Development of a positive genetic selection system for inhibition of protein splicing using mycobacterial inteins in Escherichia coli DNA gyrase subunit A.
An intein-based positive genetic selection system was developed to study protein splicing and to provide a selection system with the potential for finding splicing inhibitors. Inteins can be novel antimicrobial targets when present in essential proteins since blocking splicing would kill the organism. For example, pathogenic mycobacteria encode inteins that interrupt DNA gyrase. The gyrase selection system exploits (1) splicing of inteins out of Gyrase A and (2) the dominant lethal effect of quinolone poisoning of DNA gyrase, which in turn blocks replication. The system was adapted for whole-cell high-throughput screening using green fluorescent protein as an automatable readout of viability. To demonstrate the efficacy of this system, mutations that blocked splicing of the Mycobacterium xenopi Gyrase A intein were isolated. Splicing was then assayed at a second temperature to identify inteins with a temperature-sensitive splicing phenotype. Mutations were mapped onto a structure-based sequence alignment, which led to the rational prediction of a temperature-sensitive splicing mutation. GyrA intein subdomain relationships also provided insight into intein evolution.
Adam
Eric
E
New England BioLabs, Beverly, MA 01915, USA.
Perler
Francine B
FB
D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics; D027081:DNA Gyrase / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D007438:Introns; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009161:Mycobacterium / Q000145:classification / Q000201:enzymology / Q000235:genetics*; D019154:Protein Splicing; D017434:Protein Structure, Tertiary; D015363:Quinolones / Q000494:pharmacology; D012641:Selection, Genetic; D013696:Temperature
D002352:Carrier Proteins; D015363:Quinolones; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II