Project description:The interactions between RNA polymerase and ribosomes are critical for the coordination of transcription with translation. We report that RNA polymerase directly binds ribosomes and isolated large and small ribosomal subunits.
Project description:In this study, the native Sinapis alba plastid-encoded RNA polymerase (PEP) complex was purified and cross-linking MS was used to help in its structure determination.
Project description:Many regulatory proteins and complexes have been identified which influence transcription by RNA polymerase (pol) II with a fine precision. In comparison, only a few regulatory proteins are known for pol III, which transcribes mostly house-keeping and non-coding RNAs. Yet, pol III transcription is precisely regulated under various stress conditions like starvation. We used proteomic approaches and pol III transcription complex components TFIIIC (Tfc6), pol III (Rpc128) and TFIIIB (Brf1) as baits to find identify the potential interactors through mass spectrometry-based proteomics. A large number of proteins were found in the interactome, which includes known chromatin modifiers, factors and regulators of transcription by pol I and pol II.
Project description:RNA polymerase II (Pol II) was immunoprecipitated from Arabidopsis thaliana seedlings, using antibodies that specifically recognize: 1) C’ terminal Domain (CTD), 2) CTD Serine 5 Phosphorylation and 3) CTD Serine 2 Phosphorylation. Proteins that co-immunoprecipitate with different Pol II pools were analysed.
Project description:During eukaryotic transcription, RNA polymerase II undergoes dynamic post-translational modification on the C-terminal domain (CTD) of the largest subunit , generating a sophisticated PTM landscape for the spatiotemporal recruitment to transcriptional regulators. To delineate the protein interactomes recruited to Pol II at different stages of transcription, we in vitro reconstructed phosphorylation patterns of the CTD at Ser5 and Ser2 positions, the hallmark phosphorylation at the initation and productive elongation stages of transcription, respectively. Distinctive protein interactomes indicates different proteins are recruited to RNA polymerase II at different stages of transcription by the phosphorylation of Ser2 and Ser5 of the CTD heptads. Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP) specifically binds to the Ser2 of the heptad. The loss of the interaction between CHERP and Pol II results in broad alternative splicing events. Our method points to a new method to distinguish the PTM codes that coordinate the transcription process.
Project description:Genome-wide analysis of nascent RNA synthesis gives kinetic information on the transcriptional status of RNA polymerase II (Pol II). In parallel, immunoprecipitation of the largest subunit of Pol II (RPB1) provides the protein composition of the enzyme complex in the presence or absence of the transcriptional inhibitor Actinomycin D.
Project description:There are about 600 loci in the mammalian genome that are annotated as RNA polymerase III genes. These comprise tRNA genes, the genes encoding 5S RNA, the smallest ribosomal RNA, and genes encoding catalytic or structural RNAs involved in processes as diverse as RNA processing or transcription elongation. Most RNA polymerase III genes have similar promoter structures, yet they are transcribed with different efficiencies. Here we have explored how RNA polymerase III occupancy of these genomic loci varies in a normal tissue, the liver, during the transition from a resting state to a proliferating state. We find that after partial hepatectomy, which causes synchronous entry of remaining liver cells into the cell division cycle, there is a tremendous increase in RNA polymerase III occupancy. This increase is, however, not uniform and concerns mostly loci that were lowly occupied by RNA polymerase III in resting liver. The changes in RNA polymerase III occupancy cannot be correlated with changes in RNA polymerase II occupancy around the RNA polymerase III loci nor at nearby RNA polymerase II promoters. RNA polymerase III loci with the largest fold change tend to be located in clusters, with the cluster displaying the largest changes located on chromosome 13. This suggests that increases in RNA polymerase III occupancy during the transition from resting to proliferating state affect mostly genes whose basal rate of transcription is relatively low and which are located in clusters.
Project description:Loss of nutrient supply elicits alterations of the SUMO proteome and sumoylation is crucial to various cellular processes including transcription. However, the physiological significance of sumoylation of transcriptional regulators is unclear. To begin clarifying this, we mapped the SUMO proteome under nitrogen-limiting conditions in Saccharomyces cerevisiae. Interestingly, several RNA polymerase III (RNAPIII) components are major SUMO targets under normal growth conditions, including Rpc53, Rpc82, and Ret1, and nutrient starvation results in rapid desumoylation of these proteins. These findings are supported by ChIP-seq experiments that show that SUMO is highly enriched at tDNA genes. Furthermore, RNA-seq experiments revealed that preventing sumoylation results in significantly decreased tRNA transcription. TORC1 inhibition resulted in the same effect, and our data indicate that the SUMO and TORC1 pathways are both required for robust tDNA expression. Importantly, tRNA transcription was strongly reduced in cells expressing a non-sumoylatable Rpc82-4KR mutant, which correlated with a misassembled RNAPIII transcriptional complex. Our data suggest that in addition to TORC1 activity, sumoylation of RNAPIII is key to reaching full translational capacity under optimal growth conditions.
Project description:RNA polymerases (RNAPs) transcribe DNA into RNA and are found in all living organisms with several degrees of complexity from single polypeptide chain to multimeric enzymes. In chloroplast of angiosperms, two RNAPs are involved in plastid gene transcription: the nuclear-encoded RNA polymerase (NEP) and the plastid-encoded RNA polymerase (PEP). The PEP is a prokaryotic-type multimeric RNAP found in different states depending on light stimuli and cell identity. One of these active states requires the assembly of nuclear-encoded proteins named PEP-Associated Proteins (PAPs) with the catalytic core, triggering the timely transcription of photosynthesis-associated plastid genes in cells acquiring their photosynthetic apparatus. A purification procedure was used to enrich native PEP from Sinapis alba chloroplasts. Crosslinking coupled to mass spectrometry provided initial structural information about the relative position of PEP subunits within the complex.