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

ABSTRACT: Quantitative analysis of the sequence determinants of transcription and translation regulation is of special relevance for systems and synthetic biology applications. Here, we developed a novel generic approach for the fast and efficient analysis of these determinants in vivo. ELM-seq (expression level monitoring by DNA methylation) uses Dam coupled to high-throughput sequencing) as a reporter that can be detected by DNA-seq. We used the genome-reduced bacterium Mycoplasma pneumoniae to show that it is a quantitative reporter. We showed that the methylase activity correlates with protein expression, does not affect cell viability, and has a large dynamic range (~10,000-fold). We applied ELM-seq to randomized libraries of promoters or 5’ untranslated regions. We found that transcription is greatly influenced by the bases around the +1 of the transcript and the Pribnow box, and we also identified several epistatic interactions (including the +1 and the “extended Pribnow”). Regarding translation initiation, we confirmed that the Shine-Dalgarno motif is not relevant, but instead, that RNA secondary structure is the main governing factor. With this in hand, we developed a predictor to help tailor gene expression in M. pneumoniae. The simple ELM-seq methodology will allow identifying and optimizing key sequence determinants for promoter strength and translation. The ELM-seq methodology allows both researchers and companies to identify and optimize in an easy and comprehensive manner, key sequence determinants for promoter strength and translation.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mycoplasma Pneumoniae (strain Atcc 29342 / M129)

SUBMITTER: Eva Yus  

LAB HEAD: Eva Yus

PROVIDER: PXD005606 | Pride | 2017-08-29

REPOSITORIES: Pride