STEM-LOOP-IV OF 5S RIBOSOMAL-RNA LIES CLOSE TO THE PEPTIDYLTRANSFERASE CENTER

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 5 S-complementary sequence. The DNA was transcribed with T7 polymerase u sing a mixture of [alpha-P-32]UTP and 4-thio-UTP, yielding a transcrip t in which approximate to 18 % of the uridine residues were randomly r eplaced by thiouridine. This modified 5S RNA could be reconstituted ef ficiently 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 an d 23S RNA, involving residue U-89 of the 5S RNA (at the loop end of he lix IV) linked to nucleotide 2477 of the 23S RNA in the loop end of he lix 89, immediately adjacent to the peptidyltransferase ''ring.'' On t he basis of this result, and in combination with earlier immunoelectro n microscopic data, we propose a model for the orientation of the 5S R NA in the 50S subunit.