate of pre-small subunit (pre-SSU) rRNA is a platform for biogenesis. These pre-SSU-containing RNPs have differing ribosomal-protein and auxiliary factor association and rRNA folding. Each RNP lacks the proper architecture in functional regions, thus suggesting that checkpoints preclude immature subunits from entering the translational cycle. This work offers in vivo snapshots of SSU biogenesis and reveals that multiple pathways exist for the entire SSU biogenesis process in wild-type E. coli. These findings have implications for understanding SSU biogenesis in vivo and offer a general strategy for analysis of RNP biogenesis.

The N-terminal extension of S12 influences small ribosomal subunit assembly in Escherichia coli.

The small subunit (SSU) of the ribosome of E. coli consists of a core of ribosomal RNA (rRNA) surrounded peripherally by ribosomal proteins (r-proteins). Ten of the 15 universally conserved SSU r-proteins possess nonglobular regions called extensions. The N-terminal noncanonically structured extension of S12 traverses from the solvent to intersubunit surface of the SSU and is followed by a more C-terminal globular region that is adjacent to the decoding center of the SSU. The role of the globular region in maintaining translational fidelity is well characterized, but a role for the S12 extension in SSU structure and function is unknown. We examined the effect of stepwise truncation of the extension of S12 in SSU assembly and function in vitro and in vivo. Examination of in vitro assembly in the presence of sequential N-terminal truncated variants of S12 reveals that N-terminal deletions of greater than nine amino acids exhibit decreased tRNA-binding activity and altered 16S rRNA architecture particularly in the platform of the SSU. While wild-type S12 expressed from a plasmid can rescue a genomic deletion of the essential gene for S12, rpsl; N-terminal deletions of S12 exhibit deleterious phenotypic consequences. Partial N-terminal deletions of S12 are slow growing and cold sensitive. Strains bearing these truncations as the sole copy of S12 have increased levels of free SSUs and immature 16S rRNA as compared with the wild-type S12. These differences are hallmarks of SSU biogenesis defects, indicating that the extension of S12 plays an important role in SSU assembly.

Interdependencies govern multidomain architecture in ribosomal small subunit assembly.

The 30S subunit is composed of four structural domains, the body, platform, head, and penultimate/ultimate stems. The functional integrity of the 30S subunit is dependent upon appropriate assembly and precise orientation of all four domains. We examined 16S rRNA conformational changes during in vitro assembly using directed hydroxyl radical probing mediated by Fe(II)-derivatized ribosomal protein (r-protein) S8. R-protein S8 binds the central domain of 16S rRNA directly and independently and its iron derivatized substituents have been shown to mediate cleavage in three domains of 16S rRNA, thus making it an ideal probe to monitor multidomain orientation during assembly. Cleavages in minimal ribonucleoprotein (RNP) particles formed with Fe(II)-S8 and 16S rRNA alone were compared with that in the context of the fully assembled subunit. The minimal binding site of S8 at helix 21 exists in a structure similar to that observed in the mature subunit, in the absence of other r-proteins. However, the binding site of S8 at the junction of helices 25-26a, which is transcribed after helix 21, is cleaved with differing intensities in the presence and absence of other r-proteins. Also, assembly of the body helps establish an architecture approximating, but perhaps not identical, to the 30S subunit at helix 12 and the 5' terminus. Moreover, the assembly or orientation of the neck is dependent upon assembly of both the head and the body. Thus, a complex interrelationship is observed between assembly events of independent domains and the incorporation of primary binding proteins during 30S subunit formation.

Site-directed mutants of 16S rRNA reveal important RNA domains for KsgA function and 30S subunit assembly.

KsgA is an rRNA methyltransferase important to the process of small subunit biogenesis in bacteria. It is ubiquitously found in all life including archaea and eukarya, where the enzyme is referred to as Dim1. Despite the emergence of considerable data addressing KsgA function over the last several years, details pertaining to RNA recognition are limited, in part because the most accessible substrate for in vitro studies of KsgA is the 900000 Da 30S ribosomal subunit. To overcome challenges imposed by size and complexity, we adapted recently reported techniques to construct in vivo assembled mutant 30S subunits suitable for use in in vitro methyltransferase assays. Using this approach, numerous 16S rRNA mutants were constructed and tested. Our observations indicate that the 790 loop of helix 24 plays an important role in overall catalysis by KsgA. Moreover, the length of helix 45 also is important to catalysis. In both cases loss of catalytic function occurred without an increase in the production of N(6)-methyladenosine, a likely indication that there was no critical reduction in binding strength. Both sets of observations support a "proximity" mechanism of KsgA function. We also report that several of the mutants constructed failed to assemble properly into 30S subunits, while some others did so with reduced efficiency. Therefore, the same technique of generating mutant 30S subunits can be used to study ribosome biogenesis on the whole.

