The flexible N-terminal motif of uL11 unique to eukaryotic ribosomes interacts with P-complex and facilitates protein translation.

Eukaryotic uL11 contains a conserved MPPKFDP motif at the N-terminus that is not found in archaeal and bacterial homologs. Here, we determined the solution structure of human uL11 by NMR spectroscopy and characterized its backbone dynamics by 15N-1H relaxation experiments. We showed that these N-terminal residues are unstructured and flexible. Structural comparison with ribosome-bound uL11 suggests that the linker region between the N-terminal domain and C-terminal domain of human uL11 is intrinsically disordered and only becomes structured when bound to the ribosomes. Mutagenesis studies show that the N-terminal conserved MPPKFDP motif is involved in interacting with the P-complex and its extended protuberant domain of uL10 in vitro. Truncation of the MPPKFDP motif also reduced the poly-phenylalanine synthesis in both hybrid ribosome and yeast mutagenesis studies. In addition, G→A/P substitutions to the conserved GPLG motif of helix-1 reduced poly-phenylalanine synthesis to 9-32% in yeast ribosomes. We propose that the flexible N-terminal residues of uL11, which could extend up to ∼25 Šfrom the N-terminal domain of uL11, can form transient interactions with the uL10 that help to fetch and fix it into a position ready for recruiting the incoming translation factors and facilitate protein synthesis.

The natural bicyclic hexapeptide RA-VII is a novel inhibitor of the eukaryotic translocase eEF2.

A cyclic hexapeptide, RA-VII isolated from the Rubiaceae family of plants, has high cytotoxic activity. Although RA-VII has been shown to inhibit protein synthesis in eukaryotic cells, the molecular mode of its action is not clear. Here we investigate the mechanism of the RAVII action on the translation apparatus. Biochemical functional assays showed that RA-VII inhibits poly(U)-dependent polyphenylalanine synthesis in the presence of animal elongation factors eEF1A and eEF2. Furthermore, RAVII prevented eEF2/ribosome-dependent GTPase activity, but not eEF-1A/ribosome-dependent activity. A filter binding assay demonstrated that RA-VII markedly enhances the binding affinity of eEF2 for GTP, but not for GDP, and prevents exchange of GTP in the eEF2-GTP complex, even after addition of a large excess of GTP/GDP. Limited proteolysis experiments indicated that RA-VII prevents the digestion of eEF2 in the presence of either GTP or GMPPCP, but not with GDP. Further footprint analysis and a translocation assay showed that the eEF2•GMPPNP•RA-VII complex binds to the conserved rRNA regions at the factor-binding center of the ribosome and retains the ability to translocate the A site-bound tRNA to the P-site. These results suggest that RA-VII tightly stabilizes the GTP•eEF2 complex structure, which is able to bind to the ribosomal functional site, but seems to suppress normal turnover of eEF2 after translocation. The properties of RA-VII make it a novel ligand for probing the action of eEF2 in the process of translocation on the ribosome.

A novel ribosome-dimerization protein found in the hyperthermophilic archaeon Pyrococcus furiosus using ribosome-associated proteomics.

Ribosome dimerization is one of the bacterial events that suppresses protein synthesis in the stationary phase. Protein factors responsible for ribosome dimerization in bacteria are well characterized, whereas no information is available for the corresponding factors in archaeal and eukaryotic cells. Here we describe a protein found among the ribosome-associated proteins which dimerizes the 30S ribosomal subunit of the archaeon Pyrococcus furiosus. The ribosome-associated proteins were prepared by high-salt wash of crude ribosomes, and analyzed by nanoflow liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). Of the detected proteins we focused on a protein (PF0560) whose Protein Score was the highest of all of the function-unknown proteins. PF0560 protein had a pronounced effect on the sedimentation pattern of the 30S ribosomal subunit; addition of this protein to isolated 30S subunit reduced the 30S fraction and increased the amount of the 50S fraction. This increase presumably corresponds to the dimer of the 30S subunit. The PF0560-dependent 30S-dimerization, was also observed by gel electrophoretic analysis. This effect was not observed in EDTA-treated 30S subunit, with protein-free 16S rRNA or with bacterial/eukaryotic ribosomal small subunits. Furthermore, PF0560 protein suppressed the formation of functional 70S ribosomes. These results suggest that PF0560 is a novel 30S dimerization factor, which might participate in regulation of archaeal translation.

