Global Stabilization of rRNA Structure by Ribosomal Proteins S4, S17, and S20

Ribosomal proteins stabilize the folded structure of the ribosomal RNA and enable the recruitment of further proteins to the complex. Quantitative hydroxyl radical footprinting was used to measure the extent to which three different primary assembly proteins, S4, S17, and S20, stabilize the three-dimensional structure of the Escherichia coli 16S 5′ domain. The stability of the complexes was perturbed by varying the concentration of MgCl₂. Each protein influences the stability of the ribosomal RNA tertiary interactions beyond its immediate binding site. S4 and S17 stabilize the entire 5′ domain, while S20 has a more local effect. Multistage folding of individual helices within the 5′ domain shows that each protein stabilizes a different ensemble of structural intermediates that include nonnative interactions at low Mg²⁺ concentration. We propose that the combined interactions of S4, S17, and S20 with different helical junctions bias the free-energy landscape toward a few RNA conformations that are competent to add the secondary assembly protein S16 in the next step of assembly.     

Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies.

We have investigated protein-rRNA cross-links formed in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus at the molecular level using UV and 2-iminothiolane as cross-linking agents. We identified amino acids cross-linked to rRNA for 13 ribosomal proteins from these organisms, namely derived from S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 and L36. Several other peptide stretches cross-linked to rRNA have been sequenced in which no direct cross-linked amino acid could be detected. The cross-linked amino acids are positioned within loop domains carrying RNA binding features such as conserved basic and aromatic residues. One of the cross-linked peptides in ribosomal protein S3 shows a common primary sequence motif--the KH motif--directly involved in interaction with rRNA, and the cross-linked amino acid in ribosomal protein L36 lies within the zinc finger-like motif of this protein. The cross-linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three-dimensional models. A comparison of our structural data with mutations in ribosomal proteins that lead to antibiotic resistance, and with those from protein-antibiotic cross-linking experiments, reveals functional implications for ribosomal proteins that interact with rRNA.     

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem–loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit''s decoding groove. This configuration depends on the sequence and structure of a different stem–loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis.     

Structure of Hsp15 reveals a novel RNA-binding motif

We have solved the crystal structure of the heat shock protein Hsp15, a newly isolated and very highly inducible heat shock protein that binds the ribosome. Comparison of its structure with those of two RNA-binding proteins, ribosomal protein S4 and threonyl-tRNA synthetase, reveals a novel RNA-binding motif. This newly recognized motif is remarkably common, present in at least eight different protein families that bind RNA. The motif's surface is populated by conserved, charged residues that define a likely RNA-binding site. An intriguing pattern emerges: stress proteins, ribosomal proteins and tRNA synthetases repeatedly share a conserved motif. This may imply a hitherto unrecognized functional similarity between these three protein classes.     

Structural motifs of the bacterial ribosomal proteins S20, S18 and S16 that contact rRNA present in the eukaryotic ribosomal proteins S25, S26 and S27A,respectively

The majority of constitutive proteins in the bacterial 30S ribosomal subunit have orthologues in Eukarya and Archaea. The eukaryotic counterparts for the remainder (S6, S16, S18 and S20) have not been identified. We assumed that amino acid residues in the ribosomal proteins that contact rRNA are to be constrained in evolution and that the most highly conserved of them are those residues that are involved in forming the secondary protein structure. We aligned the sequences of the bacterial ribosomal proteins from the S20p, S18p and S16p families, which make multiple contacts with rRNA in the Thermus thermophilus 30S ribosomal subunit (in contrast to the S6p family), with the sequences of the unassigned eukaryotic small ribosomal subunit protein families. This made it possible to reveal that the conserved structural motifs of S20p, S18p and S16p that contact rRNA in the bacterial ribosome are present in the ribosomal proteins S25e, S26e and S27Ae, respectively. We suggest that ribosomal protein families S20p, S18p and S16p are homologous to the families S25e, S26e and S27Ae, respectively.     

Ribosomal protein L1 recognizes the same specific structural motif in its target sites on the autoregulatory mRNA and 23S rRNA

The RNA-binding ability of ribosomal protein L1 is of profound interest since the protein has a dual function as a ribosomal protein binding rRNA and as a translational repressor binding its mRNA. Here, we report the crystal structure of ribosomal protein L1 in complex with a specific fragment of its mRNA and compare it with the structure of L1 in complex with a specific fragment of 23S rRNA determined earlier. In both complexes, a strongly conserved RNA structural motif is involved in L1 binding through a conserved network of RNA–protein H-bonds inaccessible to the solvent. These interactions should be responsible for specific recognition between the protein and RNA. A large number of additional non-conserved RNA–protein H-bonds stabilizes both complexes. The added contribution of these non-conserved H-bonds makes the ribosomal complex much more stable than the regulatory one.     