Differential assembly of 16S rRNA domains during 30S subunit formation.

Rapid and accurate assembly of the ribosomal subunits, which are responsible for protein synthesis, is required to sustain cell growth. Our best understanding of the interaction of 30S ribosomal subunit components (16S ribosomal RNA [rRNA] and 20 ribosomal proteins [r-proteins]) comes from in vitro work using Escherichia coli ribosomal components. However, detailed information regarding the essential elements involved in the assembly of 30S subunits still remains elusive. Here, we defined a set of rRNA nucleotides that are critical for the assembly of the small ribosomal subunit in E. coli. Using an RNA modification interference approach, we identified 54 nucleotides in 16S rRNA whose modification prevents the formation of a functional small ribosomal subunit. The majority of these nucleotides are located in the head and interdomain junction of the 30S subunit, suggesting that these regions are critical for small subunit assembly. In vivo analysis of specific identified sites, using engineered mutations in 16S rRNA, revealed defective protein synthesis capability, aberrant polysome profiles, and abnormal 16S rRNA processing, indicating the importance of these residues in vivo. These studies reveal that specific segments of 16S rRNA are more critical for small subunit assembly than others, and suggest a hierarchy of importance.

Appropriate maturation and folding of 16S rRNA during 30S subunit biogenesis are critical for translational fidelity.

Ribosomal protein S5 is critical for small ribosomal subunit (SSU) assembly and is indispensable for SSU function. Previously, we identified a point mutation in S5, (G28D) that alters both SSU formation and translational fidelity in vivo, which is unprecedented for other characterized S5 mutations. Surprisingly, additional copies of an extraribosomal assembly factor, RimJ, rescued all the phenotypes associated with S5(G28D), including fidelity defects, suggesting that the effect of RimJ on rescuing the miscoding of S5(G28D) is indirect. To understand the underlying mechanism, we focused on the biogenesis cascade and observed defects in processing of precursor 16S (p16S) rRNA in the S5(G28D) strain, which were rescued by RimJ. Analyses of p16S rRNA-containing ribosomes from other strains further supported a correspondence between the extent of 5(') end maturation of 16S rRNA and translational miscoding. Chemical probing of mutant ribosomes with additional leader sequences at the 5(') end of 16S rRNA compared to WT ribosomes revealed structural differences in the region of helix 1. Thus, the presence of additional nucleotides at the 5(') end of 16S rRNA could alter fidelity by changing the architecture of 16S rRNA in translating ribosomes and suggests that fidelity is governed by accuracy and completeness of the SSU biogenesis cascade.

Structural and functional divergence within the Dim1/KsgA family of rRNA methyltransferases.

The enzymes of the KsgA/Dim1 family are universally distributed throughout all phylogeny; however, structural and functional differences are known to exist. The well-characterized function of these enzymes is to dimethylate two adjacent adenosines of the small ribosomal subunit in the normal course of ribosome maturation, and the structures of KsgA from Escherichia coli and Dim1 from Homo sapiens and Plasmodium falciparum have been determined. To this point, no examples of archaeal structures have been reported. Here, we report the structure of Dim1 from the thermophilic archaeon Methanocaldococcus jannaschii. While it shares obvious similarities with the bacterial and eukaryotic orthologs, notable structural differences exist among the three members, particularly in the C-terminal domain. Previous work showed that eukaryotic and archaeal Dim1 were able to robustly complement for KsgA in E. coli. Here, we repeated similar experiments to test for complementarity of archaeal Dim1 and bacterial KsgA in Saccharomyces cerevisiae. However, neither the bacterial nor the archaeal ortholog could complement for the eukaryotic Dim1. This might be related to the secondary, non-methyltransferase function that Dim1 is known to play in eukaryotic ribosomal maturation. To further delineate regions of the eukaryotic Dim1 critical to its function, we created and tested KsgA/Dim1 chimeras. Of the chimeras, only one constructed with the N-terminal domain from eukaryotic Dim1 and the C-terminal domain from archaeal Dim1 was able to complement, suggesting that eukaryotic-specific Dim1 function resides in the N-terminal domain also, where few structural differences are observed between members of the KsgA/Dim1 family. Future work is required to identify those determinants directly responsible for Dim1 function in ribosome biogenesis. Finally, we have conclusively established that none of the methyl groups are critically important to growth in yeast under standard conditions at a variety of temperatures.

