Structure and function of ribosomal RNA

The importance of individual nucleotides for the structure and function of rRNA molecules in E. coli

“…A number of recent reports have convincingly demonstrated that some RNAs have enzyme-like functional activities [ 1,3]. Similar observations are true for ribosomal RNAs, pointing to a functional importance of defined sequence regions for individual steps in the mechanism of translation 13-61.…”

Metal ion probing of rRNAs: Evidence for evolutionarily conserved divalent cation binding pockets

“…Metal ion hydroxide-catalyzed specific cleavage of rRNAs was applied to the characterization of the immediate environment of high-affinity cation binding pockets and to probe the fine structure of rRNAs within the active ribosome+ For the maintainance of the structural as well as the functional integrity, ribosomes strictly require counterions, such as polyamines, mono-, and divalent metal ions+ Whereas polycations and monovalent ions are interchangeable, divalent metal ions are required absolutely and cannot be substituted without destroying the catalytic activity (for a review, see Streicher & Wallis, 1996)+ Metal ion-directed cleavage using Pb 2ϩ , as well as the functionally active ions Mg 2ϩ , Mn 2ϩ , and Ca 2ϩ , resulted in the identification of specific cleavage sites with various efficiencies+ Site-specific and strong metal hydroxide cuts are caused by ions bound tightly to binding pockets in close physical proximity to the cleavage site and depend on a correct tertiary RNA folding+ In contrast, metal ion cleavage can also be applied to probe the overall structure of RNA molecules+ In this Effects of bound AcPhe-tRNA and uncharged tRNA to the P site were quantified by using a Molecular Dynamics PhosphorImager+ The lead-cleavage efficiency of poly(U) programmed ribosomes at 10 mM Pb(OAc) 2 was taken as 100%+ Quantification was performed for six independent binding experiments+ case, the metal ions act from the solution and preferentially cleave every acessible single-stranded nucleotide with low efficiency and slow cleavage rate; this is sometimes termed "unspecific cleavage" (Pan et al+, 1993)+ Site-specific metal hydroxide-induced cleavage is considered to occur in single-stranded and flexible regions, whereas double-stranded RNA helices are known to be insensitive (Gornicki et al+, 1989;Ciesiolka et al+, 1998 ; data not shown), a pH effect can be ruled out+ Therefore, it can be concluded that the native structure was probed in all cases+ In some 2D models (Noller et al+, 1981;Brimacombe & Stiege, 1985), this region is involved in forming a helix, which should make it resistant to metal ion cleavage+ In light of our data, this region is not supposed to be base paired, a conclusion that is supported further by data showing nucleotides in that region to be highly reactive to single-strandspecific chemical probes (Egebjerg et al+, 1990) and by a structure model that depicts this region unpaired (Fig+ 2A)+ Second, two Mg 2ϩ (C2794 and G2801) and two Ca 2ϩ cuts (C2793 and G2801) in domain VI of 23S rRNA could be identified (Fig+ 2A) that are located within a helical region+ Comparative sequence analysis suggests a canonical base paired helix; however, this helix apparently adopts a structure that is accessible to site-specific cleavage, at least under the Mg 2ϩ and Hairpins, being the most abundant elements of RNA secondary structure (Woese et al+, 1990), are thought to provide nucleation points for folding and are often involved in protein recognition (Varani, 1995)+ In rRNAs, the most frequent hairpins contain the so called tetraloops of the GNRA, UNCG, and CUUG families+ The four nucleotides in the loops are structurally constrained, which explains the unusual thermodynamic stability of the hairpin+ The 70S ribosomal tetraloops were resistant to metal ion cleavage, with only two exceptions+ Two UN...…”

Magnesium ions mediate contacts between phosphoryl oxygens at positions 2122 and 2176 of the 23S rRNA and ribosomal protein L1

“…The secondary structure of the L1-4, L1-7, and L1-12 RNA transcripts+ Nucleotides found in E. coli 23S rRNA are in boldface and those added to stabilize the structure of the fragments are in normal type+ Numbers correspond to the positions of the nucleotides in E. coli 23S rRNA+ Helices are designated according to Brimacombe and Stiege (1985)+ A: The L1-4 RNA fragment consists of helix 77, the lower portion of helix 78, and the conserved sequence within the loop between helices 77 and 78+ The affinity of L1 for this fragment is approximately the same as that for a similar fragment containing the nonconserved nucleotides spanning positions 2163-2166 (Zimmermann et al+, 2000)+ B: The L1-7 RNA fragment has the same sequence as the L1-4 RNA except that the U2122-A2176 base pair is replaced by C-G+ C: The L1-12 RNA fragment encompasses a stem that mimics the upper portion of helix 76, helix 77, and the lower portion of helix 78, as well as the loops that interconnect these features+ The base-paired stem that joins the 59 and 39 ends of the fragment was introduced to maintain the size and stability of the loop between helices 76 and 77+…”