Localisation of a series of intra–RNA cross-links in the secondary and tertiary structure of 23S RNA, induced by ultraviolet irradiation ofEscherichia50S ribosomal subunits

Abstract

Intra-RNA cross-links were introduced into E. coli 50S ribosomal subunits by mild ultraviolet irradiation. The subunits were partially digested with cobra venom nuclease, and the cross-linked RNA complexes were isolated by two-dimensional electrophoresis. Many of the complexes were submitted to a second partial digestion procedure. Oligonucleotide analysis of the RNA fragments obtained in this manner enabled cross-links between the following ribonuclease T1 oligonucleotides in the 23S RNA to be established: positions 292-296 and 339-350; 601-604 and 652-656; 1018-1022 and 1140-1149; 1433-1435 and 1556-1560; 1836-1839 and 1898-1903; 2832-2834 (tentative) and 2878-2885; 2849-2852 and 2865-2867 (tentative); 739-748 and 2609-2618; 571-577 and 2030-2032; 1777-1792 (tentative) and 2584-2588. The first seven of these cross-links lie within the secondary structure of the 23S RNA, whereas the last three are tertiary structural cross-links. The degree of precision of the individual determinations was variable, depending on the nucleotide sequence in the vicinity of the cross-link site concerned.

Structure and function of ribosomal RNA

“…After partial digestion, the cross-linked products are isolated by two-dimensional electrophoresis and the cross-link sites determined by classical fingerprinting techniques. The early studies Glotz et al, 1981) only led to the identification of secondary structural cross-links, but, by using different partial digestion conditions, a number of tertiary as well as secondary intra-RNA cross-links have now been localized in both 50S (Stiege et al, 1982(Stiege et al, , 1983 tRNA has also been used as a target for crosslinking studies. In one series of experiments, Prince et al (1982) showed that the hypermodified uridine at the 5'-anticodon position of tRNAval or tRNASer is cross-linked to C-1400 in the E. coli 16S RNA sequence.…”

Investigation of the tertiary folding ofEscherichia coli16s RNA byin situintra-RNA crosslinking within 30S ribosomal subunits

“…The application of intra-RNA cross-linking techniques is the most direct and obvious approach for probing the tertiary structure of a large RNA molecule, and in the case of 16S and 23S ribosomal RNA several research groups have been involved in this type of study (1)(2)(3)(4)(5)(6)(7)(8)(9). For technical reasons, many of these investigations (5-9) have been confined to an analysis of cross-links induced within isolated RNA.…”

“…For technical reasons, many of these investigations (5-9) have been confined to an analysis of cross-links induced within isolated RNA. In contrast, we have always been of the firm opinion that studies on the tertiary structure of ribosomal RNA can only be meaningful if the cross-links are induced in situ in the intact ribosomal subunits or 70S ribosomes (1)(2)(3)(4).…”

“…In some cases the venom digestion was followed by a further digestion with ribonuclease H in the presence of the hexanucleotide d-(CTTCCC) (cf. 11,12,4). Cross-linked RNA complexes were isolated from these digests by two-dimensional gel electrophoresis and subjected to oligonucleotide analysis by our established methodology (3,4), and we were able to identify a total of four tertiary structural and two secondary structural cross-links.…”

“…11,12,4). Cross-linked RNA complexes were isolated from these digests by two-dimensional gel electrophoresis and subjected to oligonucleotide analysis by our established methodology (3,4), and we were able to identify a total of four tertiary structural and two secondary structural cross-links. The significance of these results with regard to the three-dimensional organisation of the 16S RNA within the 30S subunit is discussed.…”