[show abstract][hide abstract] ABSTRACT: Gcn4p is a well-characterized bZIP transcription factor that activates more than 500 genes encoding amino acids and purine biosynthesis enzymes, and many stress-response genes under various stress conditions. Under these stresses, it had been shown that transcriptions of ribosomal protein (RP) genes were decreased. However, the detailed mechanism of this downregulation has not been elucidated. In this study, we present a novel mechanistic model for a repressive role of Gcn4p on RP transcription, especially under amino-acid starvation. It was found that Gcn4p bound directly to Rap1p, which in turn inhibited Esa1p-Rap1p binding. The inhibition of Esa1p recruitment to RP promoters ultimately reduced the level of histone H4 acetylation and RP transcription. These data revealed that Gcn4p has simultaneous dual roles as a repressor for RP genes as well as an activator for amino-acid biosynthesis genes. Moreover, our results showed evidence of a novel link between general control of amino-acid biosynthesis and ribosome biogenesis mediated by Gcn4p at an early stage of adaptation to amino-acid starvation.
The EMBO Journal 12/2010; 30(5):859-72. · 9.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: Candida albicans, the common human fungal pathogen, can switch morphology from yeast to pseudohyphal or hyphal form upon various environmental cues. It is well-known that the ability of morphological conversion and adhesive growth renders C. albicans virulent. It is noteworthy that every factor involved in the morphogenesis is known to be important for the virulence of this pathogen. To examine a functional relevance of Asc1p, a ribosomal protein, in morphogenesis and virulence, an asc1 homozygous null mutant was generated. Although a normal morphological transition of the asc1 deletion strain in liquid media was found, it did not change its morphology on solid media. Moreover, the adhesion activity and hyphal-specific gene expression were defective due to ASC1 deletion. Finally, it was found that the asc1 null mutant was avirulent in a mouse model. These results strongly suggested that Asc1p a component of the 40S ribosomal subunit and a signal transducer, plays a pivotal role in cellular adhesion and virulence through regulation of specific gene expression in C. albicans.
The Journal of Microbiology 12/2010; 48(6):842-8. · 1.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: The N-end rule pathway is a regulated proteolytic system that targets proteins containing destabilizing N-terminal residues (N-degrons) for ubiquitination and proteasomal degradation in eukaryotes. The N-degrons of type 1 substrates contain an N-terminal basic residue that is recognized by the UBR box domain of the E3 ubiquitin ligase UBR1. We describe structures of the UBR box of Saccharomyces cerevisiae UBR1 alone and in complex with N-degron peptides, including that of the cohesin subunit Scc1, which is cleaved and targeted for degradation at the metaphase-anaphase transition. The structures reveal a previously unknown protein fold that is stabilized by a novel binuclear zinc center. N-terminal arginine, lysine or histidine side chains of the N-degron are coordinated in a multispecific binding pocket. Unexpectedly, the structures together with our in vitro biochemical and in vivo pulse-chase analyses reveal a previously unknown modulation of binding specificity by the residue at position 2 of the N-degron.
[show abstract][hide abstract] ABSTRACT: Ribosomal protein S3 (RpS3) is a well-known multi-functional protein mainly involved in protein biosynthesis as a member of the small ribosomal subunit. It also plays a role in repairing various DNA damage acting as a repair UV endonuclease. Most of the rpS3 pool is located in the ribosome while the minority exists in free form in the cytoplasm. We here report an additional function of rpS3 in which it represses its own translation by binding to its cognate mRNA. Through RT-PCR of the RNAs co-immunoprecipitated with ectopically expressed rpS3, rpS3 protein was found to interact with various RNAs-endogenous rpS3, 18S rRNA. The S3-C terminal domain was shown to be the major mRNA binding domain of rpS3, independent of the KH domain. This interaction was shown to occur in cytoplasmic fractions rather than ribosomal fractions, and then is involved in its own mRNA translational inhibition by in vitro translation. Furthermore, when Flag-tagged rpS3 was transiently transfected into 293T cells, the level of endogenous rpS3 gradually decreased regardless of transcription. These results suggest that free rpS3 regulates its own translation via a feedback mechanism.
Journal of Cellular Biochemistry 03/2010; 110(2):294-303. · 3.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ribosomal protein genes (RPG), which are scattered throughout the genomes of all eukaryotes, are subjected to coordinated expression. In yeast, the expression of RPGs is highly regulated, mainly at the transcriptional level. Recent research has found that many ribosomal proteins (RPs) function in multiple processes in addition to protein synthesis. Therefore, detailed knowledge of promoter architecture as well as gene regulation is important in understanding the multiple cellular processes mediated by RPGs. In this study, we investigated the functional architecture of the yeast RPS3 promoter and identified many putative cis-elements. Using beta-galactosidase reporter analysis and EMSA, the core promoter of RPS3 containing UASrpg and T-rich regions was corroborated. Moreover, the promoter occupancy of RPS3 by three transcription factors was confirmed. Taken together, our results further the current understanding of the promoter architecture and trans-elements of the Saccharomyces cerevisiae RPS3 gene.
Biochimica et Biophysica Acta 10/2009; 1789(11-12):741-50. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: The one-carbon response regulon is essential for the biosynthesis of nucleic acids as well as several amino acids. The ADE3 gene is known to encode a crucial one-carbon regulon enzyme, tetrahydrofolate synthase, which is involved in the biosynthesis of purine and the amino acids methionine and glycine. Therefore, the mechanism through which ADE3 transcription is regulated appears to be critical for the cross-talk among these metabolic pathways. Even so, the direct involvement of ADE3 transcription through gene-specific transcription factors has not been shown clearly. In this study, the promoter structure of the ADE3 gene was investigated in detail, and a genuine Gcn4p responsive element (GCRE) was confirmed among three putative GCRE elements in vivo and in vitro. Through gene deletion studies of Gcn4p and Bas1p, it was established that both factors are involved in the transcriptional regulation of the ADE3 gene. Direct binding to this GCRE and the occupancy of the ADE3 promoter by these factors were also confirmed. Taking these results together, we concluded that Gcn4p is responsible for the basal and inducible expression of the ADE3 gene, while Bas1p is required for its basal expression.
[show abstract][hide abstract] ABSTRACT: Bacteriophage PM2 has a closed circular form of double stranded DNA as a genome. This DNA from the phage is a useful source for nick-circle endonuclease assay in the fmol range. Due to difficulties in the maintenance of viral infectivity, storage conditions of the phage should be considered for the purification of PM2 DNA. The proper condition for a short-term storage of less than 2 months is to keep the PM2 phage at 4 degrees C; whereas the proper condition for a long-term storage of the PM2 phage for over 2 months is to keep it under liquid nitrogen in 7.5% glycerol. The optimal conditions for a high yield of phage progeny were also considered with the goal to achieve a successful PM2 DNA preparation. A MOI(Multiplicity Of Infection) of 0.03, in which the OD600 of the host bacteria was between 0.3 and 0.5, turned out to be optimal for the mass production of PM2 phage with a burst size of about 214. Considerations of PM2 genome size, and the concentrations and radiospecific activities of purified PM2 DNA, are required to measure the endonuclease activity in the fmol range. This study reports the proper quantitation of radioactivity and the yield of purified DNA based on these conditions.
The Journal of Microbiology 07/2004; 42(2):99-102. · 1.28 Impact Factor