Nascent Transcript Folding Plays a Major Role in Determining RNA Polymerase Elongation Rates
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PROVIDER: PRJNA561141 | ENA |
REPOSITORIES: ENA
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Project description:Transcription elongation rates influence RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S. cerevisiae. Mapping RNAPI by Miller chromatin spreads or UV crosslinking revealed 5′ enrichment and strikingly uneven local polymerase occupancy along the rDNA, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution: folding energy and GC content in the transcription bubble. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high GC in the transcription bubble slows elongation. A mathematical model for RNAPI elongation confirmed the importance of nascent RNA folding in transcription. RNAPI from S. pombe was similarly sensitive to transcript folding, as were S. cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site use, indicating regulatory significance for transcript folding.
2020-06-24 | GSE136056 | GEO
Project description: Not available
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Project description:The folding of most proteins occurs during the course of their translation while their tRNA-bound C-termini are embedded in the ribosome. How the close proximity of nascent proteins to the ribosome influences their folding thermodynamics remains poorly understood. Here, we have developed a mass spectrometry-based approach for determining the stabilities of nascent polypeptide chains using methionine oxidation as a folding probe. This approach enables quantitative measurements sub-global folding stabilities of ribosome nascent chains (RNCs) within complex protein mixtures and extracts. To validate the methodology, we analyzed the folding thermodynamics of three model proteins (DHFR, CheY and DinB) in soluble and ribosome-bound states. The data indicated that the ribosome can significantly alter the stability of nascent polypeptides. Ribosome-induced stability modulations were highly variable among different folding domains and were dependent on localized charge distributions within nascent polypeptides. The results implicated electrostatic interactions between the ribosome surface and nascent polypeptides as the cause of ribosome-induced stability modulations. The study establishes a robust proteomic methodology for analyzing localized stabilities within ribosome-bound nascent polypeptides and sheds light on how the ribosome influences the thermodynamics of protein folding.
2023-08-09 | PXD037995 | Pride
Project description:The yeast Hsp70 chaperone Ssb interacts with ribosomes and nascent chains to co-translationally assist protein folding. Here, we present a proteome-wide analysis of Hsp70 function during translation, based on in vivo selective ribosome profiling, that reveals mechanistic principles coordinating translation with chaperone-assisted protein folding. Ssb binds most cytosolic, nuclear, and mitochondrial proteins and a subset of ER proteins, supporting its general chaperone function. Position-resolved analysis of Ssb engagement reveals compartment- and protein-specific nascent chain binding profiles that are coordinated by emergence of positively charged peptide stretches enriched in aromatic amino acids. Ssbs’ function is temporally coordinated by RAC but independent from NAC. Analysis of ribosome footprint densities along orfs reveals that ribosomes translate faster at times of Ssb binding. This is coordinated by biases in mRNA secondary structure, and codon usage as well as the action of Ssb, suggesting chaperones may allow higher protein synthesis rates by actively coordinating protein synthesis with co-translational folding.
2017-07-17 | GSE93830 | GEO
Project description:The rates at which domains fold and codons are translated are important factors in determining whether a nascent protein will co-translationally fold and function or misfold and malfunction. In this study, we develop a chemical kinetic model that calculates a protein domain’s co-translational folding curve using only the domain’s bulk folding and unfolding rates and codon translation rates. We show that this model accurately predicts the course of co-translational folding measured in vivo for four different protein molecules. As part of our chemical kinetic model, we assume that there is steady-state translation kinetics through the time-course of the experiment. To prove that this assumption is valid, we performed Ribo-Seq experiments on two biological replicates of yeast cells to compare their ribosome profiles. For genes with sufficient coverage across the open reading frames in both the replicates, we compare the ribosome profiles from both replicates and see a strong correlation. This implies there is a steady-state and our assumption is valid.
