The solution structure of ribosomal protein S17E from Methanobacterium thermoautotrophicum: a structural homolog of the FF domain

The ribosomal protein S17E from the archaeon Methanobacterium thermoautotrophicum is a component of the 30S ribosomal subunit. S17E is a 62-residue protein conserved in archaea and eukaryotes and has no counterparts in bacteria. Mammalian S17E is a phosphoprotein component of eukaryotic ribosomes. Archaeal S17E proteins range from 59 to 79 amino acids, and are about half the length of the eukaryotic homologs which have an additional C-terminal region. Here we report the three-dimensional solution structure of S17E. S17E folds into a small three-helix bundle strikingly similar to the FF domain of human HYPA/FBP11, a novel phosphopeptide-binding fold. S17E bears a conserved positively charged surface acting as a robust scaffold for molecular recognition. The structure of M. thermoautotrophicum S17E provides a template for homology modeling of eukaryotic S17E proteins in the family.     

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.     

A novel RNA-binding motif in omnipotent suppressors of translation termination, ribosomal proteins and a ribosome modification enzyme?

Using computer methods for database search, multiple alignment, protein sequence motif analysis and secondary structure prediction, a putative new RNA-binding motif was identified. The novel motif is conserved in yeast omnipotent translation termination suppressor SUP1, the related DOM34 protein and its pseudogene homologue; three groups of eukaryotic and archaeal ribosomal proteins, namely L30e, L7Ae/S6e and S12e; an uncharacterized Bacillus subtilis protein related to the L7A/S6e group; and Escherichia coli ribosomal protein modification enzyme RimK. We hypothesize that a new type of RNA-binding domain may be utilized to deliver additional activities to the ribosome.     

The zinc finger domain of the archaeal minichromosome maintenance protein is required for helicase activity

The minichromosome maintenance (MCM) proteins, a family of six conserved polypeptides found in all eukaryotes, are essential for DNA replication. The archaeon Methanobacterium thermoautotrophicum Delta H contains a single homologue of MCM with biochemical properties similar to those of the eukaryotic enzyme. The amino acid sequence of the archaeal protein contains a putative zinc-binding domain of the CX(2)CX(n)CX(2)C (C(4)) type. In this study, the roles of the zinc finger domain in MCM function were examined using recombinant wild-type and mutant proteins expressed and purified from Escherichia coli. The protein with a mutation in the zinc motif forms a dodecameric complex similar to the wild-type enzyme. The mutant enzyme, however, is impaired in DNA-dependent ATPase activity and single-stranded DNA binding, and it does not possess helicase activity. These results illustrate the importance of the zinc-binding domain for archaeal MCM function and suggest a role for zinc binding in the eukaryotic MCM complex as well, since four out of the six eukaryotic MCM proteins contain a similar zinc-binding motif.     

Archaeal ribosomal protein L7 is a functional homolog of the eukaryotic 15.5kD/Snu13p snoRNP core protein

Recent investigations have identified homologs of eukaryotic box C/D small nucleolar RNAs (snoRNAs) in Archaea termed sRNAs. Archaeal homologs of the box C/D snoRNP core proteins fibrillarin and Nop56/58 have also been identified but a homolog for the eukaryotic 15.5kD snoRNP protein has not been described. Our sequence analysis of archaeal genomes reveals that the highly conserved ribosomal protein L7 exhibits extensive homology with the eukaryotic 15.5kD protein. Protein binding studies demonstrate that recombinant Methanoccocus jannaschii L7 protein binds the box C/D snoRNA core motif with the same specificity and affinity as the eukaryotic 15.5kD protein. Identical to the eukaryotic 15.5kD core protein, archaeal L7 requires a correctly folded box C/D core motif and intact boxes C and D. Mutational analysis demonstrates that critical features of the box C/D core motif essential for 15.5kD binding are also required for L7 interaction. These include stem I which juxtaposes boxes C and D, as well as the sheared G:A pairs and protruded pyrimidine nucleotide of the asymmetric bulge region. The demonstrated presence of L7Ae in the Haloarcula marismortui 50S ribosomal subunit, taken with our demonstration of the ability of L7 to bind to the box C/D snoRNA core motif, indicates that this protein serves a dual role in Archaea. L7 functioning as both an sRNP core protein and a ribosomal protein could potentially regulate and coordinate sRNP assembly with ribosome biogenesis.     

