Ribosomal binding site of release factors RF1 and RF2. A new translational termination assay in vitro.
We have established a new in vitro assay for translational termination. It consists of 70 S ribosomes bound to a synthetic RNA minimessenger via interaction with P-site binding fMet-tRNAfMet. If the A-site codon is a stop signal, release activity can be measured by quantifying hydrolyzed formylmethionine. Characteristics of this assay in terms of reaction time, ion concentration, release factor RF1 and RF2 concentration, and competition with A-site-decoding tRNA are discussed. The new assay shows that polypeptide chain release activity is directly dependent on the presence of a stop codon in the ribosomal A-site.
Grentzmann
G
G
Eccles Institute of Human Genetics, Howard Hughes Medical Institute, Salt Lake City, Utah 84112, USA. guido@howard.genetics.utah.edu
Kelly
P J
PJ
D001665:Binding Sites; D018388:Codon, Terminator; D015636:Magnesium Chloride / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010454:Peptide Termination Factors / Q000378:metabolism*; D014176:Protein Biosynthesis; D012358:RNA, Transfer, Met / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D018388:Codon, Terminator; C489002:ETF1 protein, human; D010454:Peptide Termination Factors; D012358:RNA, Transfer, Met; D012269:Ribosomal Proteins; C089238:fMet-tRNA(fMet); C041460:peptide chain termination release factor 2; D015636:Magnesium Chloride; C016700:ribosomal protein L7-L12