Structural insights into the Switching Off of the Interaction between the Archaeal Ribosomal Stalk and aEF1A by Nucleotide Exchange Factor aEF1B.

The ribosomal stalk protein plays a crucial role in functional interactions with translational GTPase factors. It has been shown that the archaeal stalk aP1 binds to both GDP- and GTP-bound conformations of aEF1A through its C-terminal region in two different modes. To obtain an insight into how the aP1•aEF1A binding mode changes during the process of nucleotide exchange from GDP to GTP on aEF1A, we have analyzed structural changes in aEF1A upon binding of the nucleotide exchange factor aEF1B. The isolated archaeal aEF1B has nucleotide exchange ability in the presence of aa-tRNA but not deacylated tRNA, and increases activity of polyphenylalanine synthesis 4-fold. The aEF1B mutation, R90A, results in loss of its original nucleotide exchange activity but retains a remarkable ability to enhance polyphenylalanine synthesis. These results suggest an additional functional role for aEF1B other than in nucleotide exchange. The crystal structure of the aEF1A•aEF1B complex, resolved at 2.0 Å resolution, shows marked rotational movement of domain 1 of aEF1A compared to the structure of aEF1A•GDP•aP1, and this conformational change results in disruption of the original aP1 binding site between domains 1 and 3 of aEF1A. The loss of aP1 binding to the aEF1A•aEF1B complex was confirmed by native gel analysis. The results suggest that aEF1B plays a role in switching off the interaction between aP1 and aEF1A•GDP, as well as in nucleotide exchange, and promote translation elongation.

Mood Disorder in Systemic Lupus Erythematosus Induced by Antiribosomal P Protein Antibodies Associated with Decreased Serum and Brain Tryptophan.

Antiribosomal P protein (anti-P) autoantibodies commonly develop in patients with systemic lupus erythematosus. We have previously established hybridoma clones producing anti-P mAbs. In this study, we explored the pathogenesis of behavioral disorders induced by anti-P Abs using these mAbs. New Zealand Black × New Zealand White F1, New Zealand White, C57BL/6, and BALB/c mice were treated with 1 mg of anti-P Abs once every 2 wk. The behavioral disorder was evaluated by the tail suspension test, forced swim test, and open field test. Following administration of anti-P Abs, New Zealand Black × New Zealand White F1 and C57BL/6 mice developed depressive behavior and showed increased anxiety with elevated serum TNF-α and IL-6 levels. Anti-P Abs were not deposited in the affected brain tissue; instead, this mood disorder was associated with lower serum and brain tryptophan concentrations. Tryptophan supplementation recovered serum tryptophan levels and prevented the behavioral disorder. TNF-α and IL-6 were essential for the decreased serum tryptophan and disease development, which were ameliorated by treatment with anti-TNF-α neutralizing Abs or dexamethasone. Peritoneal macrophages from C57BL/6 mice produced TNF-α, IL-6, and IDO-1 via interaction with anti-P Abs through activating FcγRs, which were required for disease development. IVIg, which has an immunosuppressive effect partly through the regulation of FcγR expression, also prevented the decrease in serum tryptophan and disease development. Furthermore, serum tryptophan concentrations were decreased in the sera of systemic lupus erythematosus patients with anti-P Abs, and lower tryptophan levels correlated with disease activity. Our study revealed some of the molecular mechanisms of mood disorder induced by anti-P Abs.