A central fragment of ribosomal protein S26 containing the eukaryote-specific motif YxxPKxYxK is a key component of the ribosomal binding site of mRNA region 5' of the E site codon

The eukaryotic ribosomal protein S26e (rpS26e) lacking eubacterial counterparts is a key component of the ribosomal binding site of mRNA region 5' of the codon positioned at the exit site. Here, we determined the rpS26e oligopeptide neighboring mRNA on the human 80S ribosome using mRNA analogues bearing perfluorophenyl azide-derivatized nucleotides at designed locations. The protein was cross-linked to mRNA analogues in specific ribosomal complexes, in which the derivatized nucleotide was located at positions -3 to -9. Digestion of cross-linked rpS26e with various specific proteolytic agents followed by identification of the resulting modified oligopeptides made it possible to map the cross-links to fragment 60-71. This fragment contains the motif YxxPKxYxK conserved in eukaryotic but not in archaeal rpS26e. Analysis of X-ray structure of the Tetrahymena thermophila 40S subunit showed that this motif is not implicated in the intraribosomal interactions, implying its involvement in translation process in a eukaryote-specific manner. Comparison of the results obtained with data on positioning of ribosomal ligands on the 40S subunit lead us to suggest that this motif is involved in interaction with both the 5'-untranslated region of mRNA and the initiation factor eIF3 specific for eukaryotes, providing new insights into molecular mechanisms of translation in eukaryotes.     

Solution structure of ribosomal protein S28E from Methanobacterium thermoautotrophicum

The ribosomal protein S28E from the archaeon Methanobacterium thermoautotrophicum is a component of the 30S ribosomal subunit. Sequence homologs of S28E are found only in archaea and eukaryotes. Here we report the three-dimensional solution structure of S28E by NMR spectroscopy. S28E contains a globular region and a long C-terminal tail protruding from the core. The globular region consists of four antiparallel beta-strands that are arranged in a Greek-key topology. Unique features of S28E include an extended loop L2-3 that folds back onto the protein and a 12-residue charged C-terminal tail with no regular secondary structure and greater flexibility relative to the rest of the protein. The structural and surface resemblance to OB-fold family of proteins and the presence of highly conserved basic residues suggest that S28E may bind to RNA. A broad positively charged surface extending over one side of the beta-barrel and into the flexible C terminus may present a putative binding site for RNA.     

Nog2p, a putative GTPase associated with pre-60S subunits and required for late 60S maturation steps.

Eukaryotic ribosome maturation depends on a set of well ordered processing steps. Here we describe the functional characterization of yeast Nog2p (Ynr053cp), a highly conserved nuclear protein. Nog2p contains a putative GTP-binding site, which is essential in vivo. Kinetic and steady-state measurements of the levels of pre-rRNAs in Nog2p-depleted cells showed a defect in 5.8S and 25S maturation and a concomitant increase in the levels of both 27SB(S) and 7S(S) precursors. We found Nog2p physically associated with large pre-60S complexes highly enriched in the 27SB and 7S rRNA precursors. These complexes contained, besides a subset of ribosomal proteins, at least two additional factors, Nog1p, another putative GTP-binding protein, and Rlp24p (Ylr009wp), which belongs to the Rpl24e family of archaeal and eukaryotic ribosomal proteins. In the absence of Nog2p, the pre-60S ribosomal complexes left the nucleolus, but were retained in the nucleoplasm. These results suggest that transient, possibly GTP-dependent association of Nog2p with the pre-ribosomes might trigger late rRNA maturation steps in ribosomal large subunit biogenesis.     

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.     

