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.     

Specificity and kinetics of 23S rRNA modification enzymes RlmH and RluD

Along the ribosome assembly pathway, various ribosomal RNA processing and modification reactions take place. Stem–loop 69 in the large subunit of Escherichia coli ribosomes plays a substantial role in ribosome functioning. It contains three highly conserved pseudouridines synthesized by pseudouridine synthase RluD. One of the pseudouridines is further methylated by RlmH. In this paper we show that RlmH has unique substrate specificity among rRNA modification enzymes. It preferentially methylates pseudouridine and less efficiently uridine. Furthermore, RlmH is the only known modification enzyme that is specific to 70S ribosomes. Kinetic parameters determined for RlmH are the following: The apparent K_M for substrate 70S ribosomes is 0.51 ± 0.06 μM, and for cofactor S-adenosyl-L-methionine 27 ± 3 μM; the k_cat values are 4.95 ± 1.10 min⁻¹ and 6.4 ± 1.3 min⁻¹, respectively. Knowledge of the substrate specificity and the kinetic parameters of RlmH made it possible to determine the kinetic parameters for RluD as well. The K_M value for substrate 50S subunits is 0.98 ± 0.18 μM and the k_cat value is 1.97 ± 0.46 min⁻¹. RluD is the first rRNA pseudouridine synthase to be kinetically characterized. The determined rates of RluD- and RlmH-directed modifications of 23S rRNA are compatible with the rate of 50S assembly in vivo. The fact that RlmH requires 30S subunits demonstrates the dependence of 50S subunit maturation on the simultaneous presence of 30S subunits.     

The structural basis of macrolide-ribosome binding assessed using mutagenesis of 23S rRNA positions 2058 and 2059

Macrolides are a diverse group of antibiotics that inhibit bacterial growth by binding within the peptide tunnel of the 50S ribosomal subunit. There is good agreement about the architecture of the macrolide site from different crystallography studies of bacterial and archaeal 50S subunits. These structures show plainly that 23S rRNA nucleotides A2058 and A2059 are located accessibly on the surface of the tunnel wall where they act as key contact sites for macrolide binding. However, the molecular details of how macrolides fit into this site remain a matter of contention. Here, we have generated an isogenic set of single and dual substitutions at A2058 and A2059 in Mycobacterium smegmatis to investigate the effects of the rRNA mutations on macrolide binding. Resistances conferred to a comprehensive array of 11 macrolide compounds are used to assess models of macrolide binding predicted from the crystal structures. The data indicate that all macrolides and their derivatives bind at the same site in the tunnel with their C5 amino sugar in a similar orientation. Our data are compatible with the lactone rings of 14-membered and 16-membered macrolides adopting different conformations, enabling the latter compounds to avoid a steric clash with 2058G. This difference, together with interactions conveyed via substituents that are specific to certain ketolide and macrolide sub-classes, influences the binding to the large ribosomal subunit. Our genetic data show no support for a derivatized-macrolide binding site that has been proposed to be located further down the tunnel.     

Analysis of conformational changes in 16 S rRNA during the course of 30 S subunit assembly

Ribosome biogenesis involves an integrated series of binding events coupled with conformational changes that ultimately result in the formation of a functional macromolecular complex. In vitro, Escherichia coli 30 S subunit assembly occurs in a cooperative manner with the ordered addition of 20 ribosomal proteins (r-proteins) with 16 S rRNA. The assembly pathway for 30 S subunits has been dissected in vitro into three steps, where specific r-proteins associate with 16 S rRNA early in 30 S subunit assembly, followed by a mid-assembly conformational rearrangement of the complex that then enables the remaining r-proteins to associate in the final step. Although the three steps of 30 S subunit assembly have been known for some time, few details have been elucidated about changes that occur as a result of these three specific stages. Here, we present a detailed analysis of the concerted early and late stages of small ribosomal subunit assembly. Conformational changes, roles for base-pairing and r-proteins at specific stages of assembly, and a polar nature to the assembly process have been revealed. This work has allowed a more comprehensive and global view of E.coli 30 S ribosomal subunit assembly to be obtained.     

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.     

Sequence Analysis and Comparison of 16S rRNA, 23S rRNA and 16S/23S Intergenic Spacer Region of Greening Bacterium Associated with Yellowing Disease (Huanglongbing) of Kinnow Mandarin

High incidence (up to 40%) of symptoms of yellowing and yellow mottling was observed in 5–8 years old orchards of kinnow mandarin {Citrus reticulate Balanco (‘King’ × ‘Willow mandarin’)} in the Punjab state of India during a survey in January 2007. These symptoms are often confused with nutrient deficiency and other stress related disorders. However, a greening bacterium has been attributed to cause the disease. The disease was graft transmissible and sequencing of 16S rRNA, 16S/23S intergenic spacer region and 23S rRNA of the greening bacterium associated with yellowing disease in kinnow mandarin confirmed it to be Candidatus Liberibacter asiaticus (‘Ca L. asiaticus’) showing maximum identity of 95.9% with ‘Ca L. asiaticus’ from USA and Brazil in 16S rRNA. The study indicates definite association of ‘Ca L. asiaticus’ with yellowing/chlorotic mottling symptoms of greening disease of kinnow mandarin in Punjab state of India.     

