The anchor-away (AA) technique depletes the nucleus of Saccharomyces cerevisiae of a protein of interest (the target) by conditional tethering to an abundant cytoplasmic protein (the anchor) by appropriate gene tagging and rapamycin-dependent heterodimerization. Taking advantage of the massive flow of ribosomal proteins through the nucleus during maturation, a protein of the large subunit was chosen as the anchor. Addition of rapamycin, due to formation of the ternary complex, composed of the anchor, rapamycin, and the target, then results in the rapid depletion of the target from the nucleus. All 43 tested genes displayed on rapamycin plates the expected defective growth phenotype. In addition, when examined functionally, specific mutant phenotypes were obtained within minutes. These are genes involved in protein import, RNA export, transcription, sister chromatid cohesion, and gene silencing. The AA technique is a powerful tool for nuclear biology to dissect the function of individual or gene pairs in synthetic, lethal situations.

The adenovirus genome forms chromatin-like structure with viral core proteins. This complex supports only a low level of transcription in a cell-free system, and thus core proteins have been thought to be negative factors for transcription. The mechanism how the transcription from the viral DNA complexed with core proteins is activated in infected cells remains unclear. Here, we found that both core proteins and histones are bound with the viral DNA in early phases of infection. We also found that acetylation of histone H3 occurs at the promoter regions of viral active genes in a transcription-independent manner. In addition, when a plasmid DNA complexed with core proteins was introduced into cells, core proteins enhanced transcription. Knockdown of TAF-I, a remodeling factor for viral core protein–DNA complexes, reduces the enhancement effect by core proteins, indicating that core proteins positively regulate viral transcription through the interaction with TAF-I. We would propose a possible mechanism that core proteins ensure transcription by regulating viral chromatin structure through the interaction with TAF-I.

No abstract

The adenovirus (Ad) genome complexed with viral core proteins designated Ad core is the template for transcription of early genes and the ¢rst round of replication in Adinfected cells. A cellular protein designated template-activating factor-I (TAF-I) is found to be involved in remodeling of the Ad core in vitro. Here we found that TAF-I interacts with the Ad DNA through core protein VII in infected cells in early phases of infection. In vitro binding assays using recombinant proteins showed that TAF-I forms ternary complexes with DNA^protein VII complexes.

. 79:2474-2483, 2005). We found that TAF-I interacts and colocalizes with protein VII in adenovirus-infected cells during the early phases of infection, but pp32 does not. Although pp32 had the potential ability to interact with protein VII, pp32 did not remodel the adenovirus DNA-protein VII complex in vitro. Small interfering RNA-mediated knockdown of TAF-I expression leads to the delay of the transcription timing of early genes. These results provide evidence that TAF-I plays an important role in the early stages of the adenovirus infection cycle.The adenovirus (Ad) genome is a linear double-stranded DNA of about 36,000 base pairs that is condensed with basic core proteins V and VII and polypeptide X in virions (1, 7). The copy number of protein VII in a virion particle is estimated to be 800 to 1,000, while that of protein V is approximately 160 (36). Although the precise structure of protein VII complexes with DNA is not clear, it is believed that protein VII binds strongly to DNA to maintain the condensed structure of the Ad DNA, while protein V associates with DNA-protein VII complexes less tightly (3,6,9).The Ad genome DNA is imported into the nucleus as nucleoprotein complexes containing protein VII through nuclear pore complexes (11,33). Protein VII remains associated with the Ad DNA during early phases of infection (5,31,39). Thus, the template of transcription of early genes forms a nucleoprotein complex composed of the Ad DNA and protein VII at least. Protein VII is likely to be a negative factor of genome functions. Experiments using cell-free transcription (37) and replication systems (16) with the Ad DNA-protein VII complex prepared from virions as template indicate that protein

Going Off-Target Sulfamethoxazole is a widely used sulfa-drug often used at high doses in the treatment of Pneumocytis pneumonia (PCP) in immunocompromised individuals. Haruki et al. (p. 987 ) show that sulfamethoxazole and certain other sulfa drugs inhibit the enzyme septiapterin reductase that catalyzes the final step in the biosynthesis of tetrahydrobiopterin (BH4). BH4 is a cofactor in the biosynthesis of neurotransmitters such as serotonin and dopamine. In cell culture, sulfamethoxazole lowered neurotransmitter levels through depletion of BH4, which may explain central nervous system side effects associated with PCP treatment.

fThe proteins belonging to the WhiB superfamily are small global transcriptional regulators typical of actinomycetes. In this paper, we characterize the role of WhiB5, a Mycobacterium tuberculosis protein belonging to this superfamily. A null mutant was constructed in M. tuberculosis H37Rv and was shown to be attenuated during both progressive and chronic mouse infections. Mice infected with the mutant had smaller bacillary burdens in the lungs but a larger inflammatory response, suggesting a role of WhiB5 in immunomodulation. Most interestingly, the whiB5 mutant was not able to resume growth after reactivation from chronic infection, suggesting that WhiB5 controls the expression of genes involved in this process. The mutant was also more sensitive than the wild-type parental strain to S-nitrosoglutathione (GSNO) and was less metabolically active following prolonged starvation, underscoring the importance of GSNO and starvation in development and maintenance of chronic infection. DNA microarray analysis identified 58 genes whose expression is influenced by WhiB5, including sigM, encoding an alternative sigma factor, and genes encoding the constituents of two type VII secretion systems, namely, ESX-2 and ESX-4.

Tryptophan metabolites in the kynurenine pathway are upregulated by pro-inflammatory cytokines or glucocorticoids, and are linked to anti-inflammatory and immunosuppressive activities. In addition, they are up-regulated in pathologies such as cancer, autoimmune diseases, and psychiatric disorders. The molecular mechanisms of how kynurenine pathway metabolites cause these effects are incompletely understood. On the other hand, pro-inflammatory cytokines also up-regulate the amounts of tetrahydrobiopterin (BH 4 ), an enzyme cofactor essential for the synthesis of several neurotransmitter and nitric oxide species. Here we show that xanthurenic acid is a potent inhibitor of sepiapterin reductase (SPR), the final enzyme in de novo BH 4 synthesis. The crystal structure of xanthurenic acid bound to the active site of SPR reveals why among all kynurenine pathway metabolites xanthurenic acid is the most potent SPR inhibitor. Our findings suggest that increased xanthurenic acid levels resulting from up-regulation of the kynurenine pathway could attenuate BH 4 biosynthesis and BH 4 -dependent enzymatic reactions, linking two major metabolic pathways known to be highly up-regulated in inflammation.