Ribosomal protein-dependent orientation of the 16 S rRNA environment of S15

Ribosomal protein S15 binds specifically to the central domain of 16 S ribosomal RNA (16 S rRNA) and directs the assembly of four additional proteins to this domain. The central domain of 16 S rRNA along with these five proteins form the platform of the 30 S subunit. Previously, directed hydroxyl radical probing from Fe(II)-S15 in small ribonucleoprotein complexes was used to study assembly of the central domain of 16 S rRNA. Here, this same approach was used to understand the 16 S rRNA environment of Fe(II)-S15 in 30 S subunits and to determine the ribosomal proteins that are involved in forming the mature S15-16 S rRNA environment. We have identified additional sites of Fe(II)-S15-directed cleavage in 30S subunits compared to the binary complex of Fe(II)-S15/16 S rRNA. Along with novel targets in the central domain, sites within the 5' and 3' minor domains are also cleaved. This suggests that during the course of 30S subunit assembly these elements are positioned in the vicinity of S15. Besides the previously determined role for S8, roles for S5, S6+S18, and S16 in altering the 16 S rRNA environment of S15 were established. These studies reveal that ribosomal proteins can alter the assembly of regions of the 30 S subunit from a considerable distance and influence the overall conformation of this ribonucleoprotein particle.     

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.     

Assembly of the 30S ribosomal subunit

Ribosomes are large macromolecular complexes responsible for cellular protein synthesis. The smallest known cytoplasmic ribosome is found in prokaryotic cells; these ribosomes are about 2.5 MDa and contain more than 4000 nucleotides of RNA and greater than 50 proteins. These components are distributed into two asymmetric subunits. Recent advances in structural studies of ribosomes and ribosomal subunits have revealed intimate details of the interactions within fully assembled particles. In contrast, many details of how these massive ribonucleoprotein complexes assemble remain elusive. The goal of this review is to discuss some crucial aspects of 30S ribosomal subunit assembly.     

Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: structure of the proteins and their interactions with 16 S RNA

We present a detailed analysis of the protein structures in the 30 S ribosomal subunit from Thermus thermophilus, and their interactions with 16 S RNA based on a crystal structure at 3.05 A resolution. With 20 different polypeptide chains, the 30 S subunit adds significantly to our data base of RNA structure and protein-RNA interactions. In addition to globular domains, many of the proteins have long, extended regions, either in the termini or in internal loops, which make extensive contact to the RNA component and are involved in stabilizing RNA tertiary structure. Many ribosomal proteins share similar alpha+beta sandwich folds, but we show that the topology of this domain varies considerably, as do the ways in which the proteins interact with RNA. Analysis of the protein-RNA interactions in the context of ribosomal assembly shows that the primary binders are globular proteins that bind at RNA multihelix junctions, whereas proteins with long extensions assemble later. We attempt to correlate the structure with a large body of biochemical and genetic data on the 30 S subunit.     

Role of N-terminal helix in interaction of ribosomal protein S15 with 16S rRNA

The position and conformation of the N-terminal helix of free ribosomal protein S15 was earlier found to be modified under various conditions. This variability was supposed to provide the recognition by the protein of its specific site on 16S rRNA. To test this hypothesis, we substituted some amino acid residues in this helix and assessed effects of these substitutions on the affinity of the protein for 16S rRNA. The crystal structure of the complex of one of these mutants (Thr3Cys S15) with the 16S rRNA fragment was determined, and a computer model of the complex containing another mutant (Gln8Met S15) was designed. The available and new information was analyzed in detail, and the N-terminal helix was concluded to play no significant role in the specific binding of the S15 protein to its target on 16S rRNA.     

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.     

Localization of the trigger factor binding site on the ribosomal 50S subunit

In Escherichia coli, protein folding is undertaken by three distinct sets of chaperones, the DnaK-DnaJ and GroEL-GroES systems and the trigger factor (TF). TF has been proposed to be the first chaperone to interact with the nascent polypeptide chain as it emerges from the tunnel of the 70S ribosome and thus probably plays an important role in co-translational protein folding. We have made complexes with deuterated ribosomes (50S subunits and 70S ribosomes) and protated TF and determined the TF binding site on the respective complexes using the neutron scattering technique of spin-contrast variation. Our data suggest that the TF binds in the form of a homodimer. On both the 50S subunit and the 70S ribosome, the TF position is in proximity to the tunnel exit site, near ribosomal proteins L23 and L29, located on the back of the 50S subunit. The positions deviate from one another, such that the position on the 70S ribosome is located slightly further from the tunnel than that determined for the 50S subunit alone. Nevertheless, from both determined positions interaction between TF and a short nascent chain of 57 amino acid residues would be plausible, compatible with a role for TF participation in co-translational protein folding.     

