Biology subjects

Buratowski, S.

Publications and source records attributed to Buratowski, S..

13 recordsLinked to original sources

bioRxiv2025

Dynamics of TFIIH and Spt5 during the transition from transcription initiation to elongation

The transition of RNA polymerase II from initiation to elongation requires the timely exchange of many factors. Some initiation factors and elongation factors contact the same RNA polymerase II surfaces in an apparently mutually exclusive manner. For example, space occupied by the initiation factor TFIIH and elongation factor Spt4/5 (also known as DSIF) are predicted to overlap. To determine whether Spt4/5 actively displaces TFIIH, or binds only after TFIIH dissociates, labeled yeast nuclear extracts were combined with single-molecule fluorescence microscopy to observe their dynamics in real time. TFIIH categorically dissociates before Spt4/5 arrives, with a clear interval in between with neither factor present. Therefore, Spt4/5 binding is not required to dissociate TFIIH from RNA polymerase II.

molecular biology↗

bioRxiv2025

CDK12/13 inhibition disrupts nucleolar morphology and promotes aberrant expression of IGS transcripts

The transcription-associated cyclin dependent kinases (CDKs) 12 and 13 play important roles in the expression of RNAs involved in DNA damage repair and ribosomal proteins, yet their roles if any, in the regulation of ribosomal DNA expression are unclear. Here we show that these CDKs play critical roles in the processing and degradation of noncoding RNAs in the nucleolus and that inhibition of their function leads to the upregulation of unprocessed ribosomal RNA (rRNA) transcripts in the intergenic spacer (IGS) regions between ribosomal subunits. These upregulated RNAs are aberrantly polyadenylated by the TRAMP complex polymerase TENT4, and are represented as polyadenylated rings surrounding the nucleolus. The accumulation of these noncoding RNAs is associated with increased RNA polymerase II (Pol II)-mediated transcription coupled with failure of degradation by the MTREX (SKIV2L2) RNA helicase, a member of the RNA exosome complex whose expression is prematurely terminated by CDK12/13 inhibition. Altogether, these findings provide new insights into Pol II-mediated transcriptional regulation by CDK12/13 within the nucleolus.

cancer biology↗

bioRxiv2024

Mechanisms of synergistic Mediator recruitment in RNA polymerase II transcription activation revealed by single-molecule fluorescence

Transcription activators trigger transcript production by RNA Polymerase II (RNApII) via the Mediator coactivator complex. Here the dynamics of activator, Mediator, and RNApII binding at promoter DNA were analyzed using multi-wavelength single-molecule microscopy of fluorescently labeled proteins in budding yeast nuclear extract. Binding of Mediator and RNApII to the template required activator and an upstream activator sequence (UAS), but not a core promoter. While Mediator and RNApII sometimes bind as a pre-formed complex, more commonly Mediator binds first and subsequently recruits RNApII to form a preinitiation complex precursor (pre-PIC) tethered to activators on the UAS. Interestingly, Mediator occupancy has a highly non-linear response to activator concentration, and fluorescence intensity measurements show Mediator preferentially associates with templates having at least two activators bound. Statistical mechanical modeling suggests this "synergy" is not due to cooperative binding between activators, but instead occurs when multiple DNA-bound activator molecules simultaneously interact with a single Mediator.

biochemistry↗

bioRxiv2024

Leveraging HILIC/ERLIC Separations for Online Nanoscale LC- MS/MS Analysis of Phosphopeptide Isoforms from RNA Polymerase II C-terminal Domain