Mutations of ribosomal protein S5 suppress a defect in late-30S ribosomal subunit biogenesis caused by lack of the RbfA biogenesis factor

The in vivo assembly of ribosomal subunits requires assistance by maturation proteins that are not part of mature ribosomes. One such protein, RbfA, associates with the 30S ribosomal subunits. Loss of RbfA causes cold sensitivity and defects of the 30S subunit biogenesis and its overexpression partially suppresses the dominant cold sensitivity caused by a C23U mutation in the central pseudoknot of 16S rRNA, a structure essential for ribosome function. We have isolated suppressor mutations that restore partially the growth of an RbfA-lacking strain. Most of the strongest suppressor mutations alter one out of three distinct positions in the carboxy-terminal domain of ribosomal protein S5 (S5) in direct contact with helix 1 and helix 2 of the central pseudoknot. Their effect is to increase the translational capacity of the RbfA-lacking strain as evidenced by an increase in polysomes in the suppressed strains. Overexpression of RimP, a protein factor that along with RbfA regulates formation of the ribosome''s central pseudoknot, was lethal to the RbfA-lacking strain but not to a wild-type strain and this lethality was suppressed by the alterations in S5. The S5 mutants alter translational fidelity but these changes do not explain consistently their effect on the RbfA-lacking strain. Our genetic results support a role for the region of S5 modified in the suppressors in the formation of the central pseudoknot in 16S rRNA.     

Structural insights into the assembly of the 30S ribosomal subunit in vivo: functional role of S5 and location of the 17S rRNA precursor sequence

The in vivo assembly of ribosomal subunits is a highly complex process, with a tight coordination between protein assembly and rRNA maturation events, such as folding and processing of rRNA precursors, as well as modifications of selected bases. In the cell, a large number of factors are required to ensure the efficiency and fidelity of subunit production. Here we characterize the immature 30S subunits accumulated in a factor-null Escherichia coli strain (?rsgA?rbfA). The immature 30S subunits isolated with varying salt concentrations in the buffer system show interesting differences on both protein composition and structure. Specifically, intermediates derived under the two contrasting salt conditions (high and low) likely reflect two distinctive assembly stages, the relatively early and late stages of the 3' domain assembly, respectively. Detailed structural analysis demonstrates a mechanistic coupling between the maturation of the 5' end of the 17S rRNA and the assembly of the 30S head domain, and attributes a unique role of S5 in coordinating these two events. Furthermore, our structural results likely reveal the location of the unprocessed terminal sequences of the 17S rRNA, and suggest that the maturation events of the 17S rRNA could be employed as quality control mechanisms on subunit production and protein translation.     

The 23 S rRNA environment of ribosomal protein L9 in the 50 S ribosomal subunit

Ribosomal protein L9 consists of two globular alpha/beta domains separated by a nine-turn alpha-helix. We examined the rRNA environment of L9 by chemical footprinting and directed hydroxyl radical probing. We reconstituted L9, or individual domains of L9, with L9-deficient 50 S subunits, or with deproteinized 23 S rRNA. A footprint was identified in domain V of 23 S rRNA that was mainly attributable to N-domain binding. Fe(II) was tethered to L9 via cysteine residues introduced at positions along the alpha-helix and in the C-domain, and derivatized proteins were reconstituted with L9-deficient subunits. Directed hydroxyl radical probing targeted regions of domains I, III, IV, and V of 23 S rRNA, reinforcing the view that 50 S subunit architecture is typified by interwoven rRNA domains. There was a striking correlation between the cleavage patterns from the Fe(II) probes attached to the alpha-helix and their predicted orientations, constraining both the position and orientation of L9, as well as the arrangement of specific elements of 23 S rRNA, in the 50 S subunit.     

