Escherichia coli deletes in vivo the same domains from a double-mutant leucyl-tRNA synthetase gene that were deleted in vitro to make the LeuAC urzyme

Abstract

E. coli deletes CP1 and ABD domains from [~]40% of plasmids containing leucyl-tRNA synthetase if and only if both active-site signatures are mutated. Shortened ORFs occurred in all reading frames but form three discrete sets in the same frame. One had only the AVGA signature. Two longer ones both retained the same 24-residue segment containing the AMSAS signature. Large pre-steady-state bursts and steady-state acylation assays confirm that they encode active tRNA synthetases. In these respects, the ORFs resemble models for ancestral Class I aminoacyl-tRNA synthetases (AARS). Both signatures thus appear necessary and sufficient for aminoacylation. Growth at 4{degrees} C produced most of the middle-sized ORF, which is incompatible with the others and appears to result from a distinct mechanism. Three different linkages between the two parts of the active site acylate tRNA minihelix at similar rates. That result greatly expands the sequence space of active ancestral AARS. Widely spaced active-site mutants thus trigger deletions of modules acquired as full-length AARS evolved from simpler catalysts. We propose that the deletions survive because they limit mischarging due to disrupted coupling of active-site residues to domain motion. Such deletions may thus be a general phenomenon, opening broad access to primordial gene discovery. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/647346v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1d87333org.highwire.dtl.DTLVardef@1733f25org.highwire.dtl.DTLVardef@136a74borg.highwire.dtl.DTLVardef@12137ce_HPS_FORMAT_FIGEXP M_FIG Schematic rationale for formation and selection of LeuRS urzyme-like deletions. A. Full length LeuRS uses a complex allosteric network of interactions between Dom A (CP1), Dom B (ABD) and WT active-site catalytic histidine and lysine residues. B. Creation of the double mutant corrupts the allosteric effects of the two domains (faded colors). This creates a cytotoxic protein. C. Deletion of the domains whose functions have been corrupted by disrupting the allosteric network produces variants lacking the inactivated domains. These variants have considerably less cytotoxicity. They likely also resemble evolutionary precursors of the full-length protein. This may represent a general phenomenon for double mutant multi-domain protein genes. C_FIG