Role of conserved nucleotides in building the 16 S rRNA binding site for ribosomal protein S15

Ribosomal protein S15 recognizes a highly conserved target on 16 S rRNA, which consists of two distinct binding regions. Here, we used extensive site-directed mutagenesis on a Escherichia coli 16 S rRNA fragment containing the S15 binding site, to investigate the role of conserved nucleotides in protein recognition and to evaluate the relative contribution of the two sites. The effect of mutations on S15 recognition was studied by measuring the relative binding affinity, RNA probing and footprinting. The crystallographic structure of the Thermus thermophilus complex allowed molecular modelling of the E. coli complex and facilitated interpretation of biochemical data. Binding is essentially driven by site 1, which includes a three-way junction constrained by a conserved base triple and cross-strand stacking. Recognition is based mainly on shape complementarity, and the role of conserved nucleotides is to maintain a unique backbone geometry. The wild-type base triple is absolutely required for protein interaction, while changes in the conserved surrounding nucleotides are partially tolerated. Site 2, which provides functional groups in a conserved G-U/G-C motif, contributes only modestly to the stability of the interaction. Binding to this motif is dependent on binding at site 1 and is allowed only if the two sites are in the correct relative orientation. Non-conserved bulged nucleotides as well as a conserved purine interior loop, although not directly involved in recognition, are used to provide an appropriate flexibility between the two sites. In addition, correct binding at the two sites triggers conformational adjustments in the purine interior loop and in a distal region, which are known to be involved for subsequent binding of proteins S6 and S18. Thus, the role of site 1 is to anchor S15 to the rRNA, while binding at site 2 is aimed to induce a cascade of events required for subunit assembly.     

Interaction of Era with the 30S ribosomal subunit implications for 30S subunit assembly

Era (E. coliRas-like protein) is a highly conserved and essential GTPase in bacteria. It binds to the 16S ribosomal RNA (rRNA) of the small (30S) ribosomal subunit, and its depletion leads to accumulation of an unprocessed precursor of the 16S rRNA. We have obtained a three-dimensional cryo-electron microscopic map of the Thermus thermophilus 30S-Era complex. Era binds in the cleft between the head and platform of the 30S subunit and locks the subunit in a conformation that is not favorable for association with the large (50S) ribosomal subunit. The RNA binding KH motif present within the C-terminal domain of Era interacts with the conserved nucleotides in the 3' region of the 16S rRNA. Furthermore, Era makes contact with several assembly elements of the 30S subunit. These observations suggest a direct involvement of Era in the assembly and maturation of the 30S subunit.     

Transcriptional regulation by antitermination. Interaction of RNA with NusB protein and NusB/NusE protein complex of Escherichia coli

A recombinant heterodimeric NusB/NusE protein complex of Escherichia coli was expressed under the control of a synthetic mini operon. Surface plasmon resonance measurements showed that the heterodimer complex has substantially higher affinity for the boxA RNA sequence motif of the ribosomal RNA (rrn) operons of E.coli as compared to monomeric NusB protein. Single base exchanges in boxA RNA reduced the affinity of the protein complex up to 15-fold. The impact of base exchanges in the boxA RNA on the interaction with NusB protein was studied by (1)H,(15)N heterocorrelation NMR spectroscopy. Spectra obtained with modified RNA sequences were analysed by a novel generic algorithm. Replacement of bases in the terminal segments of the boxA RNA motif caused minor chemical shift changes as compared to base exchanges in the central part of the dodecameric boxA motif.     

长苞铁杉nrDNA内转录间隔区及5.8S的二级结构分析研究

核糖体RNA及相邻区域的二级结构研究,作为一个重要的工具,已在一些分类等级上被应用于系统发育的分析。以长苞铁杉为实验材料,通过克隆、测序,利用最小自由能原理预测nrDNA内转录间隔区及5.8S转录本的二级结构,分析它们的结构特点,探讨假基因化拷贝与功能拷贝结构上的差异。分析结果表明:(1)ITS1区的二级结构主要由几个延展的发夹结构组成,配对的亚重复单位在松科植物特有的保守序列处有部分重叠,未配对的亚重复单位通常能自身折叠,保守序列的部分碱基出现在发夹结构的环中;(2)假基因化拷贝二级结构的自由能比正常拷贝高;(3)与正常拷贝的二级结构相比,假基因化拷贝在进化速率很低的5.8S功能区发生较大的变异,且在5.8 S末端没有和26 S连接配对。     

