Accession Number:

ADA422652

Title:

Site Specific Incorporation of Amino Acid Analogues into Protiens In Vivo

Descriptive Note:

Final rept. 15 Jul 1999-14 Nov 2003

Corporate Author:

MASSACHUSETTS INST OF TECH CAMBRIDGE

Personal Author(s):

Report Date:

2004-01-14

Pagination or Media Count:

11.0

Abstract:

Our objective is to develop general methods for the site-specific incorporation of amino acid analogues into proteins in bacterial and in eukaryotic cells. The approach consists of the use of an amber suppressor transfer RNA tRNA aminoacylated with an amino acid analogue, with the help of a mutant aminoacyl-tRNA synthetase, to insert the amino acid analogue at a specific site in a protein. The site of insertion of the analogue is specified by an appropriately placed amber termination codon within the gene for the protein of interest. This approach has two key requirements 1 an amber suppressor tRNA, which can not be aminoacylated by any of the endogenous aminoacyltRNA synthetases and 2 an aminoacyi-tRNA synthetase, which aminoacylates the amber suppressor tRNA but no other tRNA in the cell. Therefore, an important first goal is to identify such a 21st aminoacyl-tRNA synthetase tRNA synthetase-amber tRNA pair. This goal has been achieved. Several mutations have been introduced into yeast tyrosyl-tRNA synthetase TyrRS for isolating mutants that incorporate iodotyrosine into tRNA instead of tyrosine. Work on an alternative approach applicable to mammalian cells has led to a possible general approach for the introduction of two different amino acid analogues into a protein.

Subject Categories:

  • Biochemistry

Distribution Statement:

APPROVED FOR PUBLIC RELEASE