There is little information as to the location of early tRNA biosynthesis. Using fluorescent in situ hybridization in the budding yeast, Saccharomyces cerevisiae, examples of nuclear pre-tRNAs are shown to reside primarily in the nucleoli. We also probed the RNA subunit of RNase P. The majority of the signal from RNase P probes was nucleolar, with less intense signals in the nucleoplasm. These results demonstrate that a major portion of the tRNA processing pathway is compartmentalized in nucleoli with rRNA synthesis and ribosomal assembly. The spatial juxtaposition suggests the possibility of direct coordination between tRNA and ribosome biosynthesis. Received May 21, 1998; revised version accepted June 23, 1998. The physical organization of transcription and RNA processing events in eukaryotic nuclei has been studied extensively in the cases of pre-rRNAs and pre-mRNAs. Different stages in the expression of these RNAs are often associated with distinctive subnuclear structures, and these associations are integral to the synthesis and processing events. There is no direct information on the sites of tRNA gene transcription, although it is generally assumed that tRNA synthesis will be distributed throughout the nucleus; unlike the tandemly repeated ribosomal genes, the tRNA genes are not usually clustered in the chromosomes. In the budding yeast Saccharomyces cerevisiae, the 275 tRNA genes are scattered throughout the linear genome map (Cherry et al. 1997). Information on the sites of the numerous pre-tRNA processing events in the nucleus is also limited and has not provided a clear model for localization of the pathway.Maturation of eukaryotic pre-tRNA primary transcripts is often an ordered process, with 5Ј-and 3Ј-terminal processing preceding splicing (Tobian et al. 1985;Lee et al. 1991) and intron removal in turn preceding exit from nucleus to cytoplasm. In addition, multiple nucleotide modifications occur in the nucleus (Etcheverry et al. 1979;Hopper and Martin 1992). Although the order of processing events is not uniform for all pre-tRNAs and is not absolutely obligatory even for individual pre-tRNAs, the ordering suggests possible spatial organization of the maturation pathway. In yeast, current information on the sites of pre-tRNA processing could be consistent with either a dispersed pathway or one that is highly localized, especially to some portion of the nuclear periphery. Immunoelectron microscopy showed at least some of the tRNA splicing ligase to be near nuclear pores, leading to the speculation that the late splicing events in the nuclear pathway occur near the time that intron-containing pre-tRNAs exit to the cytoplasm (Clark and Abelson 1987). For intron-containing pretRNAs, it is therefore assumed that most of the nuclear precursor will retain the introns and that earlier processing events, such as removal of the termini, will need to take the introns into account (Leontis et al. 1988). Immunofluorescence microscopy has also localized a base modification enzyme, N 2 ,N 2 -dimethylguanosine-s...

Murine noroviruses (MNV) are closely related to the human noroviruses (HuNoV), which cause the majority of nonbacterial gastroenteritis. Unlike HuNoV, MNV grow in culture and in a small-animal model that represents a tractable model to study norovirus biology. To begin a detailed investigation of molecular events that occur during norovirus binding to cells, the crystallographic structure of the murine norovirus 1 (MNV-1) capsid protein protruding (P) domain has been determined. Crystallization of the bacterially expressed protein yielded two different crystal forms (Protein Data Bank identifiers [PDB ID], 3LQ6 and 3LQE). Comparison of the structures indicated a large degree of structural mobility in loops on the surface of the P2 subdomain. Specifically, the A-B and E-F loops were found in open and closed conformations. These regions of high mobility include the known escape mutation site for the neutralizing antibody A6.2 and an attenuation mutation site, which arose after serial passaging in culture and led to a loss in lethality in STAT1 ؊/؊ mice, respectively. Modeling of a Fab fragment and crystal structures of the P dimer into the cryoelectron microscopy three-dimensional (3D) image reconstruction of the A6.2/ MNV-1 complex indicated that the closed conformation is most likely bound to the Fab fragment and that the antibody contact is localized to the A-B and E-F loops. Therefore, we hypothesize that these loop regions and the flexibility of the P domains play important roles during MNV-1 binding to the cell surface.Murine noroviruses (MNV) are members of the family Caliciviridae, which contains small icosahedral viruses with positive-sense, single-stranded RNA genomes (18). MNV is related to human noroviruses (HuNoV), which cause most of the sporadic cases and outbreaks of infectious nonbacterial gastroenteritis worldwide in people of all ages (4,15,28,36,38,64). However, noroviruses are an understudied group of viruses due to the previous lack of a tissue culture system and small-animal model. Since its discovery in 2003 (23), MNV has become an increasingly important model to study norovirus biology (66). The availability of a small-animal model, cell culture, and reverse-genetics system, combined with many shared characteristics of human and murine noroviruses, allows detailed studies of norovirus biology (7,23,63,65,66).The norovirus genome is organized into 3 major open reading frames (ORFs), which encode the nonstructural polyprotein (ϳ200 kDa) and the major (VP1; ϳ58-kDa) and minor (VP2; ϳ20-kDa) capsid proteins (18). Recently, a putative ORF-4 was identified in MNV, but the existence of that product and its function remain unknown (60). Norovirus capsids are formed from 180 copies of VP1 arranged with Tϭ3 icosahedral symmetry (9,25,(46)(47)(48). Each capsid protein is divided into an N-terminal arm (N), a shell (S), and a C-terminal protruding (P) domain, with the last two domains connected by a short hinge. VP1 self-assembles into virus-like particles (VLPs) in baculovirus, mammalian, and plant e...