HCV IRES interacts with the 18S rRNA to activate the 40S ribosome for subsequent steps of translation initiation

Previous analyses of complexes of 40S ribosomal subunits with the hepatitis C virus (HCV) internal ribosome entry site (IRES) have revealed contacts made by the IRES with ribosomal proteins. Here, using chemical probing, we show that the HCV IRES also contacts the backbone and bases of the CCC triplet in the 18S ribosomal RNA (rRNA) expansion segment 7. These contacts presumably provide interplay between IRES domain II and the AUG codon close to ribosomal protein S5, which causes a rearrangement of 18S rRNA structure in the vicinity of the universally conserved nucleotide G1639. As a result, G1639 becomes exposed and the corresponding site of the 40S subunit implicated in transfer RNA discrimination can select . These data are the first demonstration at nucleotide resolution of direct IRES–rRNA interactions and how they induce conformational transition in the 40S subunit allowing the HCV IRES to function without AUG recognition initiation factors.     

Attachment sites of primary binding proteins L1, L2 and L23 on 23 S ribosomal RNA of Escherichia coli

The attachment sites of the primary binding proteins L1, L2 and L23 on 23 S ribosomal RNA of Escherichia coli were examined by a chemical and ribonuclease footprinting method using several probes with different specificities. The results show that the sites are confined to localized RNA regions within the large ribonuclease-protected ribonucleoprotein fragments that were characterized earlier. They are as follows: (1) L1 recognizes a tertiary structural motif in domain V centred on two interacting internal loops; the main protein interaction sites occur at the internal loop/helix junctions. (2) The L2 site constitutes a single irregular stem/loop structure in the centre of domain IV where non-Watson-Crick pairing is likely to occur. (3) L23 recognizes a tertiary structural motif involving a single terminal loop structure and part of an adjacent internal loop at the centre of domain III. Each of the three primary binding proteins, whose presence is essential for ribosomal assembly, has been associated with important ribosomal functions: L1 lies in the E-site for deacylated tRNA binding while L2 and L23 have been implicated in the P and A substrate sites, respectively, of the peptidyl transferase centre. Moreover, each of the protein sites, but particularly those of L2 and L23, lies at the centre of RNA domains where they can maximally influence both the assembly of secondary binding proteins and the function of the RNA region.     

Organisation of the S10, spc and alpha ribosomal protein gene clusters in prokaryotic genomes

Although it is well known that there is no long range colinearity in gene order in bacterial genomes, it is thought that there are several regions that are under strong structural constraints during evolution, in which gene order is extremely conserved. One such region is the str locus, containing the S10-spc-alpha operons. These operons contain genes coding for ribosomal proteins and for a number of housekeeping genes. We compared the organisation of these gene clusters in 111 sequenced prokaryotic genomes (99 bacterial and 12 archaeal genomes). We also compared the organisation to the phylogeny based on 16S ribosomal RNA gene sequences and the sequences of the ribosomal proteins L22, L16 and S14. Our data indicate that there is much variation in gene order and content in these gene clusters, both in bacterial as well as in archaeal genomes. Our data indicate that differential gene loss has occurred on multiple occasions during evolution. We also noted several discrepancies between phylogenetic trees based on 16S rRNA gene sequences and sequences of ribosomal proteins L16, L22 and S14, suggesting that horizontal gene transfer did play a significant role in the evolution of the S10-spc-alpha gene clusters.     

Slow formation of stable complexes during coincubation of minimal rRNA and ribosomal protein S4

Ribosomal protein S4 binds and stabilizes a five-helix junction or five-way junction (5WJ) in the 5′ domain of 16S ribosomal RNA (rRNA) and is one of two proteins responsible for nucleating 30S ribosome assembly. Upon binding, both protein S4 and 5WJ reorganize their structures. We show that labile S4 complexes rearrange into stable complexes within a few minutes at 42 °C, with longer coincubation leading to an increased population of stable complexes. In contrast, prefolding the rRNA has a smaller effect on stable S4 binding. Experiments with minimal rRNA fragments show that this structural change depends only on 16S residues within the S4 binding site. SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical probing experiments showed that S4 strongly stabilizes 5WJ and the helix (H) 18 pseudoknot, which become tightly folded within the first minute of S4 binding. However, a kink in H16 that makes specific contacts with the S4 N-terminal extension, as well as a right-angle motif between H3, H4, and H18, requires a minute or more to become fully structured. Surprisingly, S4 structurally reorganizes the 530-loop and increases the flexibility of H3, which is proposed to undergo a conformational switch during 30S assembly. These elements of the S4 binding site may require other 30S proteins to reach a stable conformation.