Project description:A quantitative view of cellular functions requires precise measures of the rates of biomolecule production, especially proteins-the direct effectors of biological processes. Here we present a genome-wide approach, based on ribosome profiling, for measuring absolute protein synthesis rates. The resultant E. coli dataset transforms our understanding of the extent to which protein synthesis is precisely controlled to optimize function and efficiency. For example, members of multi-protein complexes are made in precise proportion to their stoichiometry, whereas components of functional modules are produced differentially according to their hierarchical role. Estimates of absolute protein abundance also reveal principles used to optimize design. These include how the level of different types of transcription factors is optimized for rapid response, and how a metabolic pathway (methionine biosynthesis) balances production cost with activity requirements. More broadly, our studies reveal how general principles, important both for understanding natural systems and for synthesizing new ones, emerge from global quantitative analyses of protein synthesis. 4 samples of E. coli ribosome profiling and mRNA-seq, including biological replicates
Project description:A quantitative view of cellular functions requires precise measures of the rates of biomolecule production, especially proteins-the direct effectors of biological processes. Here we present a genome-wide approach, based on ribosome profiling, for measuring absolute protein synthesis rates. The resultant E. coli dataset transforms our understanding of the extent to which protein synthesis is precisely controlled to optimize function and efficiency. For example, members of multi-protein complexes are made in precise proportion to their stoichiometry, whereas components of functional modules are produced differentially according to their hierarchical role. Estimates of absolute protein abundance also reveal principles used to optimize design. These include how the level of different types of transcription factors is optimized for rapid response, and how a metabolic pathway (methionine biosynthesis) balances production cost with activity requirements. More broadly, our studies reveal how general principles, important both for understanding natural systems and for synthesizing new ones, emerge from global quantitative analyses of protein synthesis.
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.
Project description:Proteomics and transcriptomics data of tomato fruits (Solanum lycopersicum L. var. Moneymaker) at 9 developmental stages were used to calculate with a mathematical model the rate constants of synthesis and degradation for over 1,000 proteins. Proteome and transcriptome were extracted from the pericarp tissue and analyzed using label-free LC-MS/MS (Orbitrap Q-Exactive) and RNA Sequencing (Illumina), respectively. Absolute quantification of transcriptome has been obtained by spiking-in internal standard before total-RNA extraction. Absolute quantification of the proteome has been approximated using the "Total Protein" approach. An OD equation defining the changes of protein content has been used to determine the synthesis and degradation rate constants (day -1). Almost 2,400 transcript-protein pairs were identified and the translation and degradation rate constants were determined for more than a thousand proteins. The model predicted median values of about 2 min for the translation and a lifetime of approximately 11 days. Proteins involved in protein synthesis had higher ks and kd values, indicating that the protein machinery is particularly flexible. None sequenced-based features were found that could be used to predict these rate constants.
Project description:Deciphering cellular proteomes is critical for our understanding of erythropoiesis. To better comprehend commonly used models of erythropoiesis, we obtained absolute quantification of proteins expressed in cultured primary murine bone-marrow derived erythroblasts throughout erythroid differentiation. These data were compared to proteomes from Friend murine erythroleukemia (MEL), G1ER, and MEDEP cells. Overall, more than 7200 proteins were quantified in at least one of these models of murine erythropoiesis. Protein expression during differentiation of MEDEP cells was most similar to that of differentiating primary murine erythroid cells, while proteomes of Friend MEL and G1ER cells were more distantly related. Comparison of the proteomes of murine and human erythroblasts revealed species-linked variability, but these differences were not as dramatic as those observed comparing human and murine transcriptomes during erythroid differentiation. To functionally validate the differences between transcriptional and translational control, heme synthesis was inhibited in MEDEP cells and the effects on the transcriptome and the proteome examined. While heme deficiency had minimal global effect on the cellular proteome, globin proteins were significantly down regulated, supporting observations heme regulation of globin synthesis is tightly regulated at the protein level to avoid deleterious over production of globin chains. As predicted, heme deficiency led to up regulation of -aminolevulinic acid synthase 2 (Alas2) protein. Interestingly, Alas1 was upregulated at both the transcriptional and protein levels. Characterization of cellular proteomes during erythropoiesis has potential to yield insight into mechanistic principles of human disease, as well as reveal novel therapeutic targets.
Project description:With emerging methods recently developed to capture protein-anchored 3D epigenome folding we herein report an experimental advance yielding a fundamental and systematic improvement in understanding the 3D genome: integrated normalization of orthologous chromatin for measurement of absolute changes in the landscape. With Absolute Quantification of chromatin Architecture (AQuA-HiChIP) global and local changes in the 3D epigenome can be measured, and the absolute differences in protein-anchored folding can be determined. These changes can be defined in a way that couples the relative occupancy of chromatin regulatory factors or histone marks to absolute quantification of 3D chromatin structure. While our method has intrinsic limitations, including restriction by the scope of available ChIP-grade antibodies with mouse/human cross-reactivity, the approach to measuring absolute components of chromatin folding will enable new insights into the topological determinants of transcriptional control and tissue-specific epigenetic memory.
Project description:The easyCLIP protocol describes a method for both normal CLIP library construction and the absolute quantification of RNA cross-linking rates, data which could be usefully combined to analyze RNA-protein interactions. Using these cross-linking metrics, significant interactions could be defined relative to a set of random non-RBPs. The original easyCLIP protocol did not use index reads, required custom sequencing primers, and did not have an easily reproducible analysis workflow. This short paper attempts to amend these deficiencies. It also includes some additional technical experiments and investigates the usage of alternative adapters. The results here are intended to allow more options to easily perform and analyze easyCLIP.