RNA Structural Dynamics: Pre-melting and Melting Transitions in E. coli 5S rRNA

Abstract

The temperature dependent transition from duplex to a single strand in E. coli 5S ribosomal RNA is a multistep process, and it involves intermediate states. We have analyzed these structural dynamics by chemical modification of cytidines and by single strand specific nuclease digestions. This combined approach led to the characterization of premelting and melting transitions within individual structural segments of the native macromolecule, which we feel may find general application to the structure of biological polyribonucleotides: 1) G-C base pairs at the termini of helices are relatively unstable and they readily undergo premelting transition. 2) Internal G-U/A-U rich stretches of helices exhibit dynamic premelting properties. 3) Hairpin loops have a relatively stronger destabilizing effect than internal loops. 4) Bulge loops destabilize the neighbouring base pairs. 5) Melting of helical segments occurs starting from the destabilizing structures listed above, preferentially from the helix termini. E. coli 5S rRNA has been shown to adopt different conformations. The presence of urea leads to induction of enhancement in the sensitivity for nuclease S1 at several nucleotide positions. The possibility of structural rearrangements will be discussed.

Ribosomal 5s Rna: Tertiary Structure and Interactions With Proteins

“…This is in agreement with the observation that the nucleotides in the loop D of the Saccharomyces cerevisiae 5s rRNA are resistant to chemical modification and they interact with another part of the RNA molecule (Kjems et al, 1985). On the other hand, there are many data clearly supporting the involvement of loop C in the tertiary interactions, even in prokaryotic 5s rRNAs (Pieler et al, 1985;Digweed et al, 19864 b). The close proximity of loops C and D and their interaction through hydrogen bonds is possible by slight bending of the two domains and such distortions of the domains are caused by bulges, which are important sites of protein recognition and local bending (Peattie et al, 1981 ;Puglisi et al, 1993).…”

Facilitating RNA Structure Prediction with Microarrays

“…This is likely due to the high AU content and therefore weak base pairing of probes binding to the 3′ side. Published experiments and a molecular dynamics study suggest lability of the helix III/loop B region (25,64,65). DNA analogues of probes 28 and 29 induce ribonuclease H cleavage between nucleotides 29-31 indicating that the base pairs in that region are labile.…”

Temperature dependent chemical and enzymatic probing of the tRNA-like structure of TYMV RNA

“…The optimal time of incubation and enzyme concentration for each temperature is given in Table 2. The amounts of nuclease Sj used deviate appreciably from those reported by Pieler et al (8). Digestion was stopped by the addition of 80 jl cold ethanol followed by centrifugation.…”

“…The bisulphite ion preferentially reacts with cytidine residues in a single stranded region of RNA (8,12). Cytidines with the bisulphite adduct can be converted to uridines by alkaline treatment.…”

“…Firstly, De Bruin and Klug (6) deduced the tertiary structure of a mammalian mitochondrial tRNA, lacking the dihydrouridine (DHU) stem and loop, by the use of diethyl pyrocarbonate (DEP) and dimethylsulphate (DMS) at various temperatures between 0°and 900C. Secondly, 5S ribosomal RNA from various sources was analysed by Kjems et al (7) and Pieler et al (8). The use of DEP, DMS, sodium bisulphite and nuclease Sl not only provided insight into the secondary and tertiary structure of the ribosomal RNA itself but also yielded information on RNA unfolding in general.…”