title: Site Specific Genetic Incorporation of Azidophenylalanine in Schizosaccharomyces pombe.
abstract: The diversity of protein functions is impacted in significant part by the chemical properties of the twenty amino acids, which are used as building blocks for nearly all proteins. The ability to incorporate unnatural amino acids (UAA) into proteins in a site specific manner can vastly expand the repertoire of protein functions and also allows detailed analysis of protein function. In recent years UAAs have been incorporated in a site-specific manner into proteins in a number of organisms. In nearly all cases, the amber codon is used as a sense codon, and an orthogonal tRNA/aminoacyl-tRNA synthetase (RS) pair is used to generate amber suppressing tRNAs charged with the UAA. In this work, we have developed tools to incorporate the cross-linking amino acid azido-phenylalanine (AzF) through the use of bacterial tRNA(Tyr) and a modified version of TyrRS, AzFRS, in Schizosaccharomyces pombe, which is an attractive model organism for the study of cell behavior and function. We have incorporated AzF into three different proteins. We show that the majority of AzF is modified to amino-phenyl alanine, but protein cross-linking was still observed. These studies set the stage for exploitation of this new technology for the analysis of S. pombe proteins.
authors.affiliation: Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK CV4 7AL.|Department of Biological Sciences, National University of Singapore, Singapore 117543.|Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore 117604.
authors.forename: Nan
authors.initials: N
authors.lastname: Shao
authors.affiliation: Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore 117604.
authors.forename: N Sadananda
authors.initials: NS
authors.lastname: Singh
authors.affiliation: School of Life Sciences, University of Warwick, Coventry, UK CV4 7AL.
authors.forename: Susan E
authors.initials: SE
authors.lastname: Slade
authors.affiliation: School of Life Sciences, University of Warwick, Coventry, UK CV4 7AL.
authors.forename: Alexandra M E
authors.initials: AM
authors.lastname: Jones
authors.affiliation: Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry, UK CV4 7AL.|Department of Biological Sciences, National University of Singapore, Singapore 117543.|Temasek Life Sciences Laboratory, National University of Singapore, 1 Research Link, Singapore 117604.|Mechanobiology Institute, 5A Engineering Drive 1, National University of Singapore, Singapore 117411.
authors.forename: Mohan K
authors.initials: MK
authors.lastname: Balasubramanian
mesh_terms: D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism*; D018388:Codon, Terminator; D004926:Escherichia coli / Q000235:genetics; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D015202:Protein Engineering; D012329:RNA, Bacterial / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012365:RNA, Transfer, Tyr / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001386:Azides; D018388:Codon, Terminator; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; D012365:RNA, Transfer, Tyr; D029702:Schizosaccharomyces pombe Proteins; C038012:tRNA, azidophenylalanine-; C027162:4-azidophenylalanine; D010649:Phenylalanine