A region in the C-terminal domain of ribosomal protein SA required for binding of SA to the human 40S ribosomal subunit

The human ribosomal protein SA, known also as a precursor of the cell-surface laminin receptor, LAMR, is a protein of the 40S ribosomal subunit. It is homologous to eubacterial ribosomal protein S2p, but has a eukaryote-specific C-terminal domain (CTD) that is responsible in LAMR for the binding of laminin as well as prions and several viruses. Using serial deletions in the SA CTD, we showed that region between amino acids 236-262 is required for binding of the protein to 40S ribosomal subunits. All SA mutants containing this region protected nucleotides in hairpin 40 (which is not bound to any protein in the eubacterial 30S ribosomal subunit) of the 18S rRNA from hydroxyl radical attack. Comparison of our data with the cryo-EM models of the mammalian 40S ribosomal subunit allowed us to locate the SA CTD in the spatial structure of the 40S subunit.     

The C-terminal helix in the YjeQ zinc-finger domain catalyzes the release of RbfA during 30S ribosome subunit assembly

YjeQ (also called RsgA) and RbfA proteins in Escherichia coli bind to immature 30S ribosome subunits at late stages of assembly to assist folding of the decoding center. A key step for the subunit to enter the pool of actively translating ribosomes is the release of these factors. YjeQ promotes dissociation of RbfA during the final stages of maturation; however, the mechanism implementing this functional interplay has not been elucidated. YjeQ features an amino-terminal oligonucleotide/oligosaccharide binding domain, a central GTPase module and a carboxy-terminal zinc-finger domain. We found that the zinc-finger domain is comprised of two functional motifs: the region coordinating the zinc ion and a carboxy-terminal α-helix. The first motif is essential for the anchoring of YjeQ to the 30S subunit and the carboxy-terminal α-helix facilitates the removal of RbfA once the 30S subunit reaches the mature state. Furthermore, the ability of the mature 30S subunit to stimulate YjeQ GTPase activity also depends on the carboxy-terminal α-helix. Our data are consistent with a model in which YjeQ uses this carboxy-terminal α-helix as a sensor to gauge the conformation of helix 44, an essential motif of the decoding center. According to this model, the mature conformation of helix 44 is sensed by the carboxy-terminal α-helix, which in turn stimulates the YjeQ GTPase activity. Hydrolysis of GTP is believed to assist the release of YjeQ from the mature 30S subunit through a still uncharacterized mechanism. These results identify the structural determinants in YjeQ that implement the functional interplay with RbfA.     

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)     

Cryo-electron microscopy structure of the 30S subunit in complex with the YjeQ biogenesis factor

YjeQ is a protein broadly conserved in bacteria containing an N-terminal oligonucleotide/oligosaccharide fold (OB-fold) domain, a central GTPase domain, and a C-terminal zinc-finger domain. YjeQ binds tightly and stoichiometrically to the 30S subunit, which stimulates its GTPase activity by 160-fold. Despite growing evidence for the involvement of the YjeQ protein in bacterial 30S subunit assembly, the specific function and mechanism of this protein remain unclear. Here, we report the costructure of YjeQ with the 30S subunit obtained by cryo-electron microscopy. The costructure revealed that YjeQ interacts simultaneously with helix 44, the head and the platform of the 30S subunit. This binding location of YjeQ in the 30S subunit suggests a chaperone role in processing of the 3' end of the rRNA as well as in mediating the correct orientation of the main domains of the 30S subunit. In addition, the YjeQ binding site partially overlaps with the interaction site of initiation factors 2 and 3, and upon binding, YjeQ covers three inter-subunit bridges that are important for the association of the 30S and 50S subunits. Hence, our structure suggests that YjeQ may assist in ribosome maturation by preventing premature formation of the translation initiation complex and association with the 50S subunit. Together, these results support a role for YjeQ in the late stages of 30S maturation.     

Functional studies of the 900 tetraloop capping helix 27 of 16S ribosomal RNA

The 900 tetraloop (positions 898-901) of Escherichia coli 16S rRNA caps helix 27, which is involved in a conformational switch crucial for the decoding function of the ribosome. This tetraloop forms a GNRA motif involved in intramolecular RNA-RNA interactions with its receptor in helix 24 of 16S rRNA. It is involved also in an intersubunit bridge, via an interaction with helix 67 in domain IV of 23S rRNA. Using a specialized ribosome system and an instant-evolution procedure, the four nucleotides of this loop were randomized and 15 functional mutants were selected in vivo. Positions 899 and 900, responsible for most of the tetraloop/receptor interactions, were found to be the most critical for ribosome activity. Functional studies showed that mutations in the 900 tetraloop impair subunit association and decrease translational fidelity. Computer modeling of the mutations allows correlation of the effect of mutations with perturbations of the tetraloop/receptor interactions.     

Dissociation of proteins from the human 40S ribosomal subunit in a lithium chloride gradient

Human 40S ribosomal subunits were subjected to centrifugation through a 0.3–1.5 M LiCl gradient in 0.5 M KCl, 4 mM MgCl₂. Most of the proteins started to dissociate at the initial concentration of monovalent cations (0.8 M); the last to dissociate at 1.55 M salt were the core proteins S3, S5, S7, S10, S15, S16, S17, S19, S20, and S28; among these, S7, S10, S16, and S19 were the most tightly bound to 18S rRNA.     

