Effects of release factor 1 on in vitro protein translation and the elaboration of proteins containing unnatural amino acids.
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.
Departments of Chemistry and Biology, University of Virginia, Charlottesville, Virginia 22901, USA.
G F
GF
Short
S Y
SY
Golovine
S M
SM
Hecht
D000409:Alanine / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D016333:HIV Protease / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D010454:Peptide Termination Factors / Q000235:genetics / Q000502:physiology*; D010957:Plasmids / Q000096:biosynthesis / Q000138:chemical synthesis; D014176:Protein Biosynthesis; D013045:Species Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D014158:Transcription, Genetic; D014364:Tryptophan / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; C489005:prfA protein, E coli; D001224:Aspartic Acid; D014364:Tryptophan; D013762:Tetrahydrofolate Dehydrogenase; D016333:HIV Protease; D000409:Alanine