Overexpression of the thiostrepton-resistance gene from Streptomyces azureus in Escherichia coli and characterization of recognition sites of the 23S rRNA A1067 2'-methyltransferase in the guanosine triphosphatase center of 23S ribosomal RNA.
The thiostrepton-resistance gene encoding the 23S rRNA A1067 methyltransferase from Streptomyces azureus has been overexpressed in Escherichia coli using a T7-RNA-polymerase-dependent expression vector. The protein was efficiently expressed at levels up to 20% of total soluble protein and purified to near homogeneity. Kinetic parameters for S-adenosyl-L-methionine (Km = 0.1 mM) and an RNA fragment containing nucleotides 1029-1122 of the 23S ribosomal RNA from E. coli (Km = 0.001 mM) were determined. S-Adenosyl-L-homocysteine showed competitive product inhibition (Ki = 0.013 mM). Binding of either thiostrepton or protein L11 inhibited methylation. RNA sequence variants of the RNA fragment with mutations in nucleotides 1051-1108 were tested as substrates for the methylase. The experimental data indicate that methylation is dependent on the secondary structure of the hairpin including nucleotide A1067 and the exact sequence U(1066)-A(1067)-G(1068)-A(1069)-A(1070) of the single strand.
Bechthold
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Department of Chemistry, University of Washington, Seattle 98195.
Floss
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D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D017931:DNA Primers; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004926:Escherichia coli; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D007700:Kinetics; D008780:Methyltransferases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012338:RNA, Ribosomal, 23S / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012435:S-Adenosylhomocysteine / Q000494:pharmacology; D013302:Streptomyces / Q000187:drug effects / Q000235:genetics*; D013379:Substrate Specificity; D013883:Thiostrepton / Q000494:pharmacology*
D017931:DNA Primers; D012338:RNA, Ribosomal, 23S; D011994:Recombinant Proteins; D012435:S-Adenosylhomocysteine; D008780:Methyltransferases; C035181:rRNA (adenosine-O-2'-)methyltransferase; D020558:GTP Phosphohydrolases; D013883:Thiostrepton