Attenuation-Based Dual-Fluorescent-Protein Reporter for Screening Translation Inhibitors

Abstract

A reporter construct was created on the basis of the transcription attenuator region of the Escherichia coli tryptophan operon. Dual-fluorescent-protein genes for red fluorescent protein and cerulean fluorescent protein were used as a sensor and internal control of gene expression. The sequence of the attenuator was modified to avoid tryptophan sensitivity while preserving sensitivity to ribosome stalling. Antimicrobial compounds which cause translation arrest at the stage of elongation induce the reporter both in liquid culture and on an agar plate. This reporter could be used for high-throughput screening of translation inhibitors.

A bacterial homolog YciH of eukaryotic translation initiation factor eIF1 regulates stress-related gene expression and is unlikely to be involved in translation initiation fidelity

“…9 To test the influence of YciH on translation initiation from leaderless mRNA and non-AUG initiation codons in vivo, we used a set of reporter plasmids, encoding 2 fluorescent proteins, RFP (531/595 nm) and CER (430/486 nm) with readily distinguishable spectral properties. 13 In each plasmid, the RFP gene is identical and used as an internal control, while the CER fluorescent protein gene has variable translation initiation sites. In the first set of constructs (Fig.…”

Application of reporter strains for new antibiotic screening

“…Альтернативным предствляется применение генов двух флуоресцентных белков. В нашей лаборатории создана [31] и успешно применяется [32,33] система на основе генов флуоресцентных белков Red Fluorescence protein (RFP) (красный) [34] и Cerulean (CER) (голубой) [35]. Выбор именно этих белков был обусловлен тем, что их спектры поглощения и испускания практически не перекрывались между собой, поэтому уровни их экспрессии можно детектировать независимо.…”

Overview of 17,856 Compound Screening for Translation Inhibition and DNA Damage in Bacteria

“…Unregulated RFP fluorescent protein [10] was used as a control. This reporter construct was previously successfully applied for the screening of soil microorganisms culture broths provided by Gauze Institute of new antibiotics search [12]. This work resulted in identification of the mechanism of action for antibiotic amicoumacin A [17].…”

“…Previously in our laboratory, a reporter plasmid was created that was based on the application of modified tryptophan attenuator as a sensor for translation inhibitors [12]. The attenuator of transcription preceding the genes coding for tryptophan biosynthesis pathway was discovered by C. Yanofsky group in 1970s [26].…”

“…In contrast, steady and quick translation in the presence of sufficient concentration of tryptophanyl-tRNA results in the formation of terminator hairpin in the nascent transcript, leading to premature termination of transcription preventing expression of tryptophan biosynthesis genes. We substituted tryptophan codons with those coding for abundant aminoacid alanine and introduced compensatory changes to the RNA in order to preserve a formation of alternative regulatory secondary structures [12]. These mutations made attenuator insensitive to the concentration of tryptophan.…”