Nobukazu Nameki

YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl–tRNA on stalled ribosomes

In bacteria, ribosomes often become stalled and are released by a trans-translation process mediated by transfer-messenger RNA (tmRNA). In the absence of tmRNA, however, there is evidence that stalled ribosomes are released from non-stop mRNAs. Here, we show a novel ribosome rescue system mediated by a small basic protein, YaeJ, from Escherichia coli, which is similar in sequence and structure to the catalytic domain 3 of polypeptide chain release factor (RF). In vitro translation experiments using the E. coli-based reconstituted cell-free protein synthesis system revealed that YaeJ can hydrolyze peptidyl–tRNA on ribosomes stalled by both non-stop mRNAs and mRNAs containing rare codon clusters that extend downstream from the P-site and prevent Ala-tmRNA•SmpB from entering the empty A-site. In addition, YaeJ had no effect on translation of a normal mRNA with a stop codon. These results suggested a novel tmRNA-independent rescue system for stalled ribosomes in E. coli. YaeJ was almost exclusively found in the 70S ribosome and polysome fractions after sucrose density gradient sedimentation, but was virtually undetectable in soluble fractions. The C-terminal basic residue-rich extension was also found to be required for ribosome binding. These findings suggest that YaeJ functions as a ribosome-attached rescue device for stalled ribosomes.

Three of four pseudoknots in tmRNA are interchangeable and are substitutable with single‐stranded RNAs

A novel translation, trans‐translation, is facilitated by a highly structured RNA molecule, tmRNA. This molecule has two structural domains, a tRNA domain and an mRNA domain, the latter including four pseudoknot structures (PK1 to PK4). Here, we show that replacement of each of these pseudoknots, except PK1, in Escherichia coli tmRNA with a single stranded RNA did not seriously affect the functions as an alanine tRNA and as an mRNA. Furthermore, these three pseudoknots were interchangeable with only small losses of the two functions. These findings suggest that neither PK2, PK3 nor PK4 interacts in a functional manner with ribosome during the trans‐translation process. Together with an earlier study showing the significance of PK1, it is concluded that among the four pseudoknots, PK1 is the most functional.

Solution structure of the RWD domain of the mouse GCN2 protein

GCN2 is the α‐subunit of the only translation initiation factor (eIF2α) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N‐terminus, which is termed the RWD domain after three major RWD‐containing proteins: RING finger‐containing proteins, WD‐repeat‐containing proteins, and yeast DEAD (DEXD)‐like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an α + β sandwich fold consisting of two layers: a four‐stranded antiparallel β‐sheet, and three side‐by‐side α‐helices, with an αββββαα topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive β‐turns that overlap with each other by two residues (triple β‐turn). As putative binding sites with GCN1, a structure‐based alignment allowed the identification of several surface residues in α‐helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin‐conjugating enzymes (E2s), with most of the structural differences in the region connecting β‐strand 4 and α‐helix 3. The structural architecture, including the triple β‐turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein‐binding that plays specific roles in individual RWD‐containing proteins.

Solution structure of a tmRNA-binding protein, SmpB, from Thermus thermophilus

Small protein B (SmpB) is required for trans‐translation, binding specifically to tmRNA. We show here the solution structure of SmpB from an extremely thermophilic bacterium, Thermus thermophilus HB8, determined by heteronuclear nuclear magnetic resonance methods. The core of the protein consists of an antiparallel β‐barrel twisted up from eight β‐strands, each end of which is capped with the second or third helix, and the first helix is located beside the barrel. Its C‐terminal sequence (20 residues), which is rich in basic residues, shows a poorly structured form, as often seen in isolated ribosomal proteins. The results are discussed in relation to the oligonucleotide binding fold.

