Construction of modified ribosomes for incorporation of D-amino acids into proteins.
While numerous biologically active peptides contain D-amino acids, the elaboration of such species is not carried out by ribosomal synthesis. In fact, the bacterial ribosome discriminates strongly against the incorporation of D-amino acids from D-aminoacyl-tRNAs. To permit the incorporation of D-amino acids into proteins using in vitro protein-synthesizing systems, a strategy has been developed to prepare modified ribosomes containing alterations within the peptidyltransferase center and helix 89 of 23S rRNA. S-30 preparations derived from colonies shown to contain ribosomes with altered 23S rRNAs were found to exhibit enhanced tolerance for D-amino acids and to permit the elaboration of proteins containing D-amino acids at predetermined sites. Five specific amino acids in Escherichia coli dihydrofolate reductase and Photinus pyralis luciferase were replaced with D-phenylalanine and D-methionine, and the specific activities of the resulting enzymes were determined.
Dedkova
Larisa M
LM
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.
Fahmi
Nour Eddine
NE
Golovine
Serguei Y
SY
Hecht
Sidney M
SM
D000215:Acylation; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D005818:Genetic Engineering; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000096:biosynthesis / Q000235:genetics; D012358:RNA, Transfer, Met / Q000096:biosynthesis / Q000235:genetics; D012360:RNA, Transfer, Phe / Q000096:biosynthesis / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D000596:Amino Acids; D029968:Escherichia coli Proteins; D012338:RNA, Ribosomal, 23S; D012358:RNA, Transfer, Met; D012360:RNA, Transfer, Phe