Association of coexisting anti-ribosomal P and anti-dsDNA antibodies with histology and renal prognosis in lupus nephritis patients.

OBJECTIVES: Anti-ribosomal P protein autoantibodies (anti-P) specifically develop in patients with systemic lupus erythematosus. Associations of anti-P with lupus nephritis (LN) histological subclass and renal outcome remain inconclusive. We sought to determine the association of anti-P and anti-double-stranded DNA antibody (anti-dsDNA) with renal histology and prognosis in LN patients. METHODS: Thirty-four patients with LN, having undergone kidney biopsy, were included. The 2018 revised ISN/RPS classification system was used for pathophysiological evaluation. Chronic kidney disease (CKD) was defined as an estimated glomerular filtration rate < 60 mL/min/1.73 m2 for > 3 months. RESULTS: Six patients (17.6%) were positive for anti-P and 26 (76.5%) for anti-dsDNA. Among the six patients with anti-P, one did not have anti-dsDNA, but did have anti-Sm antibody, and showed a histological subtype of class V. This patient maintained good renal function for over 14 years. The remaining five patients, who had both anti-P and anti-dsDNA, exhibited proliferative nephritis and were associated with prolonged hypocomplementemia, and the incidence of CKD did not differ from patients without anti-P. CONCLUSION: Although this study included a small number of patients, the results indicated that histology class and renal prognosis associated with anti-P depend on the coexistence of anti-dsDNA. Further studies with a large number of patients are required to confirm this conclusion.

Direct visualization of translational GTPase factor pool formed around the archaeal ribosomal P-stalk by high-speed AFM.

In translation elongation, two translational guanosine triphosphatase (trGTPase) factors EF1A and EF2 alternately bind to the ribosome and promote polypeptide elongation. The ribosomal stalk is a multimeric ribosomal protein complex which plays an essential role in the recruitment of EF1A and EF2 to the ribosome and their GTP hydrolysis for efficient and accurate translation elongation. However, due to the flexible nature of the ribosomal stalk, its structural dynamics and mechanism of action remain unclear. Here, we applied high-speed atomic force microscopy (HS-AFM) to directly visualize the action of the archaeal ribosomal heptameric stalk complex, aP0•(aP1•aP1)3 (P-stalk). HS-AFM movies clearly demonstrated the wobbling motion of the P-stalk on the large ribosomal subunit where the stalk base adopted two conformational states, a predicted canonical state, and a newly identified flipped state. Moreover, we showed that up to seven molecules of archaeal EF1A (aEF1A) and archaeal EF2 (aEF2) assembled around the ribosomal P-stalk, corresponding to the copy number of the common C-terminal factor-binding site of the P-stalk. These results provide visual evidence for the factor-pooling mechanism by the P-stalk within the ribosome and reveal that the ribosomal P-stalk promotes translation elongation by increasing the local concentration of translational GTPase factors.

Switch of the interactions between the ribosomal stalk and EF1A in the GTP- and GDP-bound conformations.

Translation elongation factor EF1A delivers aminoacyl-tRNA to the ribosome in a GTP-bound form, and is released from the ribosome in a GDP-bound form. This association/dissociation cycle proceeds efficiently via a marked conformational change in EF1A. EF1A function is dependent on the ribosomal "stalk" protein of the ribosomal large subunit, although the precise mechanism of action of the stalk on EF1A remains unclear. Here, we clarify the binding mode of archaeal stalk aP1 to GTP-bound aEF1A associated with aPelota. Intriguingly, the C-terminal domain (CTD) of aP1 binds to aEF1A•GTP with a similar affinity to aEF1A•GDP. We have also determined the crystal structure of the aP1-CTD•aEF1A•GTP•aPelota complex at 3.0 Šresolution. The structure shows that aP1-CTD binds to a space between domains 1 and 3 of aEF1A. Biochemical analyses show that this binding is crucial for protein synthesis. Comparison of the structures of aP1-CTD•aEF1A•GTP and aP1-CTD•aEF1A•GDP demonstrates that the binding mode of aP1 changes markedly upon a conformational switch between the GTP- and GDP-bound forms of aEF1A. Taking into account biochemical data, we infer that aP1 employs its structural flexibility to bind to aEF1A before and after GTP hydrolysis for efficient protein synthesis.