Solution structure of the PWWP domain of the hepatoma-derived growth factor family

Among the many PWWP‐containing proteins, the largest group of homologous proteins is related to hepatoma‐derived growth factor (HDGF). Within a well‐conserved region at the extreme N‐terminus, HDGF and five HDGF‐related proteins (HRPs) always have a PWWP domain, which is a module found in many chromatin‐associated proteins. In this study, we determined the solution structure of the PWWP domain of HDGF‐related protein‐3 (HRP‐3) by NMR spectroscopy. The structure consists of a five‐stranded β‐barrel with a PWWP‐specific long loop connecting β2 and β3 (PR‐loop), followed by a helical region including two α‐helices. Its structure was found to have a characteristic solvent‐exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the β1/β2 loop (β‐β arch) and the β3/β4 loop. A similar ligand binding cavity occurs at the corresponding position in the Tudor, chromo, and MBT domains, which have structural and probable evolutionary relationships with PWWP domains. These findings suggest that the PWWP domains of the HDGF family bind to some component of chromatin via the cavity.

Solution structure of the catalytic domain of the mitochondrial protein ICT1 that is essential for cell vitality

The ICT1 protein was recently reported to be a component of the human mitoribosome and to have codon-independent peptidyl-tRNA hydrolysis activity via its conserved GGQ motif, although little is known about the detailed mechanism. Here, using NMR spectroscopy, we determined the solution structure of the catalytic domain of the mouse ICT1 protein that lacks an N-terminal mitochondrial targeting signal and an unstructured C-terminal basic-residue-rich extension, and we examined the effect of ICT1 knockdown (mediated by small interfering RNA) on mitochondria in HeLa cells using flow cytometry. The catalytic domain comprising residues 69-162 of the 206-residue full-length protein forms a structure with a β1-β2-α1-β3-α2 topology and a structural framework that resembles the structure of GGQ-containing domain 3 of class 1 release factors (RFs). Half of the structure, including the GGQ-containing loop, has essentially the same sequence and structure as those in RFs, consistent with the peptidyl-tRNA hydrolysis activity of ICT1 on the mitoribosome, which is analogous to RFs. However, the other half of the structure differs in shape from the corresponding part of RF domain 3 in that in ICT1, an α-helix (α1), instead of a β-turn, is inserted between strand β2 and strand β3. A characteristic groove formed between α1 and the three-stranded antiparallel β-sheet was identified as a putative ICT1-specific functional site by a structure-based alignment. In addition, the structured domain that recognizes stop codons in RFs is replaced in ICT1 by a C-terminal basic-residue-rich extension. It appears that these differences are linked to a specific function of ICT1 other than the translation termination mediated by RFs. Flow cytometry analysis showed that the knockdown of ICT1 results in apoptotic cell death with a decrease in mitochondrial membrane potential and mass. In addition, cytochrome c oxidase activity in ICT1 knockdown cells was decreased by 35% compared to that in control cells. These results indicate that ICT1 function is essential for cell vitality and mitochondrial function.

Solution structure and siRNA‐mediated knockdown analysis of the mitochondrial disease‐related protein C12orf65

Loss of function of the c12orf65 gene causes a mitochondrial translation defect, leading to encephalomyopathy. The C12orf65 protein is thought to play a role similar to that of ICT1 in rescuing stalled mitoribosomes during translation. Both proteins belong to a family of Class I peptide release factors (RFs), all characterized by the presence of a GGQ motif. Here, we determined the solution structure of the GGQ‐containing domain (GGQ domain) of C12orf65 from mouse by NMR spectroscopy, and examined the effect of siRNA‐mediated knockdown of C12orf65 on mitochondria in HeLa cells using flow cytometry. The GGQ domain, comprising residues 60–124 of the 184‐residue full‐length protein, forms a structure with a 310‐β1‐β2‐β3‐α1 topology that resembles the GGQ domain structure of RF more closely than that of ICT1. Thus, the GGQ domain structures of this protein family can be divided into two types, depending on the region linking β2 and β3; the C12orf65/RF type having a 6‐residue π‐HB turn and the ICT1 type having an α‐helix. Knockdown of C12orf65 resulted in increased ROS production and apoptosis, leading to inhibition of cell proliferation. Substantial changes in mitochondrial membrane potential and mass in the C12orf65‐knockdown cells were observed compared with the control cells. These results indicate that the function of C12orf65 is essential for cell vitality and mitochondrial function. Although similar effects were observed in ICT1‐downregulated cells, there were significant differences in the range and pattern of the effects between C12orf65‐ and ICT1‐knockdown cells, suggesting different roles of C12orf65 and ICT1 in rescuing stalled mitoribosomes. Proteins 2012.