The structures of antibiotics bound to the E site region of the 50 S ribosomal subunit of Haloarcula marismortui: 13-deoxytedanolide and girodazole

Crystal structures of the 50 S ribosomal subunit from Haloarcula marismortui complexed with two antibiotics have identified new sites at which antibiotics interact with the ribosome and inhibit protein synthesis. 13-Deoxytedanolide binds to the E site of the 50 S subunit at the same location as the CCA of tRNA, and thus appears to inhibit protein synthesis by competing with deacylated tRNAs for E site binding. Girodazole binds near the E site region, but is somewhat buried and may inhibit tRNA binding by interfering with conformational changes that occur at the E site. The specificity of 13-deoxytedanolide for eukaryotic ribosomes is explained by its extensive interactions with protein L44e, which is an E site component of archaeal and eukaryotic ribosomes, but not of eubacterial ribosomes. In addition, protein L28, which is unique to the eubacterial E site, overlaps the site occupied by 13-deoxytedanolide, precluding its binding to eubacterial ribosomes. Girodazole is specific for eukarytes and archaea because it makes interactions with L15 that are not possible in eubacteria.     

Comparative analysis of the 15.5kD box C/D snoRNP core protein in the primitive eukaryote Giardia lamblia reveals unique structural and functional features

Box C/D ribonucleoproteins (RNP) guide the 2'-O-methylation of targeted nucleotides in archaeal and eukaryotic rRNAs. The archaeal L7Ae and eukaryotic 15.5kD box C/D RNP core protein homologues initiate RNP assembly by recognizing kink-turn (K-turn) motifs. The crystal structure of the 15.5kD core protein from the primitive eukaryote Giardia lamblia is described here to a resolution of 1.8 ?. The Giardia 15.5kD protein exhibits the typical α-β-α sandwich fold exhibited by both archaeal L7Ae and eukaryotic 15.5kD proteins. Characteristic of eukaryotic homologues, the Giardia 15.5kD protein binds the K-turn motif but not the variant K-loop motif. The highly conserved residues of loop 9, critical for RNA binding, also exhibit conformations similar to those of the human 15.5kD protein when bound to the K-turn motif. However, comparative sequence analysis indicated a distinct evolutionary position between Archaea and Eukarya. Indeed, assessment of the Giardia 15.5kD protein in denaturing experiments demonstrated an intermediate stability in protein structure when compared with that of the eukaryotic mouse 15.5kD and archaeal Methanocaldococcus jannaschii L7Ae proteins. Most notable was the ability of the Giardia 15.5kD protein to assemble in vitro a catalytically active chimeric box C/D RNP utilizing the archaeal M. jannaschii Nop56/58 and fibrillarin core proteins. In contrast, a catalytically competent chimeric RNP could not be assembled using the mouse 15.5kD protein. Collectively, these analyses suggest that the G. lamblia 15.5kD protein occupies a unique position in the evolution of this box C/D RNP core protein retaining structural and functional features characteristic of both archaeal L7Ae and higher eukaryotic 15.5kD homologues.     

The contribution of the zinc-finger motif to the function of Thermus thermophilus ribosomal protein S14

In the crystal structure of the 30S ribosomal subunit from Thermus thermophilus, cysteine 24 of ribosomal protein S14 (TthS14) occupies the first position in a CXXC-X12-CXXC motif that coordinates a zinc ion. The structural and functional importance of cysteine 24, which is widely conserved from bacteria to humans, was studied by its replacement with serine and by incorporating the resulting mutant into Escherichia coli ribosomes. The capability of such modified ribosomes in binding tRNA at the P and A-sites was equal to that obtained with ribosomes incorporating wild-type TthS14. In fact, both chimeric ribosomal species exhibited 20% lower tRNA affinity compared with native E. coli ribosomes. In addition, replacement of the native E. coli S14 by wild-type, and particularly by mutant TthS14, resulted in reduced capability of the 30S subunit for association with 50S subunits. Nevertheless, ribosomes from transformed cells sedimented normally and had a full complement of proteins. Unexpectedly, the peptidyl transferase activity in the chimeric ribosomes bearing mutant TthS14 was much lower than that measured in ribosomes incorporating wild-type TthS14. The catalytic center of the ribosome is located within the 50S subunit and, therefore, it is unlikely to be directly affected by changes in the structure of S14. More probably, the perturbing effects of S14 mutation on the catalytic center seem to be propagated by adjacent intersubunit bridges or the P-site tRNA molecule, resulting in weak donor-substrate reactivity. This hypothesis was verified by molecular dynamics simulation analysis.     

