Poly(4-thiouridylic acid) as messenger RNA and its application for photoaffinity labelling of the ribosomal mRNA binding site.
Poly(4-thiouridylic acid) [poly(s4U)] synthesized by polymerization of 4-thiouridine 5'-diphosphate with Escherichia coli polynucleotide phosphorylase (EC 2.7.7.8) acts as messenger RNA in vitro in a protein-synthesizing system from E. coli. It stimulates binding of Phe-tRNA to ribosomes both in the presence of EF-Tu-Ts at 5 mM Mg2+ concentration and nonenzymatically at 20 mM Mg2+ concentration. It codes for the synthesis of polyphenylalanine. Poly(s4U) competes with poly(U) for binding to E. coli ribosomes. Light of 330 nm photoactivates poly(s4U) thus making it a useful photoaffinity label for the ribosomal mRNA binding site. Upon irradiation of 70-S ribosomal complexes, photoreaction occurs with ribosomal proteins as well as 16-S RNA. Ribosomes pre-incubated with R17 RNA are protected against the photoaffinity reaction. The labelling of 16-S RNA can be reduced by treatment of ribosomes with colicin E3.
D000345:Affinity Labels; D003087:Colicins / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010445:Peptide Elongation Factors; D010649:Phenylalanine; D011072:Poly U / Q000031:analogs & derivatives* / Q000378:metabolism; D011117:Polyribonucleotide Nucleotidyltransferase / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D012269:Ribosomal Proteins / Q000096:biosynthesis; D012270:Ribosomes / Q000378:metabolism; D013873:Thionucleotides / Q000378:metabolism*
D000345:Affinity Labels; D003087:Colicins; D010445:Peptide Elongation Factors; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; D013873:Thionucleotides; D011072:Poly U; D010649:Phenylalanine; D012343:RNA, Transfer; D011117:Polyribonucleotide Nucleotidyltransferase; D008274:Magnesium