Site Specific Incorporation of Amino Acid Analogues into Proteins In Vivo
Abstract
Two critical requirements for developing methods for the site-specific incorporation of amino acid analogues into proteins in vivo are: (1) a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs), and (2) an aminoacyl-tRNA synthetase, which aminoacylates the suppressor tRNA but no other tRNA in the cell. Here, we describe two such aaRS/suppressor tRNA pairs, one for use in the yeast S. cerevisiae, and another for use in E. coli. The '21st synthetase/tRNA pairs' include E. coli glutaminyl-tRNA synthetase (GlnRS) along with an amber suppressor derived from human initiator tRNA, for use in yeast, and mutants of the yeast tyrosyl-tRNA synthetase (TyrRS) along with an amber suppressor derived from E. coli initiator tRNA, for use in E. coli. The suppressor tRNAs are aminoacylated in vivo only in the presence of the heterologous aaRSs and the aminoacylated tRNAs function efficiently in suppression of amber codons. Plasmids carrying the E. coli GlnRS gene can be stably maintained in yeast.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jan 01, 2000
- Accession Number
- ADA385877
Entities
People
- Anne K. Kowal
- Caroline Kohrer
- Uttam L. Rajbhandary
Organizations
- Massachusetts Institute of Technology