The eukaryotic RNA polymerase II (Pol II) multi-protein complex transcribes mRNA and coordinates several steps of co-transcriptional mRNA processing and chromatin modification. The largest Pol II subunit, Rpb1, has a C-terminal domain (CTD) comprising dozens of repeated heptad sequences (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), each containing five phospho-accepting amino acids. The CTD heptads are dynamically phosphorylated, creating specific patterns correlated with steps of transcription initiation, elongation, and termination. This CTD phosphorylation code choreographs dynamic recruitment of important co-regulatory proteins during gene transcription. Genetic tools were used to engineer protease cleavage sites across the CTD (msCTD), creating tryptic peptides with unique sequences amenable to mass spectrometry analysis. However, phosphorylation isoforms within each msCTD sequence are difficult to resolve by standard reversed phase chromatography typically used for LC-MS/MS applications. Here, we use a panel of synthetic CTD phosphopeptides to explore the potential of hydrophilic interaction and electrostatic repulsion hydrophilic interaction (HILIC and ERLIC) chromatography as alternatives to reversed phase separation for CTD phosphopeptide analysis. Our results demonstrate that ERLIC provides improved performance for separation of singly- and doubly-phosphorylated CTD peptides for sequence analysis by LC-MS/MS. Analysis of native yeast msCTD confirms that phosphorylation on Ser5 and Ser2 represents the major endogenous phosphoisoforms. We expect this methodology will be especially useful in the investigation of pathways where multiple protein phosphorylation events converge in close proximity.

biochemistry↗

bioRxiv2023

Uncoupling the TFIIH Core and Kinase Modules Leads To Unregulated RNA Polymerase II CTD Serine 5 Phosphorylation

TFIIH is an essential transcription initiation factor for RNA polymerase II (RNApII). This multi-subunit complex comprises two modules that are physically linked in S. cerevisiae by the subunit Tfb3 (MAT1 in metazoans). The Core Module, with two DNA-dependent ATPases and several additional subunits, promotes DNA unwinding. The Kinase Module phosphorylates the C-terminal domain (CTD) of RNApII subunit Rpb1, initiating a cycle of CTD modifications that coordinate exchange of initiation and elongation factors. Why these two disparate activities are bundled into one factor is not obvious, but the connection may provide temporal coordination during early initiation. When Tfb3 is split into two parts to uncouple the TFIIH modules, the resulting cells are viable but grow very slowly. Chromatin immunoprecipitation of the split TFIIH shows that the Core Module, but not the Kinase, is properly recruited to promoters. Instead of the normal promoter-proximal peak, high CTD Serine 5 phosphorylation is seen throughout transcribed regions. Therefore, coupling the TFIIH modules is necessary to localize and limit CTD kinase activity to early stages of transcription. These results are consistent with the idea that the two TFIIH modules began as independent functional entities that later became connected by Tfb3 during early eukaryotic evolution. Impact StatementThe TFIIH subunit Tfb3/MAT1 can be split into two parts to uncouple the TFIIH kinase and DNA translocase modules, resulting in unfocused CTD phosphorylation.

molecular biology↗

bioRxiv2023

Single-molecule analysis of transcription activation: Dynamics of SAGA co-activator recruitment

Transcription activators are said to stimulate gene expression by "recruiting" coactivators to promoters, yet this term fits several different kinetic models. To directly analyze dynamics of activator-coactivator interactions, single-molecule microscopy was used to image promoter DNA, a transcription activator, and the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex within nuclear extract. SAGA readily, but transiently, binds nucleosome-free DNA without activator, while chromatin template association occurs nearly exclusively when activator is present. On both templates, activator increases SAGA association rates by up to an order of magnitude, and dramatically extends its dwell times. These effects reflect direct interactions with the transactivation domain, as VP16 or Rap1 activation domains produce different SAGA dynamics. Despite multiple bromodomains, acetyl-CoA or histone H3/H4 tail acetylation only modestly improves SAGA binding. Unexpectedly, histone acetylation more strongly affects activator residence. Our studies thus reveal two modes of SAGA interaction with the genome: a short-lived activator-independent interaction with nucleosome-free DNA, and a state tethered to promoter-bound transcription activators that can last up to several minutes.

molecular biology↗

bioRxiv2021

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 showed activator-stimulated association with promoter DNA. Gds1 co-precipitated with the histone H4 acetyltransferase NuA4, and its levels often tracked with NuA4 in immobilized template experiments. GDS1 deletion led to reduction in H4 acetylation in vivo and other phenotypes consistent with 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 structural predictions suggest it may be distantly related to the DEK protein.

molecular biology↗

bioRxiv2021

Single molecule studies reveal branched pathways for activator-dependent pre-initiation complex assembly

