Specific binding of a chemically synthesized prokaryotic ribosome recognition site. Prospect for molecular cloning and expression of eukaryotic genes.
An icosadeoxyribonucleotide containing the several features found in prokaryotic mRNA ribosome binding sites has been synthesized. This sequence can stimulate the binding of initiator fMet-tRNAf to the ribosome to form a stable 71 S initiation complex identical with those induced by natural messengers. The binding of this synthetic ribosome binding site is absolutely dependent upon initiation factor IF3, and the bound fMet-tRNAf is sensitive to puromycin indicating the formation of a functional initiation complex. A heptadecadeoxyribonucleotide, identical with the icosanucleotide but lacking the terminal A-T-G codon, can also stimulate the stable binding of fMet-tRNAf to the ribosome, suggesting that the selection of the proper A-U-G initiation codon by fMet-tRNAf is subsequent to and a result of the recognition and binding of the fMet-tRNAf . 30 S ribosome complex to the initiation site. The prospect of ligating a similar synthetic ribosome binding site in front of a eukaryotic gene for cloning in an appropriate prokaryotic vector to assure the expresion of the protein is discussed.
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D008715:Methionine; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D012343:RNA, Transfer; D008715:Methionine