Background: Whether the mitochondrial or cytoplasmic protein synthesis inhibition predominates to aminoglycosideinduced ototoxicity is unclear. Results: The ototoxicity of an aminoglycoside correlates primarily with its ability to block mitochondrial rather than cytoplasmic protein synthesis. Conclusion: Designer aminoglycosides that selectively inhibit cytoplasmic rather than mitochondrial ribosome show decreased ototoxicity. Significance: The results are beneficial for development of aminoglycoside-based drugs to treat human genetic diseases.

In the budding yeast, Saccharomyces cerevisiae, actively transcribed tRNA genes can negatively regulate adjacent RNA polymerase II (pol II)-transcribed promoters. This tRNA gene-mediated silencing is independent of the orientation of the tRNA gene and does not require direct, steric interference with the binding of either upstream pol II factors or the pol II holoenzyme. A mutant was isolated in which this form of silencing is suppressed. The responsible point mutation affects expression of the Cbf5 protein, a small nucleolar ribonucleoprotein protein required for correct processing of rRNA. Because some early steps in the S. cerevisiae pre-tRNA biosynthetic pathway are nucleolar, we examined whether the CBF5 mutation might affect this localization. Nucleoli were slightly fragmented, and the pre-tRNAs went from their normal, mostly nucleolar location to being dispersed in the nucleoplasm. A possible mechanism for tRNA gene-mediated silencing is suggested in which subnuclear localization of tRNA genes antagonizes transcription of nearby genes by pol II.nucleolus ͉ RNA polymerase III I t has previously been shown that tRNA-class RNA polymerase III (pol III) promoters can exert negative transcriptional regulation on neighboring DNA in the budding yeast, Saccharomyces cerevisiae (1). The degree to which this tRNA genemediated silencing (tgm silencing) affects nearby RNA polymerase II-transcribed genes varies among different pol II promoters. Although complete inhibition of some nearby pol II promoters has been achieved in selected artificial juxtapositions, promoters that are found bordering the 274 tRNA genes in their normal chromosomal locations must somehow be exempt, or at least conditionally resistant to this form of negative regulation.There is a particularly close association of tRNA genes with naturally occurring copies of most classes of the Ty retrotransposons (2). One possible selective pressure for maintaining an association between tRNA genes and neighboring pol II promoters is that the proximity serves some regulatory function that is beneficial for maintenance of the retrotransposon at that position. Although the presence of a Ty3 sigma element does not strongly affect expression of a neighboring tRNA gene (3), temperature-dependent silencing of the chromosomal Ty3 and sigma elements was shown to be dependent on RNA polymerase III, suggesting possible involvement of the neighboring tRNA transcription units (1). It is interesting to note that the one class of Ty retrotransposon that is not found adjacent to tRNA genes, the Ty5 class, is found instead at other silenced locations, namely telomeres and the silent mating type loci (4, 5).At this time, it is not clear what relationship the mechanism of tgm silencing might have to silencing at silent mating type loci, telomeres, ribosomal RNA genes, or other forms of negative regulation, but several types of interference between the transcription units appear to be ruled out. It seems unlikely that either readthrough by pol III or positive supercoils propaga...