Directed hydroxyl radical probing of 16S rRNA in the ribosome: Spatial proximity of RNA elements of the 3′ and 5′ domains

Abstract

We have shown previously that directed hydroxyl radical probing of 16S rRNA from Fe(II) tethered to specific sites within the RNA gives valuable information about RNA-RNA proximities in 70S ribosomes. Here, we extend that study and present probing data from nt 424 in 16S rRNA. To tether an Fe(II) to position 424 in the rRNA we created a specific discontinuity in the RNA by in vitro transcription of the RNA as two separate fragments corresponding to nt 1-423 and 424-1542. An Fe(II)-BABE was covalently attached to a 59-guanosine-a-phosphorothioate at position 424 and 30S subunits were reconstituted from the two pieces of rRNA and the small subunit proteins. Reconstituted 30S subunits capable of associating with 50S subunits were selected by isolation of 70S ribosomes. Hydroxyl radicals, generated in situ from the tethered Fe(II), cleaved positions in the RNA backbone that were close in three-dimensional space to the Fe(II), and the sites of cleavage were identified using primer extension. Fe(II) tethered to position 424 induces cleavage around nt 424, 513, and 531 in the 59-domain of 16S rRNA and around nt 1008, 1029, 1044, and 1208 in the 39-domain of 16S ribosomal RNA. These data constrain the positions of the 420, 1015, 1030 and 1000/1040 helices, for which there is little structural information. Since the 59-and 39-domains of 16S rRNA constitute the body and head, respectively, of 30S subunits, these findings provide direct evidence for proximity of RNA elements in the body and head of 30S.

Proximity of conserved U6 and U2 snRNA elements to the 5′ splice site region in activated spliceosomes

“…Fe‐BABE was prepared from aminobenzyl‐EDTA (Dojindo, Japan; DeRiemer and Meares, 1979). Reaction with the SH was as described (Newcomb and Noller, 1999). Modified pre‐mRNA was separated from Fe‐BABE by a G50 spin column purification and EtOH precipitation of Fe‐BABE‐modified pre‐mRNA.…”

A method for differentiating proteins from nucleic acids in intermediate-resolution density maps: cryo-electron microscopy defines the quaternary structure of the Escherichia coli 70S ribosome

“…It is directly adjacent to the ribosomal A site, in agreement with tRNAdependent footprints [36,37], and is part of a connection with a 16S rRNA element within the 30S subunit head (Figure 4). Directed hydroxyl radical probing indicated close proximity between helices 16 and 18 in the body and helices 33 and 34 in the head, suggesting an interaction between the 530 loop and the 1050/1200 region [38].…”

The 5′ End of U2 snRNA Is in Close Proximity to U1 and Functional Sites of the Pre-mRNA in Early Spliceosomal Complexes

“…2.5 mM oligo was cleaved with 111 mM TCEP trialkylphosphine tris (2-carboxyethyl) phosphine (TCEP) for 1 hr at 55 C in order to reduce disulfides and then precipitated with EtOH. Fe-BABE was prepared from aminobenzyl-EDTA (Dojindo, Japan) as described (DeRiemer and Meares, 1981) and allowed to react with the SH group of the oligo as described previously (Newcomb and Noller, 1999). The Fe-BABE-modified oligo was gel purified and ligated to in vitro-transcribed RNA comprising the remaining nucleotides of the U2 snRNA as described (Do ¨nmez et al, 2004).…”