Structural and Mutagenesis Studies Evince the Role of the Extended Protuberant Domain of Ribosomal Protein uL10 in Protein Translation.

The lateral stalk of ribosomes constitutes the GTPase-associated center and is responsible for recruiting translation factors to the ribosomes. The eukaryotic stalk contains a P-complex, in which one molecule of uL10 (formerly known as P0) protein binds two copies of P1/P2 heterodimers. Unlike bacterial uL10, eukaryotic uL10 has an extended protuberant (uL10ext) domain inserted into the N-terminal RNA-binding domain. Here, we determined the solution structure of the extended protuberant domain of Bombyx mori uL10 by nuclear magnetic resonance spectroscopy. Comparison of the structures of the B. mori uL10ext domain with eRF1-bound and eEF2-bound ribosomes revealed significant structural rearrangement in a "hinge" region surrounding Phe183, a residue conserved in eukaryotic but not in archaeal uL10. 15N relaxation analyses showed that residues in the hinge region have significantly large values of transverse relaxation rates. To test the role of the conserved phenylalanine residue, we created a yeast mutant strain expressing an F181A variant of uL10. An in vitro translation assay showed that the alanine substitution increased the level of polyphenylalanine synthesis by ∼33%. Taken together, our results suggest that the hinge motion of the uL10ext domain facilitates the binding of different translation factors to the GTPase-associated center during protein synthesis.

High-resolution crystal structure of peptidyl-tRNA hydrolase from Thermus thermophilus.

Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are the products of defective translation, to recycle the tRNA for further rounds of protein synthesis. Pth is ubiquitous in nature, and its activity is essential for bacterial viability. Here, we have determined the crystal structure of Pth from Thermus thermophilus (TtPth) at 1.00 Å resolution. This is the first structure of a Pth from a thermophilic bacterium and the highest resolution Pth structure reported so far. The present atomic resolution data enabled the calculation of anisotropic displacement parameters for all atoms, which revealed the directionality of the fluctuations of key regions for the substrate recognition. Comparisons between TtPth and mesophilic bacterial Pths revealed that their structures are similar overall. However, the structures of the N- and C-terminal, loop-helix α4, and helix α6 regions are different. In addition, the helix α1 to strand ß4 region of TtPth is remarkably different from those of the mesophilic bacterial Pths, because this region is 9 or 10 amino acid residues shorter than those of the mesophilic bacterial Pths. This shortening seems to contribute to the thermostability of TtPth. To further understand the determinants for the thermostability of TtPth, we compared various structural factors of TtPth with those of mesophilic bacterial Pths. The data suggest that the decreases in accessible surface area and thermolabile amino acid residues, and the increases in ion pairs, hydrogen bonds, and proline residues cooperatively contribute to the thermostability of TtPth.

The ribosomal stalk protein is crucial for the action of the conserved ATPase ABCE1.

The ATP-binding cassette (ABC) protein ABCE1 is an essential factor in ribosome recycling during translation. However, the detailed mechanochemistry of its recruitment to the ribosome, ATPase activation and subunit dissociation remain to be elucidated. Here, we show that the ribosomal stalk protein, which is known to participate in the actions of translational GTPase factors, plays an important role in these events. Biochemical and crystal structural data indicate that the conserved hydrophobic amino acid residues at the C-terminus of the archaeal stalk protein aP1 binds to the nucleotide-binding domain 1 (NBD1) of aABCE1, and that this binding is crucial for ATPase activation of aABCE1 on the ribosome. The functional role of the stalk•ABCE1 interaction in ATPase activation and the subunit dissociation is also investigated using mutagenesis in a yeast system. The data demonstrate that the ribosomal stalk protein likely participates in efficient actions of both archaeal and eukaryotic ABCE1 in ribosome recycling. The results also show that the stalk protein has a role in the function of ATPase as well as GTPase factors in translation.

