Gds1 Interacts with NuA4 To Promote H4 Acetylation at Ribosomal Protein Genes.

In our previously published studies, RNA polymerase II transcription initiation complexes were assembled from yeast nuclear extracts onto immobilized transcription templates and analyzed by quantitative mass spectrometry. In addition to the expected basal factors and coactivators, we discovered that the uncharacterized protein Gds1/YOR355W showed activator-stimulated association with promoter DNA. Gds1 coprecipitated with the histone H4 acetyltransferase NuA4, and its levels often tracked with NuA4 in immobilized-template experiments. GDS1 deletion led to a reduction in H4 acetylation in vivo and caused other phenotypes consistent with a partial loss of NuA4 activity. Genome-wide chromatin immunoprecipitation revealed that the reduction in H4 acetylation was strongest at ribosomal protein gene promoters and other genes with high NuA4 occupancy. Therefore, while Gds1 is not a stoichiometric subunit of NuA4, we propose that it interacts with and modulates NuA4 in specific promoter contexts. Gds1 has no obvious metazoan homolog, but the Alphafold2 algorithm predicts that a section of Gds1 resembles the winged-helix/forkhead domain found in DNA-binding proteins such as the FOX transcription factors and histone H1.

Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription.

In eukaryotes, RNA polymerase II (RNApII) transcribes messenger RNA from template DNA. Decades of experiments have identified the proteins needed for transcription activation, initiation complex assembly, and productive elongation. However, the dynamics of recruitment of these proteins to transcription complexes, and of the transitions between these steps, are poorly understood. We used multiwavelength single-molecule fluorescence microscopy to directly image and quantitate these dynamics in a budding yeast nuclear extract that reconstitutes activator-dependent transcription in vitro. A strong activator (Gal4-VP16) greatly stimulated reversible binding of individual RNApII molecules to template DNA. Binding of labeled elongation factor Spt4/5 to DNA typically followed RNApII binding, was NTP dependent, and was correlated with association of mRNA binding protein Hek2, demonstrating specificity of Spt4/5 binding to elongation complexes. Quantitative kinetic modeling shows that only a fraction of RNApII binding events are productive and implies a rate-limiting step, probably associated with recruitment of general transcription factors, needed to assemble a transcription-competent preinitiation complex at the promoter. Spt4/5 association with transcription complexes was slowly reversible, with DNA-bound RNApII molecules sometimes binding and releasing Spt4/5 multiple times. The average Spt4/5 residence time was of similar magnitude to the time required to transcribe an average length yeast gene. These dynamics suggest that a single Spt4/5 molecule remains associated during a typical transcription event, yet can dissociate from RNApII to allow disassembly of abnormally long-lived (i.e., stalled) elongation complexes.

Subunits of the vacuolar H+-ATPase complex, Vma4 and Vma10, are essential for virulence and represent potential drug targets in Candida albicans.

Hyphal morphogenesis of Candida albicans is important for its pathogenesis. Here, we showed that the filamentous growth of C. albicans requires vacuolar H+-ATPase function. Results showed that levels of Vma4 and Vma10 increased in cells undergoing hyphal growth compared to those undergoing yeast growth. Deleting VMA4 or VMA10 abolished vacuolar functions and hyphal morphogenesis. These deletion mutants were also characterized as avirulent in a mouse model of systemic infection. Furthermore, VMA4 and VMA10 deletion strains showed hypersensitivity to fluconazole, terbinafine, and amphotericin B. Based on these findings, Vma4 and Vma10 are not only involved in vacuole biogenesis and hyphal formation, but also are good targets for antifungal drug development in C. albicans.

Cross-talk between Tor1 and Sch9 regulates hyphae-specific genes or ribosomal protein genes in a mutually exclusive manner in Candida albicans.

The human fungal pathogen Candida albicans switches its morphology from yeast to hyphal forms. The morphological transition may render C. albicans virulent. Several signaling cascades, including those of the cyclic AMP-protein kinase A and mitogen-activated protein kinase pathways, are responsible for morphogenesis. In this study, we observed a reduction in gene transcription of ribosomal proteins during true hyphae formation. Moreover, morphogenesis-dependent decrease in ribosomal protein gene transcription was confirmed in constitutive yeast or filamentous growing strains. We consistently observed that polysome and monosome levels were decreased by hyphal stimuli through TORC1 and Sch9 kinases. Taken together, these results provide several lines of evidence to show that the Tor1-Sch9 kinase cascade, which stimulates transcription of ribosomal protein genes, exists in C. albicans. Thus, the present study revealed a novel link between ribosome biogenesis and morphogenesis in C. albicans that is mediated by Tor1 and Sch9.

Selective Kinase Inhibition Shows That Bur1 (Cdk9) Phosphorylates the Rpb1 Linker In Vivo.

