Bulges and internal loops

Internal loops occur where two helices are separated by non-Watson-Crick base pairs. Bulges refer to cases where all bases on one strand can base pair while one or more bases on the opposite strand cannot (Fig. 1.1). Both bulges and internal loops are potential, and often used, targets for site-specific recognition of the RNA. 

Internal loops are optimal structures for specific interactions. The 

The bulged loops can either provide unique protein recognition sites by a direct interaction with the unpaired bases or by exposing the major groove at the termini of the adjacent helices. Moreover, a bulge may generate a bend in the RNA helix which can add to the specificity of a protein interaction. 

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RNA duplexes have A-form geometry due to the presence of the hydroxyl group at the 2 -position of the ribose sugar. Although RNA can hybridize with another RNA strand to form a double helix, RNA is predominantly single stranded with a secondary structure consisting of hairpin loops, bulges, and internal loops. As a result, RNA intrinsically has a more complex structure than duplex DNA. 

FIGURE 8-26 Secondary structure of RNAs (a) Bulge, internal loop, and hairpin loop, (b) The paired regions generally have an A-form right-handed helix, as shown for a hairpin. 

Among studies of RNA folding using 2AP fluorescence, only two are used here as examples of what can be learned by steady-state fluorescence. These are large RNAs ( 150-200 nt) that have many duplex regions, loops, and internal bulges that adopt a unique active tertiary fold in the... 

Paromomycin binds in the major groove of the A-site rRNA, within the internal loop (see Fig. 1). Distortion of the RNA backbone by the presence of the bulged nucleotide A and noncanonical A A base pair leads to the formation of a distinct binding pocket for paromomycin. 

In the protein-DNA complexes described in the last section, the DNA molecules adopt essentially B-type conformation. However, double-stranded RNA adopts the A conformation, and, more important, RNA serves many more functions than DNA and therefore requires a conformational flexibility similar to that of protein folding. The secondary structural motifs in RNA include stem (double-stranded) regions, hairpin loops, single- or multiple-base bulges, internal loops and multiple-way junctions (see reviews [121, 122] and the description of tRNA secondary structure below, which includes some of the mentioned motifs). The conformational flexibility of RNA suggests that other types of recognition are operative in protein-RNA complexes than in complexes with DNA. 

Figure 12-3 Conformation of the ferritin IRE, (A) Secondary structure. (B) Superposition of models of the internal loop/bulge showing the proximity of G26 to both U5 or U6 for base pairing. (C) The effect of pH on intemal/loop bulge conformation (2D-NMR) G26-U5 and G26-U6 base pairs.

Figure 12-4 Differential binding of IRPl and IRP2 to natural IREs (Iron Responsive Elements). P-5 -RNAs (n = 29-30 nucleotides) were melted and annealed before mixing with recombinant IRP proteins (The proteins were kindly provided by E. A. Leibold, University of Utah, and W. E. Walden, University of Illinois). RNA-protein complexes were separated from RNA by electrophoresis in non-denatuiing polyacrylamide gels [20]. Per contains an internal loop/bulge (Figure 12-3), and TfR, eALAS, and m-aconitase IREs have C-bulges. Per mutation AU6 converts the Per internal loop/bulge to a C-bulge. Per ferritin TfR transferrin receptor eALAS erythroid amino-levulinate synthase and m-aconitase. No IRE/IRP complex was detectable.

Hehcal segments are interspersed with bulge, internal and hairpin loops that contain noncanonical base pairs and unstacked bases. 

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