Important note: Information in this article was accurate in 1999. The state of the art may have changed since the publication date.
Effects of release factor 1 on in vitro protein translation and the elaboration of proteins containing unnatural amino acids.
Biochemistry. 1999 Jul 6;38(27):8808-19. Unique Identifier : AIDSLINE MED/99321518 Short GF 3rd; Golovine SY; Hecht SM; Departments of Chemistry and Biology, University of Virginia,; Charlottesville, Virginia 22901, USA.
Abstract:
An in vitro protein synthesizing system was modified to facilitate the improved, site-specific incorporation of unnatural amino acids into proteins via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs. The modified system included an S-30 extract derived from Escherichia coli that expresses a temperature-sensitive variant of E. coli release factor 1 (RF1). Mild heat treatment of the S-30 extract partially deactivated RF1 and improved UAG codon readthrough by as much as 11-fold, as demonstrated by the incorporation of unnatural amino acids into positions 25 and 125 of HIV-1 protease and positions 10 and 22 of E. coli dihydrofolate reductase. The increases in yields were the greatest for those amino acids normally incorporated poorly in the in vitro protein synthesizing system, thus significantly enhancing the repertoire of modified amino acids that can be incorporated into the proteins of interest. The substantial increase in mutant protein yields over those obtained with an S-30 extract derived from an RF1 proficient E. coli strain is proposed to result from a relaxed stringency of termination by RF1 at the stop codon (UAG). When RF1 levels were depleted further, the intrinsic rate of DHFR synthesis increased, consistent with the possibility that RF1 competes not only at stop codons but also at other mRNA codons during peptide elongation. It thus seems possible that in addition to its currently accepted role as a protein factor involved in peptide termination, RF1 is also involved in functions that control the rate at which protein synthesis proceeds.
Keywords: JOURNAL ARTICLE Alanine/ANALOGS & DERIVATIVES/GENETICS/METABOLISM Amino Acids/GENETICS/*METABOLISM Aspartic Acid/ANALOGS & DERIVATIVES/GENETICS/METABOLISM Bacterial Proteins/*BIOSYNTHESIS/GENETICS Comparative Study Dimerization Escherichia coli/*GENETICS Heat HIV Protease/CHEMICAL SYNTHESIS/GENETICS/METABOLISM Peptide Termination Factors/GENETICS/*PHYSIOLOGY Plasmids/BIOSYNTHESIS/CHEMICAL SYNTHESIS Species Specificity Support, U.S. Gov't, P.H.S. Tetrahydrofolate Dehydrogenase/METABOLISM Transcription, Genetic *Translation, Genetic Tryptophan/ANALOGS & DERIVATIVES/GENETICS/METABOLISM 991030
A99A0971
AEGiS presents published material, reprinted with permission and neither endorses nor opposes any material. All information contained on this website, including information relating to health conditions, products, and treatments, is for informational purposes only. It is often presented in summary or aggregate form. It is not meant to be a substitute for the advice provided by your own physician or other medical professionals. Always discuss treatment options with a doctor who specializes in treating HIV.
Important note: Information in this article was accurate in 1999. The state of the art may have changed since the publication date.