The Interaction between the Ribosomal Stalk Proteins and Translation Initiation Factor 5B Promotes Translation Initiation.

Ribosomal stalk proteins recruit translation elongation GTPases to the factor-binding center of the ribosome. Initiation factor 5B (eIF5B in eukaryotes and aIF5B in archaea) is a universally conserved GTPase that promotes the joining of the large and small ribosomal subunits during translation initiation. Here we show that aIF5B binds to the C-terminal tail of the stalk protein. In the cocrystal structure, the interaction occurs between the hydrophobic amino acids of the stalk C-terminal tail and a small hydrophobic pocket on the surface of the GTP-binding domain (domain I) of aIF5B. A substitution mutation altering the hydrophobic pocket of yeast eIF5B resulted in a marked reduction in ribosome-dependent eIF5B GTPase activity in vitro In yeast cells, the eIF5B mutation affected growth and impaired GCN4 expression during amino acid starvation via a defect in start site selection for the first upstream open reading frame in GCN4 mRNA, as observed with the eIF5B deletion mutant. The deletion of two of the four stalk proteins diminished polyribosome levels (indicating defective translation initiation) and starvation-induced GCN4 expression, both of which were suppressible by eIF5B overexpression. Thus, the mutual interaction between a/eIF5B and the ribosomal stalk plays an important role in subunit joining during translation initiation in vivo.

The C-terminal helix of ribosomal P stalk recognizes a hydrophobic groove of elongation factor 2 in a novel fashion.

Archaea and eukaryotes have ribosomal P stalks composed of anchor protein P0 and aP1 homodimers (archaea) or P1•P2 heterodimers (eukaryotes). These P stalks recruit translational GTPases to the GTPase-associated center in ribosomes to provide energy during translation. The C-terminus of the P stalk is known to selectively recognize GTPases. Here we investigated the interaction between the P stalk and elongation factor 2 by determining the structures of Pyrococcus horikoshii EF-2 (PhoEF-2) in the Apo-form, GDP-form, GMPPCP-form (GTP-form), and GMPPCP-form bound with 11 C-terminal residues of P1 (P1C11). Helical structured P1C11 binds to a hydrophobic groove between domain G and subdomain G' of PhoEF-2, where is completely different from that of aEF-1α in terms of both position and sequence, implying that such interaction characteristic may be requested by how GTPases perform their functions on the ribosome. Combining PhoEF-2 P1-binding assays with a structural comparison of current PhoEF-2 structures and molecular dynamics model of a P1C11-bound GDP form, the conformational changes of the P1C11-binding groove in each form suggest that in response to the translation process, the groove has three states: closed, open, and release for recruiting and releasing GTPases.

Binding of translation elongation factors to individual copies of the archaeal ribosomal stalk protein aP1 assembled onto aP0.

Ribosomes in all organisms contain oligomeric and flexible proteins called stalks, which are responsible for the recruitment of translational GTPase factors to the ribosome. Archaeal ribosomes have three stalk homodimers (aP1)2 that constitute a heptameric complex with the anchor protein aP0. We investigated the factor binding ability of aP1 proteins assembled onto aP0, by gel-retardation assays. The isolated aP0(aP1)2(aP1)2(aP1)2 complex, as well as the form bound to the Escherichia coli 50S core, as a hybrid 50S particle, interacted strongly with elongation factor aEF2, but weakly with aEF1A. These interactions were disrupted by a point mutation, F107S, at the C-terminus of aP1. To examine the ability of each copy of aP0-associated aP1 to bind to elongation factors, we constructed aP0·aP1 variant trimers, composed of an aP0 mutant and a single (aP1)2 dimer. Biochemical and quantitative analyses revealed that the resultant three trimers, aP0(aP1)2I, aP0(aP1)2II, and aP0(aP1)2III, individually bound two molecules of aEF2, suggesting that each copy of the aP1 C-terminal region in the aP0·aP1 trimers can bind tightly to aEF2. Interestingly, the unstable binding of aEF1A to each of the three aP0·aP1 trimers was remarkably stabilized in the presence of aEF2. The stability of the aEF1A binding to the stalk complex may be affected by the presence of aEF2 bound to the complex, by an unknown mechanism.

Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute.

Argonaute proteins are key players in the gene silencing mechanisms mediated by small nucleic acids in all domains of life from bacteria to eukaryotes. However, little is known about the Argonaute protein that recognizes guide RNA/target DNA. Here, we determine the 2 Å crystal structure of Rhodobacter sphaeroides Argonaute (RsAgo) in a complex with 18-nucleotide guide RNA and its complementary target DNA. The heteroduplex maintains Watson-Crick base-pairing even in the 3'-region of the guide RNA between the N-terminal and PIWI domains, suggesting a recognition mode by RsAgo for stable interaction with the target strand. In addition, the MID/PIWI interface of RsAgo has a system that specifically recognizes the 5' base-U of the guide RNA, and the duplex-recognition loop of the PAZ domain is important for the DNA silencing activity. Furthermore, we show that Argonaute discriminates the nucleic acid type (RNA/DNA) by recognition of the duplex structure of the seed region.

Involvement of ribosomal protein L6 in assembly of functional 50S ribosomal subunit in Escherichia coli cells.

Ribosomal protein L6, an essential component of the large (50S) subunit, primarily binds to helix 97 of 23S rRNA and locates near the sarcin/ricin loop of helix 95 that directly interacts with GTPase translation factors. Although L6 is believed to play important roles in factor-dependent ribosomal function, crucial biochemical evidence for this hypothesis has not been obtained. We constructed and characterized an Escherichia coli mutant bearing a chromosomal L6 gene (rplF) disruption and carrying a plasmid with an arabinose-inducible L6 gene. Although this ΔL6 mutant grew more slowly than its wild-type parent, it proliferated in the presence of arabinose. Interestingly, cell growth in the absence of arabinose was biphasic. Early growth lasted only a few generations (LI-phase) and was followed by a suspension of growth for several hours (S-phase). This suspension was followed by a second growth phase (LII-phase). Cells harvested at both LI- and S-phases contained ribosomes with reduced factor-dependent GTPase activity and accumulated 50S subunit precursors (45S particles). The 45S particles completely lacked L6. Complete 50S subunits containing L6 were observed in all growth phases regardless of the L6-depleted condition, implying that the ΔL6 mutant escaped death because of a leaky expression of L6 from the complementing plasmid. We conclude that L6 is essential for the assembly of functional 50S subunits at the late stage. We thus established conditions for the isolation of L6-depleted 50S subunits, which are essential to study the role of L6 in translation.

Crystal structure of translation initiation factor 5B from the crenarchaeon Aeropyrum pernix.

Initiation factor 5B (IF5B) is a universally conserved translational GTPase that catalyzes ribosomal subunit joining. In eukaryotes, IF5B directly interacts via a groove in its domain IV with initiation factor 1A (IF1A), another universally conserved initiation factor, to accomplish efficient subunit joining. Here, we have determined the first structure of a crenarchaeal IF5B, which revealed that the archaea-specific region of IF5B (helix α15) binds and occludes the groove of domain IV. Therefore, archaeal IF5B cannot access IF1A in the same manner as eukaryotic IF5B. This fact suggests that different relationships between IF5B and IF1A exist in archaea and eukaryotes.

Characterization of silk gland ribosomes from a bivoltine caddisfly, Stenopsyche marmorata: translational suppression of a silk protein in cold conditions.