RNA polymerase II (Pol II) transcription reconstituted from purified factors suggests pre-initiation complexes (PICs) can assemble by sequential incorporation of factors at the TATA box. However, these basal transcription reactions are generally independent of activators and co-activators. To study PIC assembly under more realistic conditions, we used single-molecule microscopy to visualize factor dynamics during activator-dependent reactions in nuclear extracts. Surprisingly, Pol II, TFIIF, and TFIIE can pre-assemble on enhancer-bound activators before loading into PICs, and multiple Pol II complexes can bind simultaneously to create a localized cluster. Unlike TFIIF and TFIIE, TFIIH binding is singular and dependent on the basal promoter. Activator-tethered factors exhibit dwell times on the order of seconds. In contrast, PICs can persist on the order of minutes in the absence of nucleotide triphosphates, although TFIIE remains unexpectedly dynamic even after TFIIH incorporation. Our kinetic measurements lead to a new branched model for activator-dependent PIC assembly. HIGHLIGHTSSingle molecule microscopy reveals unexpected dynamics of RNA Pol II and GTFs Multiple Pol IIs cluster on UAS/enhancer-bound activators before binding the core promoter Pol II, TFIIF, and TFIIE, but not TFIIH, can pre-assemble at the UAS/enhancer Activators increase the rates of Pol II and GTF association with DNA eTOC BlurbSingle-molecule microscopy experiments by Baek et al. show that RNA polymerase II and basal transcription factors TFIIF and TFIIE preassemble on UAS/enhancer-bound activators, poised for loading into initiation complexes with TFIIH at the core promoter. Transcription activators kinetically enhance factor recruitment, creating a localized cluster of polymerases at the UAS/enhancer.

molecular biology↗

bioRxiv2020

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 multi-wavelength 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, with no detectable involvement of RNApII-containing condensates. 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 pre-initiation 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. Significance StatementThe synthesis of a eukaryotic messenger RNA molecule involves the association of RNA polymerase and dozens of accessory proteins on DNA. We used differently colored fluorescent dyes to tag DNA, RNA polymerase II, and the elongation factor Spt4/5 in yeast nuclear extract, and then observed the assembly and dynamics of individual molecules of the proteins with single DNA molecules by microscopy. The observations quantitatively define an overall pathway by which transcription complexes form and evolve in response to an activator protein. They suggest how molecular complex dynamics may be tuned to optimize efficient RNA production.

biochemistry↗

bioRxiv2019

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 levels 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 post-initiation time, rather than distance elongated. Chemical inhibition of Kin28/Cdk7 blocks both Serine 5 and Serine 2 phosphorylation, affects initiation site choice, and inhibits elongation efficiency. EC components dependent on CTD phosphorylation include capping enzyme, Cap Binding Complex, Set2, and the PAF1 complex. By recapitulating many known features of in vivo elongation, this system reveals new details that clarify how EC-associated core elongation factors, chromatin regulators, and RNA processing factors change at each step of transcription.

biochemistry↗

bioRxiv2018

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 Cdkl2/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, which are known target sites, but also at Serine 2. 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 tSH2 domain, and here we show that Bur1/Cdk9 is the kinase responsible for this modification in vivo.

molecular biology↗

bioRxiv2018

Identification of three sequence motifs in the transcription termination factor Sen1 that mediate direct interactions with Nrd1

The Nrd1-Nab3-Sen1 (NNS) complex carries out the transcription termination of noncoding RNAs (ncRNAs) in yeast, although the detailed interactions among its subunits remain obscure. Here we have identified three sequence motifs in Sen1 that mediate direct interactions with the RNA polymerase II CTD interaction domain (CID) of Nrd1, determined the crystal structures of these Nrd1 interaction motifs (NIMs) bound to the CID, and characterized the interactions in vitro and in yeast.

biochemistry↗

bioRxiv2017

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 co-activators, and interact with nucleosomes. Here we show that transcription induces stable TAF binding to downstream promoter DNA, independent of upstream contacts, TBP, or other basal transcription factors. This transcription-dependent TAF complex promotes subsequent activator-independent transcription, and promoter response to TAF mutations in vivo correlates with the level of downstream, rather than overall, Taf1 crosslinking. We propose a new model in which TAFs function as reinitiation factors, accounting for the differential responses of promoters to various transcription factor mutations.

molecular biology↗