RNA secondary structure diagrams familiar to molecular biologists summarize at a glance the folding of RNA chains to form Watson–Crick paired double helices. However, they can be misleading: First of all, they imply that the nucleotides in loops and linker segments, which can amount to 35% to 50% of a structured RNA, do not significantly interact with other nucleotides. Secondly, they give the impression that RNA molecules are loosely organized in three-dimensional (3D) space. In fact, structured RNAs are compactly folded as a result of numerous long-range, sequence-specific interactions, many of which involve loop or linker nucleotides. Here, we provide an introduction for students and researchers of RNA on the types, prevalence, and sequence variations of inter-nucleotide interactions that structure and stabilize RNA 3D motifs and architectures, using Escherichia coli (E. coli) 16S ribosomal RNA as a concrete example. The picture that emerges is that almost all nucleotides in structured RNA molecules, including those in nominally single-stranded loop or linker regions, form specific interactions that stabilize functional structures or mediate interactions with other molecules. The small number of noninteracting, ‘looped-out’ nucleotides make it possible for the RNA chain to form sharp turns. Base-pairing is the most specific interaction in RNA as it involves edge-to-edge hydrogen bonding (H-bonding) of the bases. Non-Watson–Crick base pairs are a significant fraction (30% or more) of base pairs in structured RNAs.

Preparing conventional DNA templates for in vitro RNA transcription involves PCR amplification of the DNA gene coding for the RNA of interest from plasmid or genomic DNA, subsequent amplification with primers containing a 5' T7 promoter region, and confirmation of the amplified DNA sequence. Complications arise in applications where long, nonnative sequences are desired in the final RNA transcript. Here we describe a ligase-independent method for the preparation of long synthetic DNA templates for in vitro RNA transcription. In Recursive PCR, partially complementary DNA oligonucleotides coding for the RNA sequence of interest are annealed, extended into the full-length double-stranded DNA, and amplified in a single PCR. Long insertions, mutations, or deletions are accommodated prior to in vitro transcription by simple substitution of oligonucleotides.

Mammalian mitochondrial ribosomes evolved from bacterial ribosomes by reduction of ribosomal RNAs, increase of ribosomal protein content, and loss of guanine nucleotides. Guanine is the base most sensitive to oxidative damage. By systematically comparing high-quality, small ribosomal subunit RNA sequence alignments and solved 3D ribosome structures from mammalian mitochondria and bacteria, we deduce rules for folding a complex RNA with the remaining guanines shielded from solvent. Almost all conserved guanines in both bacterial and mammalian mitochondrial ribosomal RNA form guanine-specific, local or long-range, RNA–RNA or RNA–protein interactions. Many solvent-exposed guanines conserved in bacteria are replaced in mammalian mitochondria by bases less sensitive to oxidation. New guanines, conserved only in the mitochondrial alignment, are strategically positioned at solvent inaccessible sites to stabilize the ribosomal RNA structure. New mitochondrial proteins substitute for truncated RNA helices, maintain mutual spatial orientations of helices, compensate for lost RNA–RNA interactions, reduce solvent accessibility of bases, and replace guanines conserved in bacteria by forming specific amino acid–RNA interactions.

IscU, the central scaffold protein in the bacterial ISC iron-sulfur (Fe-S) cluster biosynthesis system, has long been recognized to bind a Zn ion at its active site. While initially regarded as an artifact, Zn binding has been shown to induce stabilization of the IscU structure that may mimic a state biologically relevant to IscU's role in Fe-S cluster biosynthesis. More recent studies have revealed that SufU, a homologous protein involved in Fe-S cluster biosynthesis in Gram-positive bacteria, also binds a Zn ion with structural implications. Given the widespread occurrence of the "IscU-like" protein fold, particularly among Fe-S cluster biosynthesis systems, an interesting question arises as to whether Zn ion binding and the resulting structural alterations are common properties in IscU-like proteins. Interactions between IscU and specific metal ions are investigated and compared side-by-side with those of SufU from a representative Gram-positive bacterium in the phylum Firmicutes. These studies were extended with additional transition metal ions chosen to investigate the influence of coordination geometry on selectivity for binding at the active sites of IscU and SufU. Monitoring and comparing the conformational behavior and stabilization afforded by different transition metal ions upon IscU and SufU revealed similarities between the two proteins and suggest that metal-dependent conformational transitions may be characteristic of U-type proteins involved in Fe-S cluster biosynthesis.

We have proposed that the ancient ribosome increased in size during early evolution by addition of small folding-competent RNAs. In this Accretion Model, small RNAs and peptides were subsumed onto subunit surfaces, gradually encasing and freezing previously acquired components. The model predicts that appropriate rRNA fragments have inherited local autonomy of folding and local autonomy of assembly with ribosomal proteins (rProteins), and that the rProtein and rRNA are co-chaperones. To test these predictions, we investigate the rRNA interactions of rProtein uL23 and its tail, uL23, which is a β-hairpin that penetrates deep into the core of the large ribosomal subunit. In the assembled ribosome, uL23 associates with Domain III of the rRNA and a subdomain called "DIII". Here using band shift assays, fluorescence Job plots, and yeast three-hybrid assays, we investigate the interactions of rProtein uL23 and its tail with Domain III and with DIII rRNA. We observe rRNA-uL23 complexes in the absence of Mg ions and rRNA-uL23 (n > 1) complexes in the presence of Mg ions. By contrast, the intact uL23 rProtein binds in slightly anticooperative complexes of various stoichiometries. The globular and tail regions of rProtein uL23 are distinctive in their folding behaviors and the ion dependences of their association with rRNA. For the globular region of the rProtein, folding is independent of rRNA, and rRNA association is predominantly by nonelectrostatic mechanisms. For the tail region of the protein, folding requires rRNA, and association is predominantly by electrostatic mechanisms. We believe these protein capabilities could have roots in ancient evolution and could be mechanistically important in co-chaperoning the assembly of the ribosome.