Cyclin-dependent kinases play multiple roles in RNA polymerase II transcription. Cdk7/Kin28, Cdk9/Bur1, and Cdk12/Ctk1 phosphorylate the polymerase and other factors to drive the dynamic exchange of initiation and elongation complex components over the transcription cycle. We engineered strains of the yeast Saccharomyces cerevisiae for rapid, specific inactivation of individual kinases by addition of a covalent inhibitor. While effective, the sensitized kinases can display some idiosyncrasies, and inhibition can be surprisingly transient. As expected, inhibition of Cdk7/Kin28 blocked phosphorylation of the Rpb1 C-terminal domain heptad repeats at serines 5 and 7, the known target sites. However, serine 2 phosphorylation was also abrogated, supporting an obligatory sequential phosphorylation mechanism. Consistent with our previous results using gene deletions, Cdk12/Ctk1 is the predominant kinase responsible for serine 2 phosphorylation. Phosphorylation of the Rpb1 linker enhances binding of the Spt6 tandem SH2 domain, and here we show that Bur1/Cdk9 is the kinase responsible for these modifications in vivo.

In vitro analysis of RNA polymerase II elongation complex dynamics.

RNA polymerase II elongation complexes (ECs) were assembled from nuclear extract on immobilized DNA templates and analyzed by quantitative mass spectrometry. Time-course experiments showed that initiation factor TFIIF can remain bound to early ECs, while levels of core elongation factors Spt4-Spt5, Paf1C, Spt6-Spn1, and Elf1 remain steady. Importantly, the dynamic phosphorylation patterns of the Rpb1 C-terminal domain (CTD) and the factors that recognize them change as a function of postinitiation time rather than distance elongated. Chemical inhibition of Kin28/Cdk7 in vitro blocks both Ser5 and Ser2 phosphorylation, affects initiation site choice, and inhibits elongation efficiency. EC components dependent on CTD phosphorylation include capping enzyme, cap-binding complex, Set2, and the polymerase-associated factor (PAF1) complex. By recapitulating many known features of in vivo elongation, this system reveals new details that clarify how EC-associated factors change at each step of transcription.

In vitro assembly and proteomic analysis of RNA polymerase II complexes.

The RNA polymerase II (RNApII) transcription cycle consists of multiple steps involving dozens of protein factors. Here we describe a useful approach to study the dynamics of initiation and early elongation, comprising an in vitro transcription system in which complexes are assembled on immobilized DNA templates and analyzed by quantitative mass spectrometry. This unbiased screening system allows quantitation of RNApII complex components on either naked DNA or chromatin templates. In addition to transcription, the system reproduces co-transcriptional mRNA capping and multiple transcription-related histone modifications. In combination with other biochemical and genetic methods, this approach can provide insights into the mechanistic details of gene expression by RNApII.

Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation.

TFIID binds promoter DNA to recruit RNA polymerase II and other basal factors for transcription. Although the TATA-binding protein (TBP) subunit of TFIID is necessary and sufficient for in vitro transcription, the TBP-associated factor (TAF) subunits recognize downstream promoter elements, act as coactivators, and interact with nucleosomes. In yeast nuclear extracts, transcription induces stable TAF binding to downstream promoter DNA, promoting subsequent activator-independent transcription reinitiation. In vivo, promoter responses to TAF mutations correlate with the level of downstream, rather than overall, Taf1 cross-linking. We propose a new model in which TAFs function as reinitiation factors, accounting for the differential responses of promoters to various transcription factor mutations.

Proteomic analysis demonstrates activator- and chromatin-specific recruitment to promoters.

In-depth characterization of RNA polymerase II preinitiation complexes remains an important and challenging goal. We used quantitative mass spectrometry to explore context-dependent Saccharomyces cerevisiae preinitiation complex formation at the HIS4 promoter reconstituted on naked and chromatinized DNA templates. The transcription activators Gal4-VP16 and Gal4-Gcn4 recruited a limited set of chromatin-related coactivator complexes, namely the chromatin remodeler Swi/Snf and histone acetyltransferases SAGA and NuA4, suggesting that transcription stimulation is mediated through these factors. Moreover, the two activators differentially recruited the coactivator complexes, consistent with specific activator-coactivator interactions. Chromatinized templates suppressed recruitment of basal transcription factors, thereby amplifying the effect of activators, compared with naked DNA templates. This system is sensitive, highly reproducible, and easily applicable to mapping the repertoire of proteins found at any promoter.

Gcn4p-mediated transcriptional repression of ribosomal protein genes under amino-acid starvation.

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.

Structural basis for the recognition of N-end rule substrates by the UBR box of ubiquitin ligases.

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.

RpS3 translation is repressed by interaction with its own mRNA.

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.

Asc1p, a ribosomal protein, plays a pivotal role in cellular adhesion and virulence in Candida albicans.

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.

Determination of the core promoter regions of the Saccharomyces cerevisiae RPS3 gene.

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.

Cooperative regulation of ADE3 transcription by Gcn4p and Bas1p in Saccharomyces cerevisiae.

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.

Biochemical quantitation of PM2 phage DNA as a substrate for endonuclease assay.

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.