A reporter system coupled with high-throughput sequencing unveils key bacterial transcription and translation determinants

Nat Commun. 2017 Aug 28;8(1):368. doi: 10.1038/s41467-017-00239-7.

Abstract

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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Methylation
  • Escherichia coli / genetics
  • Gene Expression Profiling / methods*
  • Gene Expression Regulation
  • Genes, Reporter
  • Genome
  • High-Throughput Nucleotide Sequencing / methods
  • Mycoplasma pneumoniae / genetics*
  • Protein Biosynthesis*
  • Site-Specific DNA-Methyltransferase (Adenine-Specific) / chemistry
  • Site-Specific DNA-Methyltransferase (Adenine-Specific) / genetics
  • Transcription, Genetic*

Substances

  • Dam methyltransferase
  • Site-Specific DNA-Methyltransferase (Adenine-Specific)