Nucleolar binding sequences of the ribosomal protein S6e family reside in evolutionary highly conserved peptide clusters

Proteomic analyses of the nucleolus have revealed almost 700 functionally diverse proteins implicated in ribosome biogenesis, nucleolar assembly, and regulation of vital cellular processes. However, this nucleolar inventory has not unveiled a specific consensus motif necessary for nucleolar binding. The ribosomal protein family characterized by their basic nature should exhibit distinct binding sequences that enable interactions with the rRNA precursor molecules facilitating subunit assembly. We succeeded in delineating 2 minimal nucleolar binding sequences of human ribosomal protein S6 by fusing S6 cDNA fragments to the 5' end of the LacZ gene and subsequently detecting the intracellular localization of the beta-galactosidase fusion proteins. Nobis1 (nucleolar binding sequence 1), comprising of 4 highly conserved amino acid clusters separated by glycine or proline, functions independently of the 3 authentic nuclear localization signals (NLSs). Nobis2 consists of 2 conserved peptide clusters and requires the authentic NLS2 in its native context. Similarly, we deduced from previous publications that the single Nobis of ribosomal protein S25 is also highly conserved. The functional protein domain organization of the ribosomal protein S6e family consists of 3 modules: NLS, Nobis, and the C-terminal serine cluster of the phosphorylation sites. This modular structure is evolutionary conserved in vertebrates, invertebrates, and fungi. Remarkably, nucleolar binding sequences of small and large ribosomal proteins reside in peptide clusters conserved over millions of years.     

S6:S18 ribosomal protein complex interacts with a structural motif present in its own mRNA

Prokaryotic ribosomal protein genes are typically grouped within highly conserved operons. In many cases, one or more of the encoded proteins not only bind to a specific site in the ribosomal RNA, but also to a motif localized within their own mRNA, and thereby regulate expression of the operon. In this study, we computationally predicted an RNA motif present in many bacterial phyla within the 5′ untranslated region of operons encoding ribosomal proteins S6 and S18. We demonstrated that the S6:S18 complex binds to this motif, which we hereafter refer to as the S6:S18 complex-binding motif (S6S18CBM). This motif is a conserved CCG sequence presented in a bulge flanked by a stem and a hairpin structure. A similar structure containing a CCG trinucleotide forms the S6:S18 complex binding site in 16S ribosomal RNA. We have constructed a 3D structural model of a S6:S18 complex with S6S18CBM, which suggests that the CCG trinucleotide in a specific structural context may be specifically recognized by the S18 protein. This prediction was supported by site-directed mutagenesis of both RNA and protein components. These results provide a molecular basis for understanding protein-RNA recognition and suggest that the S6S18CBM is involved in an auto-regulatory mechanism.     

Interplay between ribosomal protein S27a and MDM2 protein in p53 activation in response to ribosomal stress

Ribosomal proteins play a critical role in tightly coordinating p53 signaling with ribosomal biogenesis. Several ribosomal proteins have been shown to induce and activate p53 via inhibition of MDM2. Here, we report that S27a, a small subunit ribosomal protein synthesized as an 80-amino acid ubiquitin C-terminal extension protein (CEP80), functions as a novel regulator of the MDM2-p53 loop. S27a interacts with MDM2 at the central acidic domain of MDM2 and suppresses MDM2-mediated p53 ubiquitination, leading to p53 activation and cell cycle arrest. Knockdown of S27a significantly attenuates the p53 activation in cells in response to treatment with ribosomal stress-inducing agent actinomycin D or 5-fluorouracil. Interestingly, MDM2 in turn ubiquitinates S27a and promotes proteasomal degradation of S27a in response to actinomycin D treatment, thus forming a mutual-regulatory loop. Altogether, our results reveal that S27a plays a non-redundant role in mediating p53 activation in response to ribosomal stress via interplaying with MDM2.     