Mutual induced fit binding of Xenopus ribosomal protein L5 to 5S rRNA

A library of random mutations in Xenopus ribosomal protein L5 was generated by error-prone PCR and used to delineate the binding domain for 5S rRNA. All but one of the amino acid substitutions that affected binding affinity are clustered in the central region of the protein. Several of the mutations are conservative substitutions of non-polar amino acid residues that are unlikely to form energetically significant contacts to the RNA. Thermal denaturation, monitored by circular dichroism (CD), indicates that L5 is not fully structured and association with 5S rRNA increases the t(m) of the protein by 16 degrees C. L5 induces changes in the CD spectrum of 5S rRNA, establishing that the complex forms by a mutual induced fit mechanism. Deuterium exchange reveals that a considerable amount of L5 is unstructured in the absence of 5S rRNA. The fluorescence emission of W266 provides evidence for structural changes in the C-terminal region of L5 upon binding to 5S rRNA; whereas, protection experiments demonstrate that the N terminus remains highly sensitive to protease digestion in the complex. Analysis of the amino acid sequence of L5 by the program PONDR predicts that the N and C-terminal regions of L5 are intrinsically disordered, but that the central region, which contains three essential tyrosine residues and other residues important for binding to 5S rRNA, is likely to be structured. Initial interaction of the protein with 5S rRNA likely occurs through this region, followed by induced folding of the C-terminal region. The persistent disorder in the N-terminal domain is possibly exploited for interactions between the L5-5S rRNA complex and other proteins.     

Internucleotide movements during formation of 16 S rRNA-rRNA photocrosslinks and their connection to the 30 S subunit conformational dynamics

UV light-induced RNA photocrosslinks are formed at a limited number of specific sites in the Escherichia coli and in other eubacterial 16 S rRNAs. To determine if unusually favorable internucleotide geometries could explain the restricted crosslinking patterns, parameters describing the internucleotide geometries were calculated from the Thermus thermophilus 30 S subunit X-ray structure and compared to crosslinking frequencies. Significant structural adjustments between the nucleotide pairs usually are needed for crosslinking. Correlations between the crosslinking frequencies and the geometrical parameters indicate that nucleotide pairs closer to the orientation needed for photoreaction have higher crosslinking frequencies. These data are consistent with transient conformational changes during crosslink formation in which the arrangements needed for photochemical reaction are attained during the electronic excitation times. The average structural rearrangement for UVA-4-thiouridine (s4U)-induced crosslinking is larger than that for UVB or UVC-induced crosslinking; this is associated with the longer excitation time for s4U and is also consistent with transient conformational changes. The geometrical parameters do not completely predict the crosslinking frequencies, implicating other aspects of the tertiary structure or conformational flexibility in determining the frequencies and the locations of the crosslinking sites. The majority of the UVB/C and UVA-s4U-induced crosslinks are located in four regions in the 30 S subunit, within or at the ends of RNA helix 34, in the tRNA P-site, in the distal end of helix 28 and in the helix 19/helix 27 region. These regions are implicated in different aspects of tRNA accommodation, translocation and in the termination reaction. These results show that photocrosslinking is an indicator for sites where there is internucleotide conformational flexibility and these sites are largely restricted to parts of the 30 S subunit associated with ribosome function.     

16S rRNA序列分析法在医学微生物鉴定中的应用

１６Ｓ ｒＲＮＡ序列分析作为微生物系统分类的主要依据已得到了广泛认同,随着微生物核糖体数据库的日益完善,该技术成为细菌分类和鉴定的一个有力工具。本文概述了 １６５ ｒＲＮＡ序列分析法的技术步骤以及该技术在医学微生物研究中的应用,总结了目前文献报导的各种致病微生物种属特异性 １６５ ｒＲＮＡ引物和探针序列,同时分析了该技术在应用中存在的一些问题。     

The binding interface between Bacillus stearothermophilus ribosomal protein S15 and its 5'-translational operator mRNA

The Bacillus stearothermophilus ribosomal protein S15 (BS15) binds a purine-rich three-helix junction motif in the central domain of 16S ribosomal RNA (rRNA) as well as a translational operator located in the 5'-untranslated region (5'-UTR) of its cognate messenger RNA (mRNA). An in-frame fusion between the 5'-UTR of the BS15 gene and beta-galactosidase (lacZ) was prepared, and tested for BS15-dependent translational repression of lacZ activity in Escherichia coli. The presence of BS15 in trans represses lacZ activity 24-fold. A series of detailed point mutations in BS15 were tested for their effects upon translational repression of lacZ activity. These point mutations demonstrated that the 5'-UTR-BS15 binding interface utilizes many of the same conserved amino acid residues implicated in the binding of BS15 to 16S rRNA. The data demonstrate that the S15 protein can bind to an RNA target motif based primarily upon appropriate minor groove and sugar-phosphate backbone contacts, irrespective of the specific RNA sequence.