Forschung

Our group is interested in the biochemistry and evolution of RNA-directed nucleotidyltransferases and the design of artificial riboswitches for control of gene expression.

1. tRNA Nucleotidyltransferases

These nucleotidyltransferases add the nucleotide triplet C-C-A to the 3’-end of tRNAs, where this sequence represents the site for aminoacylation. Using a single nucleotide binding pocket in the N-terminal catalytic core, the CCA-adding enzymes incorporate two C residues and subsequently switch their specificity towards A addition. In the NTP binding site, a set of highly conserved amino acids forms Watson-Crick-like hydrogen bonds to the incoming nucleotides (amino acid template). A reorientation of these residues is responsible for the mentioned change in nucleotide specificity.

Besides the CCA-adding enzymes, closely related nucleotidyltransferases with partial activities exist, adding only two C residues (CC-adding enzymes) or the terminal A (A-adding enzymes). In addition, poly(A) polymerases are further close relatives of the CCA-adding enzymes. Using a reciprocal exchange strategy, we are trying to dissect individual enzyme regions and determine their specific functions to understand the molecular basis for the specificity of the individual types of enzymes. In the case of CC-adding enzymes, we could demonstrate that a hinge region required for the specificity switch of the amino acid template is missing due to a small deletion. When such a hinge element is reinserted, the CC-adding enzyme regains the full CCA-adding activity.

With a similar strategy, we are identifying functional domains in a series of other related nucleotidyltransferases and poly(A) polymerases.

2. Synthetic riboswitches as regulatory devices for gene expression

In synthetic biology and metabolic engineering, there is a strong demand for orthogonal or externally controlled regulation of gene expression. RNA-based regulatory devices like riboswitches represent a promising alternative to proteins. These regulatory elements, in nature primarily found in bacteria, are usually embedded in the 5′-untranslated region of the mRNA. They allow a fast and direct control of gene expression, as no synthesis of regulatory proteins is required. Riboswitches show a modular composition of an aptamer domain as sensor and an adjacent expression platform as response element. The aptamer specifically interacts with a small molecule as a ligand, modulating the secondary structure of the response domain. As a result, expression of the downstream located genes is either turned on or off. In most cases, this regulation occurs at the level of transcription or translation.

In principle, the riboswitch composition allows for a free combination of different modules in a plug-and-play-like mode to generate new artificial regulatory devices responding to a ligand of choice. In a bottom-up approach, we generate such synthetic riboswitches by combining in vitro selected aptamers with computationally predicted expression platforms (collaboration with the Bioinformatics Group of Peter Stadler at our university). The design strategy can be extended for the construction of more sophisticated regulatory principles like logic gates. Furthermore, the detailed biochemical and structural investigation of such elements is an essential prerequisite to understand RNA structure – function relation and enables us to dramatically increase the success rate of designing novel RNA regulatory elements as building blocks for various applications in synthetic biology.

letzte Änderung: 09.10.2018

Kontakt

Prof. Dr. Mario Mörl
Institut für Biochemie
Brüderstraße 34
D-04103 Leipzig

Telefon: +49 341 97-36910
Telefax: +49 341 97-36919
E-Mail

Sekretariat
Petra Hartung

Telefon: +49 341 97-36910
Telefax: +49 341 97-36919
E-Mail