Project Details
Projekt
Directed evolution to understand enzyme function of almost protein independent bacterial RNase P RNA versus largely protein-dependent archaeal RNase P RNA
Applicant
Professor Dr. Roland K. Hartmann
Subject Area
Biochemistry
Term
from 2004 to 2011
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 5429323
Ribonuclease P (RNase P) catalyzes tRNA 5'-end maturation in all organisms and organelles. The RNA subunit (P RNA) of RNase P from Bacteria is an active catalyst in vitro in the absence of the protein cofactor. P RNAs from eucaryotes, organelles and most Archaea have entirely lost this capacity during evolution, and all rational attempts to re-engineer RNA-alone catalysis have failed. Thus, to understand the structural and conformational requirements for P ribozyme function, approaches of directed evolution are needed. To realize such an evolutionary strategy, we plan: (a) sequence randomization and mutagenesis of selected genes encoding P RNAs which have almost or completely lost their RNA-alone activity during evolution, to generate large pools of molecules with sequence and structure variation; (b) in vitro selection of variants with P ribozyme activity and (c) in vivo selection of variants that can functionally replace the native P RNA in bacterial complementation strains. We expect answers to the following questions: (1) Can we retro-evolve ribozyme activity? (2) What are the requirements for specific P RNA ribozyme function in terms of conformational flexibility and stability? (3) Which structural changes are required to convert a non-bacterial P RNA to a bacterial-like P RNA ribozyme? (4) How extensive is sequence variation among the group of functional RNA variants selected in vitro and in vivo? (5) Does a gain of function of mutagenized RNA variants in the bacterial host mimic natural evolution? Will these RNAs be efficient ribozymes in vitro?
DFG Programme
Priority Programmes
