Takaomi Nomura

Replacement of L7/L12.L10 Protein Complex in Escherichia coli Ribosomes with the Eukaryotic Counterpart Changes the Specificity of Elongation Factor Binding

The L8 protein complex consisting of L7/L12 and L10 in Escherichia coli ribosomes is assembled on the conserved region of 23 S rRNA termed the GTPase-associated domain. We replaced the L8 complex in E. coli 50 S subunits with the rat counterpart P protein complex consisting of P1, P2, and P0. The L8 complex was removed from the ribosome with 50% ethanol, 10 mm MgCl2, 0.5 m NH4Cl, at 30 °C, and the rat P complex bound to the core particle. Binding of the P complex to the core was prevented by addition of RNA fragment covering the GTPase-associated domain of E. coli 23 S rRNA to which rat P complex bound strongly, suggesting a direct role of the RNA domain in this incorporation. The resultant hybrid ribosomes showed eukaryotic translocase elongation factor (EF)-2-dependent, but not prokaryotic EF-G-dependent, GTPase activity comparable with rat 80 S ribosomes. The EF-2-dependent activity was dependent upon the P complex binding and was inhibited by the antibiotic thiostrepton, a ligand for a portion of the GTPase-associated domain of prokaryotic ribosomes. This hybrid system clearly shows significance of binding of the P complex to the GTPase-associated RNA domain for interaction of EF-2 with the ribosome. The results also suggest that E. coli 23 S rRNA participates in the eukaryotic translocase-dependent GTPase activity in the hybrid system.

A Mode of Assembly of P0, P1, and P2 Proteins at the GTPase-associated Center in Animal Ribosome IN VITRO ANALYSES WITH P0 TRUNCATION MUTANTS

Ribosomal P0, P1, and P2 proteins, together with the conserved domain of 28 S rRNA, constitute a major part of the GTPase-associated center in eukaryotic ribosomes. We investigated the mode of assembly in vitro by using various truncation mutants of silkworm P0. When compared with wild type (WT)-P0, the C-terminal truncation mutants CΔ65 and CΔ81 showed markedly reduced binding ability to P1 and P2, which was offset by the addition of an rRNA fragment covering the P0·P1-P2 binding site. The mutant CΔ107 lost the P1/P2 binding activity, whereas it retained the rRNA binding. In contrast, the N-terminal truncation mutants NΔ21-NΔ92 completely lost the rRNA binding, although they retained P1/P2 binding capability, implying an essential role of the N terminus of P0 for rRNA binding. The P0 mutants NΔ6, NΔ14, and CΔ18-CΔ81, together with P1/P2 and eL12, bound to the Escherichia coli core 50 S subunits deficient in L10·L7/L12 complex and L11. Analysis of incorporation of 32P-labeled P1/P2 into the 50 S subunits with WT-P0 and CΔ81 by sedimentation analysis indicated that WT-P0 bound two copies of P1 and P2, but CΔ81 bound only one copy each. The hybrid ribosome with CΔ81 that appears to contain one P1-P2 heterodimer retained lower but considerable activities dependent on eukaryotic elongation factors. These results suggested that two P1-P2 dimers bind to close but separate regions on the C-terminal half of P0. The results were further confirmed by binding experiments using chimeric P0 mutants in which the C-terminal 81 or 107 amino acids were replaced with the homologous sequences of the archaebacterial P0.

In vitro reconstitution of the GTPase-associated centre of the archaebacterial ribosome: The functional features observed in a hybrid form with Escherichia coli 50S subunits

We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0.Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0.Ph-L12 complex and Ph-L11 could replace L10.L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryotic, as well as archaebacterial elongation factors, but not for prokaryotic elongation factors. The GTPase and polyphenylalanine-synthetic activity that is dependent on eukaryotic elongation factors was comparable with that of the hybrid ribosomes carrying the eukaryotic ribosomal proteins. The results suggest that the archaebacterial proteins, including the Ph-L12 homodimer, are functionally accessible to eukaryotic translation factors.