Fragmentation heterogeneity of 23S ribosomal RNA in Haemophilus species

The fragmentation of 23S rRNA of 22 Haemophilus influenzae strains and eight strains belonging to other Haemophilus species was investigated. Instead of intact molecules, the 23S rRNA molecules were found to be cleaved into two to five smaller conserved fragments in most strains examined, especially in H. influenzae type b (5/6) and nontypeable strains (5/5). One or two conserved potential cleavage sites were identified by PCR analysis of the strains showing a fragmented 23S rRNA pattern. The relevant nucleotide sequences were determined and compared to H. influenzae Rd, which contains intact 23S rRNA molecules. An identical 112 bp long intervening sequence (IVS) at position 542 and a conserved 121–123 bp IVS sequence at position 1171 were found in two H. influenzae type b strains and one nontypeable strain. Among the strains with fragmented 23S rRNA, nearly half showed a heterogeneous cleavage pattern due to the dispersion of IVSs among different 23S rRNA operons. The localization of the conserved H. influenzae IVSs coincided well with the extensively studied IVSs among other bacteria, but differed in nucleotide sequence from any other reported IVSs. Therefore, the IVSs of Haemophilus 23S rRNA may originate from a common source that is independent of other bacteria.     

Site-specific cleavage of the 40S ribosomal subunit reveals eukaryote-specific ribosomal protein S28 in the subunit head

After resolving the crystal structure of the prokaryotic ribosome, mapping the proteins in the eukaryotic ribosome is a challenging task. We applied RNase H digestion to split the human 40S ribosomal subunit into head and body parts. Mass spectrometry of the proteins in the 40S subunit head revealed the presence of eukaryote-specific ribosomal protein S28e. Recombinant S28e was capable of specific binding to the 3′ major domain of the 18S rRNA (K_a = 8.0 ± 0.5 × 10⁹ M⁻¹). We conclude that S28e has a binding site on the 18S rRNA within the 40S subunit head.

Structured summary

MINT-8044084: S8 (uniprotkb:P62241) and S19 (uniprotkb:P39019) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044095: S8 (uniprotkb:P62241), S19 (uniprotkb:P39019) and S13 (uniprotkb:P62277) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044024: S29 (uniprotkb:P62273), S28 (uniprotkb:P62857), S21 (uniprotkb:P63220), S20 (uniprotkb:P60866), S26 (uniprotkb:P62854), S25 (uniprotkb:P62851), S12 (uniprotkb:P25398), S17 (uniprotkb:P08708), S19 (uniprotkb:P39019), S14 (uniprotkb:P62263), S16 (uniprotkb:P62249) and S11 (uniprotkb:P62280) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044065: S29 (uniprotkb:P62273), S28 (uniprotkb:P62857), S19 (uniprotkb:P39019), S14 (uniprotkb:P62263) and S16 (uniprotkb:P62249) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)     

Definition of bases in 23S rRNA essential for ribosomal subunit association

The ribosome is a two-subunit molecular machine, sporting a working cycle that involves coordinated movements of the subunits. Recent structural studies of the 70S ribosome describe a rather large number of intersubunit contacts, some of which are dynamic during translocation. We set out to determine which intersubunit contacts are functionally indispensable for the association of ribosome subunits by using a modification interference approach. Modification of the N-1 position of A715, A1912, or A1918 in Escherichia coli 50S subunits is strongly detrimental to 70S ribosome formation. This result points to 23S rRNA helices 34 and 69, and thus bridges B2a and B4, as essential for ensuring stability of the 70S ribosome.     

23S rRNA甲基转移酶RrmJ促进细菌50S亚基后期组装

核糖体是所有细胞中负责蛋白质合成的分子机器。它自身在细胞内的组装成熟过程受到严密调控,需要诸多组装因子的参与。RrmJ是原核生物中一类保守的甲基转移酶,能够甲基化修饰核糖体上肽基转移酶中心（peptidyl transferase center, PTC）内A环的U2552位点。敲除rrmJ基因的大肠杆菌表现出显著的生长缺陷及50S亚基组装前体的累积,因而RrmJ在50S亚基组装中具有重要作用。本研究对细菌生长实验与核糖体图谱分析表明,回补表达RrmJ的质粒对于ΔrrmJ菌株生长缺陷有显著改善,50S前体累积现象也得到有效缓解。通过共沉淀实验证明,RrmJ与ΔrrmJ菌株中提取的50S前体结合能力显著强于缺失型或野生型菌株中纯化的成熟50S;当加入S-腺苷甲硫氨酸时,该酶与50S前体结合能力显著下降。冷冻电镜三维重构数据进一步阐明,缺失型菌株50S前体主要停滞在组装晚期两个PTC区域成熟程度不同的特定时段。综合上述结果表明,U2552位点的修饰发生在50S亚基组装晚期特定阶段,这一事件不仅会加速A环的RNA螺旋折叠,另有可能促进附近PTC区域结构成熟。     

Characterization of 16S rRNA mutations that decrease the fidelity of translation initiation

In bacteria, initiation of translation is kinetically controlled by factors IF1, IF2, and IF3, which work in conjunction with the 30S subunit to ensure accurate selection of the initiator tRNA (fMet-tRNA(fMet)) and the start codon. Here, we show that mutations G1338A and A790G of 16S rRNA decrease initiation fidelity in vivo and do so in distinct ways. Mutation G1338A increases the affinity of tRNA(fMet) for the 30S subunit, suggesting that G1338 normally forms a suboptimal Type II interaction with fMet-tRNA(fMet). By stabilizing fMet-tRNA(fMet) in the preinitiation complex, G1338A may partially compensate for mismatches in the codon-anti-codon helix and thereby increase spurious initiation. Unlike G1338A, A790G decreases the affinity of IF3 for the 30S subunit. This may indirectly stabilize fMet-tRNA(fMet) in the preinitiation complex and/or promote premature docking of the 50S subunit, resulting in increased levels of spurious initiation.     

