We have determined that lOSa RNA (one of the small stable RNAs found in Escherichia cobl) has an interesting structural feature: the 5' end and the 3' end of lOSa RNA can be arranged in a s ture that is equivalent to a half-molecule (acceptor stem and TFC stem-loop) of alanine tRNA of E. coli. Primer-extension analysis of lOSa RNA extracted from a bacterial mutant with temperature-sensitive RNase P function revealed that the precursor to lOSa RNA (pre-lOSa RNA) is folded into a pre-tRNA-like structure in vivo such that it can be cleaved by RNase P to generate the 5' end of the mature 10Sa RNA. The purified 10Sa RNA can be charged with alanine in vitro. Disruption of the gene encoding lOSa RNA (ssrA) caused a reduction in the rate of cell growth, which was especaly apparent at 45C, and a reduction in motility on se d agar. These phenotypic characteristics of the deletion strain (AssrA) allowed us to investigate the effects of some mutations in lOSa RNA in vivo, although the exact function of lOSa RNA still remains unclear. When the G-U pair (G3-U357) in lOSa RNA, which may be equivalent to the determinant G-U pair of alanine tRNA, was changed to a GA or G-C pair, the ability to complement the phenotypic mutations of the AssrA strain was lost. Furthermore, this inability to complement the mutant phenotypes that was caused by the substitution of the determinant bases by a GA pair could be overcome by the introduction of a gene encoding alanyl-tRNA synthetase (aaS) on a multicopy plasmid. The evidence suggests that the proposed structural features of lOSa RNA are indeed manifested in vivo.lOSa RNA is one of the small stable RNAs that is found in Escherichia coli. It was first identified as lOS RNA, which was actually a mixture of lOSa RNA and Ml RNA (originally designated lOSb RNA) (1, 2). Subsequently, Ml RNA was shown to be a "catalytic RNA" (3), and it has been extensively studied from both a structural and a functional perspective (4). However, in contrast to Ml RNA, lOSa RNA has not been the focus of much recent attention, and very little is known about either its structure or its function.In our previous study, we found that the ssrA gene that encodes lOSa RNA of E. coli is included in the genomes of phages 438, 439, and 440 of Kohara's library (5), which corresponds to 56.5 minutes on the linkage map of the chromosome (6). We cloned a 2.2-kb fragment that covers the overlapping region of these phages into a plasmid vector and determined the nucleotide sequence of the fragment. § We also purified lOSa RNA from E. coli strain W3110 and determined its nucleotide sequence from both the 5' and the 3' end.The nucleotide sequence of the ssrA gene from E. coli has already been reported by Chauhan and Apirion (7). In comparing our sequences with the published sequence, we noticed an interesting structural feature of lOSa RNA, as presented in Fig. 2-namely, 7 nucleotides at the 5' end and 28 nucleotides at the 3' end can be arranged in a structure that is equivalent to a half-molecule of tRNA. This half-mole...

A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.

Quality control mechanisms operate in various steps of ribosomal biogenesis to ensure the production of functional ribosome particles. It was reported previously that mature ribosome particles containing nonfunctional mutant rRNAs are also recognized and selectively removed by a cellular quality control system (nonfunctional rRNA decay [NRD]). Here, we show that the NRD of 25S rRNA requires a ubiquitin E3 ligase component Rtt101p and its associated protein Mms1p, identified previously as factors involved in DNA repair. We revealed that a group of proteins associated with nonfunctional ribosome particles are ubiquitinated in a Rtt101-Mms1-dependent manner. 25S NRD was disrupted when ubiquitination was inhibited by the overexpression of modified ubiquitin molecules, demonstrating a direct role for ubiquitin in this pathway. These results uncovered an unexpected connection between DNA repair and the quality control of rRNAs. Our findings support a model in which responses to DNA and rRNA damages are triggered by a common ubiquitin ligase complex during genotoxic stress harmful to both molecules.[Keywords: Ubiquitin; ribosome; genotoxic stress; quality control; rRNA] Supplemental material is available at http://www.genesdev.org. Gene mutations often result in the production of nonfunctional RNA molecules. In addition, RNA itself is continuously damaged by endogenous and exogenous stress, including ionizing radiation, exposure to certain chemical compounds, and the intracellular generation of reactive oxygen species (Bregeon and Sarasin 2005). Rare but measurable errors in transcription also produce mutant RNAs that do not properly fulfill their roles and aims. In order to avoid a breakdown of cellular order, it is important for cells to detect and selectively dismantle such irregular RNA molecules continuously. It is well documented that various types of aberrant RNAs are selectively removed in eukaryotic cells (Doma and Parker 2007). Three pathways requiring distinct factors degrade different classes of aberrant mRNAs, including mRNAs with a nonsense mutation in their ORFs (nonsensemediated mRNA decay) (Isken and Maquat 2007), mRNAs with no termination codon (nonstop mRNA decay) van Hoof et al. 2002), and mRNAs with a highly stable structure that prevents ribosomal progression (no-go mRNA decay) (Doma and Parker 2006). Recently, it has been reported that tRNAs with hypomodifications are also selectively degraded in vivo, indicating that stable RNAs are monitored by cellular quality control systems (Kadaba et al. 2004;Chernyakov et al. 2008). However, it is not clear how the quality control of ribosomal RNAs (rRNAs), another species of stable RNAs, is achieved, although rRNAs are highly abundant and essential for life.The eukaryotic ribosome is a massive ribonucleoprotein (RNP) complex that consists of four rRNAs and about 80 ribosomal proteins (Venema and Tollervey 1999). The precursor 35S rRNA transcribed by RNA polymerase (Pol) I is processed into three parts; 18S, 5.8S, and 25S rRNA. 5S rRNA is synthesized indep...