2016-01-30 | GSE75322 | GEO
Project description:The oxidation of protein-bound methionines to form methionine sulfoxides has a broad range of biological ramifications and it is therefore important to delineate factors that influence methionine oxidation rates within a protein. Previously, neighboring residue effects and solvent accessibility (SA) had been shown to impact the susceptibility of methionine residues to oxidation. In this study, we provide proteome-wide evidence that oxidation rates of buried methionine residues are also strongly influenced by the thermodynamic folding stability of the domains where they reside. We surveyed the E. coli proteome using several proteomic methodologies and globally measured oxidation rates of methionines in the presence and absence of tertiary structure as well as folding stabilities of methionione containing domains. The data indicate that buried methionines have a wide range of protection factors (PFs) against oxidation that correlate strongly with folding stabilities. Concordantly, we show that in comparison to E. coli, the proteome of the thermophile T. thermophilus is significantly more stable and thus more resistant to methionine oxidation. These results indicate that oxidation rates of buried methionines from the native state of proteins can be used as a metric of folding stability. To demonstrate the utility of this correlation, we used native methionine oxidation rates to survey the folding stabilities of E. coli and T. thermophilus proteomes at various temperatures and suggest a model that relates the temperature dependence of the folding stabilities of these two species to their optimal growth temperatures.
2022-04-22 | PXD030245 | Pride
Project description:Transcription elongation rate affects nascent histone pre-mRNA folding and 3’ end processing
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Project description:Defining the in vivo folding pathway of cellular RNAs is essential to understand how they reach their final native conformation. We here introduce a novel method, named Structural Probing of Elongating Transcripts (SPET-seq), that permits single-base resolution analysis of transcription intermediates’ secondary structures on a transcriptome-wide scale, enabling base-resolution analysis of the RNA folding events. Our results suggest that cotranscriptional RNA folding in vivo is a mixture of cooperative folding events, in which local RNA secondary structure elements are formed as they get transcribed, and non-cooperative events, in which 5΄-halves of long-range helices get sequestered into transient non-native interactions until their 3΄ counterparts have been transcribed. Together our work provides the first transcriptome-scale overview of RNA cotranscriptional folding in a living organism.
2017-07-19 | GSE95567 | GEO
Project description:The protein secretory pathway must maintain homoeostasis while producing a wide assortment of proteins in different conditions. It is also used extensively to produce many useful proteins in biotechnology. As such, secretory pathway dysfunction can be highly detrimental to the cell, resulting in the molecular basis for many human diseases, and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. To better understand some of these dysfunctions, we measured multiple systems-level states of the cell (physiology, transcriptome, metabolism) while secreting a small protein (insulin precursor) or a large protein (?-amylase). This was carried out in the presence and absence of HAC1, a key transcription factor in maintaining secretory homeostasis. Clear trends in cellular stress were apparent across multiple data resulting from our perturbations. In particular, processes involving (1) degradation of protein / recycling amino acids, (2) overall transcription/translation repression, and (3) oxidative stress. Apparent runaway oxidative radical production was explained by a thermodynamic model that we put forward for disulfide formation in the endoplasmic reticulum. This model predicts that balancing the relative rates of protein folding and disulfide bond formation are key to easing oxidative stress. These predictions have direct implications in how to engineer a broad range of recombinant proteins for secretion and provide potential hypotheses for the root causes of several secretory-associated diseases. Yeast strains were constructed that produce and secrete (a) IP or (b) ?-amylase and were compared to yeast strains containing (c) an empty vector in both wild-type and HAC1 deletion backgrounds. These strains are named WN (WT with empty vector), WI (WT secreting IP), WA (WT secreting ?-amylase), dN (?hac1 with empty vector), dI (?hac1 secreting IP), and dA (?hac1 secreting ?-amylase). Strains were characterized in batch fermentation and samples were taken in mid-exponential phase. Triplicate fermentations were carried out for each strain.
2011-12-01 | E-GEOD-27062 | biostudies-arrayexpress
Project description: Not available
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