Chemical probing of RNA and RNA/protein complexes.

Chemical probing is widely used as a rapid approach for assessing RNA structure, folding, and function. In this chapter, we outline procedures for handling and using chemicals commonly used to probe nucleic acids. Detailed experimental conditions and design for footprinting and modification interference are presented herein. Protocols for RNA extraction, normalization, primer extension, and data evaluation are also provided. The methods described are designed to aid in the study of large RNAs, but with slight modifications are applicable to smaller RNAs.

Sequence and structural evolution of the KsgA/Dim1 methyltransferase family.

BACKGROUND: One of the 60 or so genes conserved in all domains of life is the ksgA/dim1 orthologous group. Enzymes from this family perform the same post-transcriptional nucleotide modification in ribosome biogenesis, irrespective of organism. Despite this common function, divergence has enabled some family members to adopt new and sometimes radically different functions. For example, in S. cerevisiae Dim1 performs two distinct functions in ribosome biogenesis, while human mtTFB is not only an rRNA methyltransferase in the mitochondria but also a mitochondrial transcription factor. Thus, these proteins offer an unprecedented opportunity to study evolutionary aspects of structure/function relationships, especially with respect to our recently published work on the binding mode of a KsgA family member to its 30S subunit substrate. Here we compare and contrast KsgA orthologs from bacteria, eukaryotes, and mitochondria as well as the paralogous ErmC enzyme. RESULTS: By using structure and sequence comparisons in concert with a unified ribosome binding model, we have identified regions of the orthologs that are likely related to gains of function beyond the common methyltransferase function. There are core regions common to the entire enzyme class that are associated with ribosome binding, an event required in rRNA methylation activity, and regions that are conserved in subgroups that are presumably related to non-methyltransferase functions. CONCLUSION: The ancient protein KsgA/Dim1 has adapted to cellular roles beyond that of merely an rRNA methyltransferase. These results provide a structural foundation for analysis of multiple aspects of ribosome biogenesis and mitochondrial transcription.

Suppression of a cold-sensitive mutation in ribosomal protein S5 reveals a role for RimJ in ribosome biogenesis.

A specific mutation of Escherichia coli ribosomal protein S5, in which glycine is changed to aspartate at position 28 [S5(G28D)], results in cold sensitivity and defects in ribosome biogenesis and translational fidelity. In an attempt to understand the roles of S5 in these essential cellular functions, we selected extragenic suppressors and identified rimJ as a high-copy suppressor of the cold-sensitive phenotype associated with the S5(G28D) mutation. Our studies indicate that RimJ overexpression suppresses the growth defects, anomalous ribosome profiles and mRNA misreading exhibited by the S5(G28D) mutant strain. Although previously characterized as the N-acetyltransferase of S5, our data indicate that RimJ, when devoid of acetyltransferase activity, can suppress S5(G28D) defects thus indicating that the suppression activity of RimJ is not dependent on its acetyltransferase activity. Additionally, RimJ appears to associate with pre-30S subunits indicating that it acts on the ribonucleoprotein particle. These findings suggest that RimJ has evolved dual functionality; it functions in r-protein acetylation and as a ribosome assembly factor in E. coli.

A conserved rRNA methyltransferase regulates ribosome biogenesis.

In contrast to the diversity of most ribosomal RNA modification patterns and systems, the KsgA methyltransferase family seems to be nearly universally conserved along with the modifications it catalyzes. Our data reveal that KsgA interacts with small ribosomal subunits near functional sites, including Initiation factor 3 and 50S subunit binding sites. These findings suggest a checkpoint role for this modification system and offer a functional rationale for the unprecedented level of conservation.

Assembly of the 30S Ribosomal Subunit.