Three Binding Sites for Stalk Protein Dimers Are Generally Present in Ribosomes from Archaeal Organism

Ribosomes have a characteristic protuberance termed the stalk, which is indispensable for ribosomal function. The ribosomal stalk has long been believed to be a pentameric protein complex composed of two sets of protein dimers, L12-L12, bound to a single anchor protein, although ribosomes carrying three L12 dimers were recently discovered in a few thermophilic bacteria. Here we have characterized the stalk complex from Pyrococcus horikoshii, a thermophilic species of Archaea. This complex is known to be composed of proteins homologous to eukaryotic counterparts rather than bacterial ones. In truncation experiments of the C-terminal regions of the anchor protein Ph-P0, we surprisingly observed three Ph-L12 dimers bound to the C-terminal half of Ph-P0, and the binding site for the third dimer was unique to the archaeal homologs. The stoichiometry of the heptameric complex Ph-P0(Ph-L12)2(Ph-L12)2(Ph-L12)2 was confirmed by mass spectrometry of the intact complex. In functional tests, ribosomes carrying a single Ph-L12 dimer had significant activity, but the addition of the second and third dimers increased the activity. A bioinformatics analysis revealed the evidence that ribosomes from all archaeal and also from many bacterial organisms may contain a heptameric complex at the stalk, whereas eukaryotic ribosomes seem to contain exclusively a pentameric stalk complex, thus modifying our view of the stalk structure significantly.

Long-range periodic sequence of the cement/silk protein of Stenopsyche marmorata: purification and biochemical characterisation

The long-range periodic amino acid sequence of the bifunctional silk/cement protein from larvae of the caddisfly, Stenopsyche marmorata, is discussed in this study. The protein, named the S. marmorata silk protein (Smsp-1), was first purified to electrophoretic homogeneity. The results of Edman-based sequencing of Smsp-1 tryptic digests were consistent with the amino acid sequence deduced from a cDNA clone of the Smsp-1 gene. All undetected amino acids in the Edman-based sequencing were encoded as Ser, suggesting the presence of O-phospho-Ser. 31P-NMR and an O-phospho-amino acid analysis successfully showed that the O-phospho-Ser residue occurred in a clustered manner, serving a cement function for Smsp-1. Two patterns of non-phosphorylated repeats, –SLGPYGDPRGDXLGPYGG– (X = V, G or D) and –GVGPYGDGLGPYGG–, were enriched in Smsp-1 compared with the O-phospho-Ser cluster, and have fibre-forming functions.

A Covariant Change of the Two Highly Conserved Bases in the GTPase-associated Center of 28 S rRNA in Silkworms and Other Moths

The GTPase-associated center in 23/28 S rRNA is one of the most conserved functional domains throughout all organisms. We detected a unique sequence of this domain in Bombyx morispecies in which the bases at positions 1094 and 1098 (numbering fromEscherichia coli 23 S rRNA) are C and G instead of the otherwise universally conserved bases U and A, respectively. These changes were also observed in four other species of moths, but not in organisms other than the moths. Characteristics of the B. mori rRNA domain were investigated by native polyacrylamide gel electrophoresis using RNA fragments containing residues 1030–1128. Although two bands of protein-free RNA appeared on gel, they shifted to a single band when bound to Bombyx ribosomal proteins Bm-L12 and Bm-P complex, equivalent to E. coli L11 and L8, respectively. Bombyx RNA showed lower binding capacity than rat RNA for the ribosomal proteins and anti-28 S autoantibody, specific for a folded structure of the eukaryotic GTPase-associated domain. When the C1094/G1098bases in Bombyx RNA were replaced by the conserved U/A bases, the protein-free RNA migrated as a single band, and the complex formation with Bm-L12, Bm-P complex, and anti-28 S autoantibody was comparable to that of rat RNA. The results suggest that the GTPase-associated domain of moth-type insects has a labile structural feature that is caused by an unusual covariant change of the U1094/A1098 bases to C/G.

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.

