New assays for riboswitch ligand discovery and a novel lysine degradation pathway in bacteria

Abstract

The impact of information is strongly dependent on its processing. This holds true for biological systems. The processing of genetic information is regulated in multiple ways. From DNA encoded promoter strength over RNA stability to protein modifications cells modulate the impact of the genetic information according to their physiological state, cellular environment and in response to stress. At every stage there are various possibilities to interfere with information processing. Herein, two ways of regulation of the information flow were studied. First, I will present the establishment of methods to allow the unbiased discovery of novel riboswitches. Riboswitches are RNA encoded cis-regulatory elements. Upon the binding of a ligand, they undergo structural rearrangement and therefore alter the expression of the adjacent gene. Two independent methods were used. In-line probing, a method often used to characterize riboswitches was optimized to work in a reliable and reproducible way. Results are presented for the characterization of GEMM-I riboswitch variants against all ten canonical c-di-nucleotides. This technique is valuable for the characterization of riboswitches. However, it turned out to be not promising for the screening of novel riboswitch ligands, because it is time-consuming and laborious. Therefore, a coupled transcription-translation assay was developed allowing fast and easy screening of even complex mixtures of small molecules. A huge advantage of this system is the kinetic coupling of transcription and translation as it takes place in prokaryotes and often influences riboswitch performance. Second, CsiR, the repressor of the csiD operon, was characterized together with the genes of the controlled operon. We could show that CsiR itself is allosterically regulated by glutarate. This lead to the discovery of a novel widespread lysine degradation pathway in bacteria being the first described in E. coli. The degradation proceeds via cadaverine, 5-aminovalerate, glutarate and ultimately yields succinate. The pathway is coupled to the central carbon and energy metabolism.