Quantitative analysis of the sequence determinants of transcription and translation regulation is relevant for systems and synthetic biology. To identify these determinants, researchers have developed different methods of screening random libraries using fluorescent reporters or antibiotic resistance genes. Here, we have implemented a generic approach called ELM-seq (expression level monitoring by DNA methylation) that overcomes the technical limitations of such classic reporters. ELM-seq uses DamID (Escherichia coli DNA adenine methylase as a reporter coupled with methylation-sensitive restriction enzyme digestion and high-throughput sequencing) to enable in vivo quantitative analyses of upstream regulatory sequences. Using the genome-reduced bacterium Mycoplasma pneumoniae, we show that ELM-seq has a large dynamic range and causes minimal toxicity. We use ELM-seq to determine key sequences (known and putatively novel) of promoter and untranslated regions that influence transcription and translation efficiency. Applying ELM-seq to other organisms will help us to further understand gene expression and guide synthetic biology.Quantitative analysis of how DNA sequence determines transcription and translation regulation is of interest to systems and synthetic biologists. Here the authors present ELM-seq, which uses Dam activity as reporter for high-throughput analysis of promoter and 5'-UTR regions.

We thank the CRG/UPF Proteomics Unit that is part of the Plataforma de Recursos Biomoleculares y Bioinformáticos (Instituto de Salud Carlos III), supported by grant PT13/0001. The project was supported by funds from the Fundación Marcelino Botin and the Spanish Ministerio de Economía y Competitividad (BIO2007-61762). This project was financed by Instituto de Salud Carlos III and co-financed by Federación Española de Enfermedades Raras under grant agreement PI10/01702 and the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program, under grant agreement Nos 634942 (MycoSynVac) and 670216 (MYCOCHASSIS). We acknowledge support from the Spanish Ministry of Economy and Competitiveness, Centro de Excelencia Severo Ochoa 2013-2017 and the support given by Juan de la Cierva-Incorporación Program (IJCI‐2014‐22070) to J.S.Y. We also acknowledge the support of the CERCA Program/Generalitat de Catalunya.