A DNA fragment containing the Escherichia coli 5S rDNA sequence linked to a T7 promoter was prepared by PCR from an M13 clone carrying the 5S- complementary sequence. The DNA was transcribed with T7 polymerase using a mixture of [α-32P]UTP and 4-thio-UTP, yielding a transcript in which ≃18% of the uridine residues were randomly replaced by thiouridine. This modified 5S RNA could be reconstituted efficiently into 50S ribosomal subunits or 70S functional complexes. The reconstituted particles were irradiated at wavelengths above 300 nm, and the crosslinked ribosomal components were identified. A crosslink in high yield was reproducibly observed between the modified 5S RNA and 23S RNA, involving residue U-89 of the 5S RNA (at the loop end of helix IV) linked to nucleotide 2477 of the 23S RNA in the loop end of helix 89, immediately adjacent to the peptidyltransferase 'ring.' On the basis of this result, and in combination with earlier immunoelectron microscopic data, we propose a model for the orientation of the 5S RNA in the 50S subunit.
|Number of pages||5|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|Publication status||Published - 10 May 1994|
- 5S-23S RNA contact
- ribosome structure
- thiouridine crosslinking