Engineering and Characterization of the Ribosomal L10·L12 Stalk Complex : A STRUCTURAL ELEMENT RESPONSIBLE FOR HIGH TURNOVER OF THE ELONGATION FACTOR G-DEPENDENT GTPase

The ribosomal stalk protein L12 is essential for events dependent on the GTP-binding translation factors. It has been recently shown that ribosomes from Thermus thermophilus contain a heptameric complex L10·(L12)2·(L12)2·(L12)2, rather than the conventional pentameric complex L10·(L12)2·(L12)2. Here we describe the reconstitution of the heptameric complex from purified L10 and L12 and the characterization of its role in elongation factor G-dependent GTPase activity using a hybrid system with Escherichia coli ribosomes. The T. thermophilus heptameric complex resulted in a 2.5-fold higher activity than the E. coli pentameric complex. The structural element of the T. thermophilus complex responsible for the higher activity was investigated using a chimeric L10 protein (Ec-Tt-L10), in which the C-terminal L12-binding site in E. coli L10 was replaced with the same region from T. thermophilus, and two chimeric L12 proteins: Ec-Tt-L12, in which the E. coli N-terminal domain was fused with the T. thermophilus C-terminal domain, and Tt·Ec-L12, in which the T. thermophilus N-terminal domain was fused with the E. coli C-terminal domain. High GTPase turnover was observed with the pentameric chimeric complex formed from E. coli L10 and Ec-Tt-L12 but not with the heptameric complex formed from Ec-Tt-L10 and Tt·Ec-L12. This suggested that the C-terminal region of T. thermophilus L12, rather than the heptameric nature of the complex, was responsible for the high GTPase turnover. Further analyses with other chimeric L12 proteins identified helix α6 as the region most likely to contain the responsible element.

Biochemical evidence for the heptameric complex L10(L12)6 in the Thermus thermophilus ribosome: in vitro analysis of its molecular assembly and functional properties.

The stalk protein L12 is the only multiple component in 50S ribosomal subunit. In Escherichia coli, two L12 dimers bind to the C-terminal domain of L10 to form a pentameric complex, L10[(L12)(2)](2), while the recent X-ray crystallographic study and tandem MS analyses revealed the presence of a heptameric complex, L10[(L12)(2)](3), in some thermophilic bacteria. We here characterized the complex of Thermus thermophilus (Tt-) L10 and Tt-L12 stalk proteins by biochemical approaches using C-terminally truncated variants of Tt-L10. The C-terminal 44-residues removal (Delta44) resulted in complete loss of interactions with Tt-L12. Quantitative analysis of Tt-L12 assembled onto E. coli 50S core particles, together with Tt-L10 variants, indicated that the wild-type, Delta13 and Delta23 variants bound three, two and one Tt-L12 dimers, respectively. The hybrid ribosomes that contained the T. thermophilus proteins were highly accessible to E. coli elongation factors. The progressive removal of Tt-L12 dimers caused a stepwise reduction of ribosomal activities, which suggested that each individual stalk dimer contributed to ribosomal function. Interestingly, the hybrid ribosomes showed higher EF-G-dependent GTPase activity than E. coli ribosomes, even when two or one Tt-L12 dimer. This result seems to be due to a structural characteristic of Tt-L12 dimer.

Characterization of Underwater Silk Proteins from Caddisfly Larva, Stenopsyche marmorata

The underwater silk threads of a caddisfly larva, Stenopsyche marmorata, is composed of four kinds of proteins, which are designated as Smsp-1 to -4. Smsp-1 has been purified in a electrophoretic homogeneity, and the peptide sequencing of the Smsp-1 tryptic digests indicated the presence of eight kinds of characteristic segments, most of which are enriched in Pro residues. The segments 3 and 4 are the 14-residue sequences of SLGPYGDXLGPYGG (X = G, D, or V) and GVGPYGDGLGPYGG, respectively. Partial molecular cloning of the Smsp-1-C-end region suggested that the hydrophobic segment 3,4-repeating moieties occupy mostly a half of the entire primary sequence of Smsp-1 gene. The segments 7 and 8 contain highly phosphorylated regions, where the Ser residues are exclusively modified in the cluster-like sequences as –(Xaa S) n – (Xaa = G, A, V, or I; S = O-phospho-L-serine; n = 3 or 4). The O-phosphorylation of Smsp-1 may switch the protein function as the fiber-forming components to the underwater cements for building the pupal case in the late 5th instar stage of the larvae.