RESUMO
A major goal of biology is to provide a quantitative description of cellular behaviour. This task, however, has been hampered by the difficulty in measuring protein abundances and their variation. Here we present a strategy that pairs high-throughput flow cytometry and a library of GFP-tagged yeast strains to monitor rapidly and precisely protein levels at single-cell resolution. Bulk protein abundance measurements of >2,500 proteins in rich and minimal media provide a detailed view of the cellular response to these conditions, and capture many changes not observed by DNA microarray analyses. Our single-cell data argue that noise in protein expression is dominated by the stochastic production/destruction of messenger RNAs. Beyond this global trend, there are dramatic protein-specific differences in noise that are strongly correlated with a protein's mode of transcription and its function. For example, proteins that respond to environmental changes are noisy whereas those involved in protein synthesis are quiet. Thus, these studies reveal a remarkable structure to biological noise and suggest that protein noise levels have been selected to reflect the costs and potential benefits of this variation.
Assuntos
Proteoma/metabolismo , Proteômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Meios de Cultura/farmacologia , Citometria de Fluxo , Proteoma/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Processos Estocásticos , Fatores de TempoRESUMO
Techniques for systematically monitoring protein translation have lagged far behind methods for measuring messenger RNA (mRNA) levels. Here, we present a ribosome-profiling strategy that is based on the deep sequencing of ribosome-protected mRNA fragments and enables genome-wide investigation of translation with subcodon resolution. We used this technique to monitor translation in budding yeast under both rich and starvation conditions. These studies defined the protein sequences being translated and found extensive translational control in both determining absolute protein abundance and responding to environmental stress. We also observed distinct phases during translation that involve a large decrease in ribosome density going from early to late peptide elongation as well as widespread regulated initiation at non-adenine-uracil-guanine (AUG) codons. Ribosome profiling is readily adaptable to other organisms, making high-precision investigation of protein translation experimentally accessible.
Assuntos
Códon , Genoma Fúngico , Biossíntese de Proteínas , RNA Fúngico/genética , RNA Mensageiro/genética , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/genética , Regiões 5' não Traduzidas , Biblioteca Gênica , Íntrons , Elongação Traducional da Cadeia Peptídica , Iniciação Traducional da Cadeia Peptídica , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Análise de Sequência de DNARESUMO
In eukaryotes, the combinatorial association of sequence-specific DNA binding proteins is essential for transcription. We have used protein arrays to test 492 pairings of a nearly complete set of coiled-coil strands from human basic-region leucine zipper (bZIP) transcription factors. We find considerable partnering selectivity despite the bZIPs' homologous sequences. The interaction data are of high quality, as assessed by their reproducibility, reciprocity, and agreement with previous observations. Biophysical studies in solution support the relative binding strengths observed with the arrays. New associations provide insights into the circadian clock and the unfolded protein response.