Articles you may be interested inTunneling magnetoresistance effect in a few-electron quantum-dot spin valve Appl. Phys. Lett. 93, 222107 (2008); 10.1063/1.3042098 High Kondo temperature ( T K 80 K ) in self-assembled InAs quantum dots laterally coupled to nanogap electrodes Appl. Phys. Lett. 93, 062101 (2008); 10.1063/1.2968206 Electric-field control of tunneling magnetoresistance effect in a Ni ∕ In As ∕ Ni quantum-dot spin valve Appl. Phys. Lett. 91, 022107 (2007); 10.1063/1.2759264 Kondo effect in quantum dots coupled to ferromagnetic leads: effect of noncollinear magnetization AIP Conf.

We demonstrate an electric-field control of tunneling magnetoresistance (TMR) effect in a semiconductor quantum-dot spin-valve device. By using ferromagnetic Ni nano-gap electrodes, we observe the Coulomb blockade oscillations at a small bias voltage. In the vicinity of the Coulomb blockade peak, the TMR effect is significantly modulated and even its sign is switched by changing the gate voltage, where the sign of the TMR value changes at the resonant condition.PACS numbers:

We experimentally study the tunneling magnetoresistance ͑TMR͒ effect as a function of bias voltage ͑V SD ͒ in lateral Ni/ InAs/ Ni quantum-dot ͑QD͒ spin valves showing Coulomb blockade characteristics. With varying V SD , the TMR value oscillates and the oscillation period corresponds to conductance changes observed in the current-voltage ͑I-V SD ͒ characteristics. We also find an inverse TMR effect near V SD values where negative differential conductance is observed. A possible mechanism of the TMR oscillation is discussed in terms of spin accumulation on the QD and spin-dependent transport properties via excited states.

Eukaryotic cells have quality control systems that eliminate nonfunctional rRNAs with deleterious mutations (nonfunctional rRNA decay, NRD). We have previously reported that 25S NRD requires an E3 ubiquitin ligase complex, which is involved in ribosomal ubiquitination. However, the degradation process of nonfunctional ribosomes has remained unknown. Here, using genetic screening, we identified two ubiquitin-binding complexes, the Cdc48-Npl4-Ufd1 complex (Cdc48 complex) and the proteasome, as the factors involved in 25S NRD. We show that the nonfunctional 60S subunit is dissociated from the 40S subunit in a Cdc48 complex-dependent manner, before it is attacked by the proteasome. When we examined the nonfunctional 60S subunits that accumulated under proteasome-depleted conditions, the majority of mutant 25S rRNAs retained their full length at a single-nucleotide resolution. This indicates that the proteasome is an essential factor triggering rRNA degradation. We further showed that ribosomal ubiquitination can be stimulated solely by the suppression of the proteasome, suggesting that ubiquitin-proteasome-dependent RNA degradation occurs in broader situations, including in general rRNA turnover.

Aminoacyl-tRNA synthetases, a family of enzymes essential for protein synthesis, are promising targets of antimicrobials. Indolmycin, a secondary metabolite of Streptomyces griseus and a selective inhibitor of prokaryotic tryptophanyl-tRNA synthetase (TrpRS), was used to explore the mechanism of inhibition and to explain the resistance of a naturally occurring strain. Streptomyces coelicolor A3(2), an indolmycin-resistant strain, contains two trpS genes encoding distinct TrpRS enzymes. We show that TrpRS1 is indolmycin-resistant in vitro and in vivo, whereas TrpRS2 is sensitive. The lysine (position 9) in the enzyme tryptophan binding site is essential for making TrpRS1 indolmycin-resistant. Replacement of lysine 9 by glutamine, which at this position is conserved in most bacterial TrpRS proteins, abolished the ability of the mutant trpS gene to confer indolmycin resistance in vivo. Molecular modeling suggests that lysine 9 sterically hinders indolmycin binding to the enzyme. Tryptophan recognition (assessed by k cat / K M ) by TrpRS1 is 4-fold lower than that of TrpRS2. Examination of the mRNA for the two enzymes revealed that only TrpRS2 mRNA is constitutively expressed, whereas mRNA for the indolmycin-resistant TrpRS1 enzyme is induced when the cells are exposed to indolmycin.