Structural and functional studies of the Thermus thermophilus 16S rRNA methyltransferase RsmG

The RsmG methyltransferase is responsible for N⁷ methylation of G527 of 16S rRNA in bacteria. Here, we report the identification of the Thermus thermophilus rsmG gene, the isolation of rsmG mutants, and the solution of RsmG X-ray crystal structures at up to 1.5 Å resolution. Like their counterparts in other species, T. thermophilus rsmG mutants are weakly resistant to the aminoglycoside antibiotic streptomycin. Growth competition experiments indicate a physiological cost to loss of RsmG activity, consistent with the conservation of the modification site in the decoding region of the ribosome. In contrast to Escherichia coli RsmG, which has been reported to recognize only intact 30S subunits, T. thermophilus RsmG shows no in vitro methylation activity against native 30S subunits, only low activity with 30S subunits at low magnesium concentration, and maximum activity with deproteinized 16S rRNA. Cofactor-bound crystal structures of RsmG reveal a positively charged surface area remote from the active site that binds an adenosine monophosphate molecule. We conclude that an early assembly intermediate is the most likely candidate for the biological substrate of RsmG.     

Specific contacts between protein S4 and ribosomal RNA are required at multiple stages of ribosome assembly

Assembly of bacterial 30S ribosomal subunits requires structural rearrangements to both its 16S rRNA and ribosomal protein components. Ribosomal protein S4 nucleates 30S assembly and associates rapidly with the 5′ domain of the 16S rRNA. In vitro, transformation of initial S4–rRNA complexes to long-lived, mature complexes involves refolding of 16S helix 18, which forms part of the decoding center. Here we use targeted mutagenesis of Geobacillus stearothermophilus S4 to show that remodeling of S4–rRNA complexes is perturbed by ram alleles associated with reduced translational accuracy. Gel mobility shift assays, SHAPE chemical probing, and in vivo complementation show that the S4 N-terminal extension is required for RNA binding and viability. Alanine substitutions in Y47 and L51 that interact with 16S helix 18 decrease S4 affinity and destabilize the helix 18 pseudoknot. These changes to the protein–RNA interface correlate with no growth (L51A) or cold-sensitive growth, 30S assembly defects, and accumulation of 17S pre-rRNA (Y47A). A third mutation, R200A, over-stabilizes the helix 18 pseudoknot yet results in temperature-sensitive growth, indicating that complex stability is finely tuned by natural selection. Our results show that early S4–RNA interactions guide rRNA folding and impact late steps of 30S assembly.     

Crystal structure of the RluD pseudouridine synthase catalytic module, an enzyme that modifies 23S rRNA and is essential for normal cell growth of Escherichia coli

Pseudouridine (5-beta-D-ribofuranosyluracil, Psi) is the most commonly found modified base in RNA. Conversion of uridine to Psi is performed enzymatically in both prokaryotes and eukaryotes by pseudouridine synthases (EC 4.2.1.70). The Escherichia coli Psi-synthase RluD modifies uridine to Psi at positions 1911, 1915 and 1917 within 23S rRNA. RluD also possesses a second function related to proper assembly of the 50S ribosomal subunit that is independent of Psi-synthesis. Here, we report the crystal structure of the catalytic module of RluD (residues 68-326; DeltaRluD) refined at 1.8A to a final R-factor of 21.8% (R(free)=24.3%). DeltaRluD is a monomeric enzyme having an overall mixed alpha/beta fold. The DeltaRluD molecule consists of two subdomains, a catalytic subdomain and C-terminal subdomain with the RNA-binding cleft formed by loops extending from the catalytic sub-domain. The catalytic sub-domain of DeltaRluD has a similar fold as in TruA, TruB and RsuA, with the location of the RNA-binding cleft, active-site and conserved, catalytic Asp residue superposing in all four structures. Superposition of the crystal structure of TruB bound to a T-stem loop with RluD reveals that similar RNA-protein interactions for the flipped-out uridine base would exist in both structures, implying that base-flipping is necessary for catalysis. This observation also implies that the specificity determinants for site-specific RNA-binding and recognition likely reside in parts of RluD beyond the active site.