Establishment of Escherichia coli Cell System to Investigate glmS Riboswitch Activity

Abstract

Antibiotic resistant has become a long concern in medical world for decades. To address this concern, new drug targets are needed. One promising drug target is glmS riboswitch, a RNA regulatory element, which is activated upon binding to glucosamine-6-phosphate (GlcN6P) and catalyzes self-cleavage. However, finding glmS riboswitch activating compounds requires reliable screening system. Herein we established Escherichia coli cell screening system to identify compounds that can activate glmS riboswitch activity. To establish the system, we incorporated RNaseJ1, a ribonuclease that digest 5’ hydroxyl cleaved glmS riboswitch fragment, to E. coli. RNaseJ1 activity to digest 5’ hydroxyl fragment was tested by utilizing hammerhead riboswitch, a constitutive self-cleaving riboswitch, based on trimethoprim (TMP) selection and reporter protein production. Lastly, we validated the established E. coli system containing RNaseJ1 and glmS riboswitch in presence of glucosamine (GlcN), to activate glmS riboswitch, based on TMP selection and reporter protein production. The results demonstrated RNaseJ1 can be produced in E. coli. Activity of RNaseJ1 and glmS riboswitch system in E. coli was not found and requires further investigation. It is recommended to study further at RNA level, optimize RNaseJ1 production in the established system, and change E. coli strain from BL21(DE3) to yhbj mutant.