The tertiary folding of Escherichia coli 16S RNA, as studied by in situ intra-RNA cross-linking of 30S ribosomal subunits with bis-(2-chloroethyI)-methylamine

Abstract

Intra-RNA cross-links were introduced into E. coli 30S ribosomal subunits by treatment with bis-(2-chloroethyl)methylamine. The subunits were partially digested with cobra venom nuclease, and the cross-linked complexes were separated by two-dimensional electrophoresis and analysed according to our published procedures. Tertiary structural cross-links in the 16S RNA were identified between nucleotides 31 and 306, and between the tetranucleotide 693-696 and nucleotides 794 or 799. Secondary structural cross-links, lying at the ends of double-helical regions, were found between nucleotides 46 and the trinucleotide 362-364, and between the dinucleotide 148-149 and nucleotide 174. Cross-links within double-helical elements were identified between the tetranucleotide 128-131 and nucleotide 232, between nucleotide 250 and the dinucleotide 274-275, and between nucleotides 1413 and 1486. Adenine as well as guanine residues were involved in the cross-links.

Structure of a bacterial 30S ribosomal subunit at 5.5 Å resolution

“…The 690 and 790 loops are indeed in close proximity (Fig. 3c), as has been predicted by on the basis of intra-site crosslinking 40 and footprinting experiments using P-site tRNA or antibiotics 28 . Moreover, the 690 and 790 stems protrude into the intersubunit interface, with their minor grooves available for association with the 50S subunit, in agreement with hydroxy-radical footprinting data on subunit association 30 .…”

A UV-crosslinkable interaction in human U6 snRNA

“…Here we have described a specific UV-crosslinkable interaction between residues G44 and C81 in human U6 snRNA+ Although most of our studies were performed with a truncated version of U6 snRNA, the same crosslink was detected with a full-length molecule (unpubl+ data)+ This crosslink is similar to other tertiary RNA crosslinks described previously (e+g+, Atmadja & Brimacombe, 1985;Branch et al+, 1985Branch et al+, , 1989Downs & Cech, 1990;Butcher & Burke, 1994) in that it results from a folded structure of the U6 molecule, because it is detectable only after denaturing and annealing+ The potential significance of this crosslink is enhanced because it juxtaposes G44 in the phylogenetically invariant ACAGAGA sequence and C81, which is opposite G54 in the invariant trinucleotide AGC+ Thus, this interaction has the potential of bringing these two important regions of U6 snRNA into close proximity+ Deletion mutations that remove either ACAGAGA or U6 sequences immediately downstream of C81 eliminated crosslinking, but single-nucleotide substitutions at either position unexpectedly had little effect (results not shown)+ These findings suggest that the overall structure of the region is more important for crosslinking than are the identities of the bases involved+ We discuss below other FIGURE 2. RNA sequence requirements for crosslinking+ Three U6 RNA fragments, U6 25-99, 37-99, and 48-99, were UV irradiated under the conditions indicated at the top of the panel (plus 40 mM Tris HCl, pH 7+5)+ Crosslinking was performed for the times indicated and RNAs resolved by denaturing gel electrophoresis as above+ XL and UNXL indicate the positions of crosslinked and uncrosslinked U6 RNAs, respectively+ FIGURE 3.…”

Base changes at position 792 of Escherichia coli 16S rRNA affect assembly of 70S ribosomes.

“…subunit assembly (8), which suggests that a number of bases in this region of 16S rRNA are all essential for 30S-50S interaction. Positions 791 and 792 appear to be on the platform area of the 30S subunit, which places them at the interface between the 30S and 50S subunits (28)(29)(30)(31). This region also appears to be an IF3 binding region (32).…”