Identification of intermolecular RNA cross-links at the subunit interface of the Escherichia coli ribosome.
32P-Labeled 70S ribosomes and polysomes were isolated from cultures of Escherichia coli and treated with the cross-linking reagent bis(2-chloroethyl)methylamine. Intermolecular 16S-23S RNA cross-linked complexes were separated from other products of the cross-linking reactions by a two-step sucrose density gradient centrifugation procedure and subjected to oligodeoxynucleotide-directed partial nuclease digestions with RNase H. Cross-linked RNA fragments released by such directed digests were resolved by two-dimensional gel electrophoresis and analyzed using classical oligonucleotide fingerprinting techniques. Two distinct intermolecular cross-links between the 16S and 23S RNA could be localized in this manner, involving positions 1408-1411 and 1518-1520 in the 16S RNA sequence and positions 1912-1920 in the 23S RNA sequence. These data provide the first direct topographical links between the RNA of the 30S and 50S subunits in the functional ribosome and, together with previous topographical data concerning the three-dimensional folding of the rRNA, demonstrate that there is a tight cluster at the ribosomal interface both of sites implicated in ribosomal function and of posttranscriptionally modified nucleotides in the rRNA.
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Federal Republic of Germany.
P
P
Mitchell
M
M
Osswald
R
R
Brimacombe
D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D003198:Computer Simulation; D003432:Cross-Linking Reagents; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics*; D008466:Mechlorethamine / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D012329:RNA, Bacterial / Q000737:chemistry*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000302:isolation & purification; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000302:isolation & purification; D016914:Ribonuclease H / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry*
D003432:Cross-Linking Reagents; D009841:Oligonucleotides; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; D008466:Mechlorethamine; D016914:Ribonuclease H