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. This review provides an overview of the current understanding of the assembly of the Escherichia coli 30S ribosomal subunit. The E. coli 30S subunit contains one rRNA molecule (16S) and 21 ribosomal proteins (r-proteins; S1 to S21). 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. In vitro reconstitution of functional 30S subunits is carried out by using a mixture of TP30, individually purified natural or recombinant r-proteins, and natural 16S rRNA. 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. The potential roles for r-proteins in 30S subunit assembly were determined by omitting single proteins in reconstitution experiments. The RNPs resulting from single protein omissions were examined in terms of their composition and function to determine the roles of the absent proteins. 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 (1, 2) and the 70S ribosome (3) reveal details of the r-protein and rRNA interactions.

Assembly of the 5' and 3' minor domains of 16S ribosomal RNA as monitored by tethered probing from ribosomal protein S20.

The ribosomal protein (r-protein) S20 is a primary binding protein. As such, it interacts directly and independently with the 5' domain as well as the 3' minor domain of 16S ribosomal RNA (rRNA) in minimal particles and the fully assembled 30S subunit. The interactions observed between r-protein S20 and the 5' domain of 16S rRNA are quite extensive, while those between r-protein S20 and the 3' minor domain are significantly more limited. In this study, directed hydroxyl radical probing mediated by Fe(II)-derivatized S20 proteins was used to monitor the folding of 16S rRNA during r-protein association and 30S subunit assembly. An analysis of the cleavage patterns in the minimal complexes [16S rRNA and Fe(II)-S20] and the fully assembled 30S subunit containing the same Fe(II)-derivatized proteins shows intriguing similarities and differences. These results suggest that the two domains, 5' and 3' minor, are organized relative to S20 at different stages of assembly. The 5' domain acquires, in a less complex ribonucleoprotein particle than the 3' minor domain, the same architecture as observed in mature subunits. These results are similar to what would be predicted of subunit assembly by the 5'-to-3' direction assembly model.

Breaking the cycle of translation.

In a recent issue of Molecular Cell, Trobro and Aqvist (2007) reported mechanistic insight into release factor-induced peptide hydrolysis. Now, in this issue, the Green research group establishes unexpected complexity in decoding translation stop codons (Youngman et al., 2007).

Temperature-dependent RNP conformational rearrangements: analysis of binary complexes of primary binding proteins with 16 S rRNA.

Ribonucleoprotein particles (RNPs) are important components of all living systems, and the assembly of these particles is an intricate, often multistep, process. The 30 S ribosomal subunit is composed of one large RNA (16 S rRNA) and 21 ribosomal proteins (r-proteins). In vitro studies have revealed that assembly of the 30 S subunit is a temperature-dependent process involving sequential binding of r-proteins and conformational changes of 16 S rRNA. Additionally, a temperature-dependent conformational rearrangement was reported for a complex of primary r-protein S4 and 16 S rRNA. Given these observations, a systematic study of the temperature-dependence of 16 S rRNA architecture in individual complexes with the other five primary binding proteins (S7, S8, S15, S17, and S20) was performed. While all primary binding r-proteins bind 16 S rRNA at low temperature, not all r-proteins/16 S rRNA complexes undergo temperature-dependent conformational rearrangements. Some RNPs achieve the same conformation regardless of temperature, others show minor adjustments in 16 S rRNA conformation upon heating and, finally, others undergo significant temperature-dependent changes. Some of the architectures achieved in these rearrangements are consistent with subsequent downstream assembly events such as assembly of the secondary and tertiary binding r-proteins. The differential interaction of 16 S rRNA with r-proteins illustrates a means for controlling the sequential assembly pathway for complex RNPs and may offer insights into aspects of RNP assembly in general.

A novel single amino acid change in small subunit ribosomal protein S5 has profound effects on translational fidelity.

S5 is a small subunit ribosomal protein (r-protein) linked to the functional center of the 30S ribosomal subunit. In this study we have identified a unique amino acid mutation in Escherichia coli S5 that produces spectinomycin-resistance and cold sensitivity. This mutation significantly alters cell growth, folding of 16S ribosomal RNA, and translational fidelity. While translation initiation is not affected, both +1 and -1 frameshifting and nonsense suppression are greatly enhanced in the mutant strain. Interestingly, this S5 ribosome ambiguity-like mutation is spatially remote from previously identified S5 ribosome ambiguity (ram) mutations. This suggests that the mechanism responsible for ram phenotypes in the novel mutant strain is possibly distinct from those proposed for other known S5 (and S4) ram mutants. This study highlights the importance of S5 in ribosome function and cell physiology, and suggests that translational fidelity can be regulated in multiple ways.