Larval Stenopsyche marmorata constructs food capture nets and fixed retreats underwater using self-produced proteinaceous silk fibers. In the Chikuma River (Nagano Prefecture, Japan) S. marmorata has a bivoltine life cycle; overwintering larvae grow slowly with reduced net spinning activity in winter. We recently reported constant transcript abundance of S. marmorata silk protein 1 (Smsp-1), a core S. marmorata silk fiber component, in all seasons, implying translational suppression in the silk gland during winter. Herein, we prepared and characterized silk gland ribosomes from seasonally collected S. marmorata larvae. Ribosomes from silk glands immediately frozen in liquid nitrogen (LN2) after dissection exhibited comparable translation elongation activity in spring, summer, and autumn. Conversely, silk glands obtained in winter did not contain active ribosomes and Smsp-1. Ribosomes from silk glands immersed in ice-cold physiological saline solution for approximately 4 h were translationally inactive, despite summer collection and Smsp-1 expression. The ribosomal inactivation occurs because of defects in the formation of 80S ribosomes, presumably due to splitting of 60S subunits containing 28S rRNA with central hidden break, in response to cold stress. These results suggest a novel-type ribosome-regulated translation control mechanism.

Functional role of the C-terminal tail of the archaeal ribosomal stalk in recruitment of two elongation factors to the sarcin/ricin loop of 23S rRNA.

Two types of elongation factors alternate in their binding to the factor-binding center of the ribosome. Both binding events are accompanied by GTP hydrolysis and drive the translation elongation cycle. The multicopy ribosomal protein family, termed the stalk, contributes actively to the elongation process. Recent evidence indicates that the mobile C-terminal tail of archaeal stalk aP1 directly interacts with both the elongation factors aEF1A and aEF2. To investigate the functional significance of these interactions in recruitment of elongation factors to the factor-binding center of the ribosome, we substituted the archaeal stalk complex aL10•aP1 for the bL10•bL12 stalk complex in the Escherichia coli 50S subunit. The resultant hybrid ribosome accessed archaeal aEF1A and aEF2 in a manner dependent on the C-terminal tail containing the hydrophobic residues Leu103, Leu106 and Phe107. Bases G2659 and A2660 in the sarcin/ricin loop (SRL) of 23S rRNA were protected against DMS modification by both factors as was A1067 by aEF2. Mutagenesis indicated that this protection was dependent on the intact C-terminal tail of aP1. The results suggest a crucial role for the interactions between the stalk C-terminal tail and elongation factors in their recruitment to the SRL of 23S rRNA within the ribosome.

Molecular insights into the interaction of the ribosomal stalk protein with elongation factor 1α.

In all organisms, the large ribosomal subunit contains multiple copies of a flexible protein, the so-called 'stalk'. The C-terminal domain (CTD) of the stalk interacts directly with the translational GTPase factors, and this interaction is required for factor-dependent activity on the ribosome. Here we have determined the structure of a complex of the CTD of the archaeal stalk protein aP1 and the GDP-bound archaeal elongation factor aEF1α at 2.3 Å resolution. The structure showed that the CTD of aP1 formed a long extended α-helix, which bound to a cleft between domains 1 and 3 of aEF1α, and bridged these domains. This binding between the CTD of aP1 and the aEF1α•GDP complex was formed mainly by hydrophobic interactions. The docking analysis showed that the CTD of aP1 can bind to aEF1α•GDP located on the ribosome. An additional biochemical assay demonstrated that the CTD of aP1 also bound to the aEF1α•GTP•aminoacyl-tRNA complex. These results suggest that the CTD of aP1 interacts with aEF1α at various stages in translation. Furthermore, phylogenetic perspectives and functional analyses suggested that the eukaryotic stalk protein also interacts directly with domains 1 and 3 of eEF1α, in a manner similar to the interaction of archaeal aP1 with aEF1α.