Signature amino acids enable the archaeal L7Ae box C/D RNP core protein to recognize and bind the K-loop RNA motif

The archaeal L7Ae and eukaryotic 15.5kD protein homologs are members of the L7Ae/15.5kD protein family that characteristically recognize K-turn motifs found in both archaeal and eukaryotic RNAs. In Archaea, the L7Ae protein uniquely binds the K-loop motif found in box C/D and H/ACA sRNAs, whereas the eukaryotic 15.5kD homolog is unable to recognize this variant K-turn RNA. Comparative sequence and structural analyses, coupled with amino acid replacement experiments, have demonstrated that five amino acids enable the archaeal L7Ae core protein to recognize and bind the K-loop motif. These signature residues are highly conserved in the archaeal L7Ae and eukaryotic 15.5kD homologs, but differ between the two domains of life. Interestingly, loss of K-loop binding by archaeal L7Ae does not disrupt C′/D′ RNP formation or RNA-guided nucleotide modification. L7Ae is still incorporated into the C′/D′ RNP despite its inability to bind the K-loop, thus indicating the importance of protein–protein interactions for RNP assembly and function. Finally, these five signature amino acids are distinct for each of the L7Ae/L30 family members, suggesting an evolutionary continuum of these RNA-binding proteins for recognition of the various K-turn motifs contained in their cognate RNAs.     

Solution structure of ribosomal protein L40E, a unique C4 zinc finger protein encoded by archaeon Sulfolobus solfataricus

The ribosomal protein L40E from archaeon Sulfolobus solfataricus is a component of the 50S ribosomal subunit. L40E is a 56-residue, highly basic protein that contains a C4 zinc finger motif, CRKC_X(10)_CRRC. Homologs are found in both archaea and eukaryotes but are not present in bacteria. Eukaryotic genomes encode L40E as a ubiquitin-fusion protein. L40E was absent from the crystal structure of euryarchaeota 50S ribosomal subunit. Here we report the three-dimensional solution structure of L40E by NMR spectroscopy. The structure of L40E is a three-stranded beta-sheet with a simple beta2beta1beta3 topology. There are two unique characteristics revealed by the structure. First, a large and ordered beta2-beta3 loop twists to pack across the one side of the protein. L40E contains a buried polar cluster comprising Lys19, Lys20, Cys22, Asn29, and Cys36. Second, the surface of L40E is almost entirely positively charged. Ten conserved basic residues are positioned on the two sides of the surface. It is likely that binding of zinc is essential in stabilizing the tertiary structure of L40E to act as a scaffold to create a broad positively charged surface for RNA and/or protein recognition.     

Cryo-EM structure of the archaeal 50S ribosomal subunit in complex with initiation factor 6 and implications for ribosome evolution

Translation of mRNA into proteins by the ribosome is universally conserved in all cellular life. The composition and complexity of the translation machinery differ markedly between the three domains of life. Organisms from the domain Archaea show an intermediate level of complexity, sharing several additional components of the translation machinery with eukaryotes that are absent in bacteria. One of these translation factors is initiation factor 6 (IF6), which associates with the large ribosomal subunit. We have reconstructed the 50S ribosomal subunit from the archaeon Methanothermobacter thermautotrophicus in complex with archaeal IF6 at 6.6?? resolution using cryo-electron microscopy (EM). The structure provides detailed architectural insights into the 50S ribosomal subunit from a methanogenic archaeon through identification of the rRNA expansion segments and ribosomal proteins that are shared between this archaeal ribosome and eukaryotic ribosomes but are mostly absent in bacteria and in some archaeal lineages. Furthermore, the structure reveals that, in spite of highly divergent evolutionary trajectories of the ribosomal particle and the acquisition of novel functions of IF6 in eukaryotes, the molecular binding of IF6 on the ribosome is conserved between eukaryotes and archaea. The structure also provides a snapshot of the reductive evolution of the archaeal ribosome and offers new insights into the evolution of the translation system in archaea.