Probing functions of the ribosomal peptidyl transferase center by nucleotide analog interference.
The ribosome is a huge ribonucleoprotein complex in charge of protein synthesis in every living cell. The catalytic center of this dynamic molecular machine is entirely built up of 23S ribosomal RNA and therefore the ribosome can be referred to as the largest natural ribozyme known so far. The in vitro reconstitution approach of large ribosomal subunits described herein allows nucleotide analog interference studies to be performed. The approach is based on the site-specific introduction of nonnatural nucleotide analogs into the peptidyl transferase center, the active site located on the interface side of the large ribosomal subunit. This method combined with standard tests of ribosomal functions broadens the biochemical repertoire to investigate the mechanism of diverse aspects of translation considerably and adds another layer of molecular information on top of structural and mutational studies of the ribosome.
Division of Genomics and RNomics, Innsbruck Biocenter, Medical University of Innsbruck, Innsbruck, Austria.
Matthias D
MD
Erlacher
Norbert
N
Polacek
D001483:Base Sequence; D020134:Catalytic Domain; D005818:Genetic Engineering / Q000379:methods*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry*; D010455:Peptides / Q000378:metabolism; D010458:Peptidyl Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011072:Poly U / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis / Q000187:drug effects; D011691:Puromycin / Q000494:pharmacology; D012338:RNA, Ribosomal, 23S / Q000235:genetics / Q000378:metabolism; D012341:RNA, Ribosomal, 5S / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D013824:Thermus / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects
D009711:Nucleotides; D010455:Peptides; D012338:RNA, Ribosomal, 23S; D012341:RNA, Ribosomal, 5S; D011072:Poly U; C012001:polyphenylalanine; D011691:Puromycin; D010458:Peptidyl Transferases