Identification of residues required for stalled-ribosome rescue in the codon-independent release factor YaeJ

The YaeJ protein is a codon-independent release factor with peptidyl-tRNA hydrolysis (PTH) activity, and functions as a stalled-ribosome rescue factor in Escherichia coli. To identify residues required for YaeJ function, we performed mutational analysis for in vitro PTH activity towards rescue of ribosomes stalled on a non-stop mRNA, and for ribosome-binding efficiency. We focused on residues conserved among bacterial YaeJ proteins. Additionally, we determined the solution structure of the GGQ domain of YaeJ from E. coli using nuclear magnetic resonance spectroscopy. YaeJ and a human homolog, ICT1, had similar levels of PTH activity, despite various differences in sequence and structure. While no YaeJ-specific residues important for PTH activity occur in the structured GGQ domain, Arg118, Leu119, Lys122, Lys129 and Arg132 in the following C-terminal extension were required for PTH activity. All of these residues are completely conserved among bacteria. The equivalent residues were also found in the C-terminal extension of ICT1, allowing an appropriate sequence alignment between YaeJ and ICT1 proteins from various species. Single amino acid substitutions for each of these residues significantly decreased ribosome-binding efficiency. These biochemical findings provide clues to understanding how YaeJ enters the A-site of stalled ribosomes.

Identity elements of Thermus thermophilus tRNAThr

In this study, we identified nucleotides that specify aminoacylation of tRNAThr by Thermus thermophilus threonyl‐tRNA synthetase (ThrRS) using in vitro transcripts. Mutation studies showed that the first base pair in the acceptor stem as well as the second and third positions of the anticodon are major identity elements of T. thermophilus tRNAThr, which are essentially the same as those of Escherichia coli tRNAThr. The discriminator base, U73, also contributed to the specific aminoacylation, but not the second base pair in the acceptor stem. These findings are in contrast to E. coli tRNAThr, where the second base pair is required for threonylation, with the discriminator base, A73, playing no roles. In addition, among several mutations at the third base pair in the acceptor stem, only the G3‐U70 mutant was a poor substrate for ThrRS, suggesting that the G3‐U70 wobble pair, which is the identity determinant of tRNAAla, acts as a negative element for ThrRS. Similar results were obtained in E. coli and yeast. Thus, this manner of rejection of tRNAAla is also likely to have been retained in the threonine system throughout evolution.

Inhibitory effects of choline-O-sulfate on amyloid formation of human islet amyloid polypeptide.

Choline‐O‐sulfate (2‐(trimethylammonio)ethyl sulfate, COS) is a naturally occurring osmolyte that is synthesized by plants, lichens, algae, fungi, and several bacterial species. We examined the inhibitory effects of COS on amyloid formation of the human islet amyloid polypeptide (hIAPP or amylin) using a thioflavin T (ThT) fluorescence assay, circular dichroism spectroscopy and transmission electron microscopy. The results showed that COS suppresses a conformational change of hIAPP from a random coil to a β‐sheet structure, resulting in the inhibition of amyloid formation. Comparisons with various structural analogs including carnitine, acetylcholine and non‐detergent sulfobetaines (NDSBs) using the ThT fluorescence assay showed that COS is the most effective inhibitor of hIAPP amyloid formation, suggesting that the sulfate group, which is unique to COS, significantly contributes to the inhibition.