The crystal structure of the dimerization initiation site of genomic HIV-1 RNA reveals an extended duplex with two adenine bulges

Abstract

These crystal structures represent the putative matured form of the initial kissing-loop complex. They show the ability of this self-complementary RNA hairpin loop to acquire a more stable extended duplex structure. Both bulged adenines form a striking 'base grip' that could be a recognition signal, either in cis for another viral RNA sequence, or in trans for a protein, possibly the NCp. Magnesium binding might be important to promote and stabilize the observed extrahelical conformation of these bulges.

Dissecting the Protein–RNA and RNA–RNA Interactions in the Nucleocapsid-mediated Dimerization and Isomerization of HIV-1 Stemloop 1

“…According to the high-resolution structures available for this dimeric conformer, A255 can alternatively participate in a zipper-like motif with the adenines on the opposite strand 42; 43 , or assume a bulged-out conformation that could promote protein-RNA or RNA-RNA interactions. 41 In this context, our results provide strong experimental support to the hypothesis that the A-bulges may indeed act as 'base-grips' for NC recognition. Furthermore, considering that the zipperlike motif increases the stability of strand association, protein binding could induce local destabilization by locking unpaired nucleobases in less favorable, bulged-out conformations.…”

Crystal structure of a model branchpoint–U2 snRNA duplex containing bulged adenosines

“…The effects of the bulged adenosines on base stacking in the RNA helix were evaluated using the program Freehelix (Dickerson, 1998) to determine rise, twist, and inclination for the base pairs+ The bulged adenosines were omitted from the duplex for these calculations+ The average rise is 2+7 Å per base pair with a range of 2+5 to 3+0 Å per base pair; the average twist is 33+68 with a range of 29+78 to 39+48; and the average inclination is 15+78 with a range of 11+48 and 19+58+ These average values are very close to those of A-form RNA, which has a rise of 2+8 Å per base pair, a twist of 32+78, and an inclination of 168+ The closely related A9-form RNA has a rise of 3+0 Å per base pair, a twist of 30+08, and an inclination of 10+08 (Arnott et al+, 1973)+ Even the base pairs on either side of the bulged nucleotides are very close to the A-form values+ The base step flanking X5 has a rise of 2+5 Å, a twist of 39+48, and inclinations of 15+78 and 11+48, and the base pairs flanking Y5 have a rise of 2+8 Å, a twist of 36+98, and inclinations of 13+28 and 15+98+ This analysis shows that although the phosphate backbone is quite flexible and can easily accommodate a bulged nucleotide, base stacking is robust and maintains a conformation that is strikingly similar to A-form RNA+ This ability of RNA to incorporate bulges with only local distortions appears to be a common theme, because similar results have been obtained in other duplexes with bulges (Cate et al+, 1996;Portmann et al+, 1996;Ennifar et al+, 1999)+…”

“…In addition to direct hydrogen bonding and stacking interactions, two water molecules (W1, W2) bridge the three-helix interaction observed here (Fig+ 6A, red spheres)+ One of these waters, W1, sits above the bulged adenosines and bridges helix II (orange) and helix III (gray) via four hydrogen bonds in a geometry close to tetrahedral+ This water (W1) is well ordered in its pocket (Fig+ 7) and has a crystallographic B-value that is comparable to those of the surrounding RNA The bulged adenosines from the structure described here are shown in aqua and orange as previously and are thicker compared to the other bulged adenosines+ The other bulged adenosines are two from the P4P6 crystal structure (red and yellow), two from the RNA/DNA chimera crystal structure (fucia and purple), one from the MS2 complex crystal structure (green), and one from the RNA spliced leader NMR structure (black)+ C: The superposition is rotated 908 in comparison to the view in B and again shown in stereo+ (35-40 Å 2 )+ W2 also bridges helix II via the N7 group of Y5 (bulged adenosine) and helix III via the 29 OH group of Y8 (guanosine)+ These hydrogen bonds are listed in Table 2+ The other 20 ordered water molecules in this structure form hydrogen bonds with only one helix+ Although divalent metals frequently mediate RNA packing and tertiary interactions (Laing et al+, 1994;Cate et al+, 1997;Ennifar et al+, 1999), the structure of the bulged adenosine helix described here is free of divalent metal ions, and no ordered monovalent ions appear to be involved in the packing interactions+ Crystallization is dependent upon the presence of spermine, but there is no apparent electron density for ordered spermine in this structure, suggesting that it is either disordered or exists at low occupancy+ Perhaps RNA tertiary interactions involving bulged nucleotides are less dependent upon the presence of metals than other types of RNA tertiary interactions as they can maintain a greater than usual distance between the phosphate backbones of adjacent helices+ The three-helix interaction described here brings the phosphate groups to within 5 Å in one location, but otherwise maintains spacing of greater than 7 Å+ The type of three-way RNA interaction described here may provide a generally useful scheme for folding in RNAs by bringing together three helical regions+ Crystal packing interactions have previously served as models for relevant tertiary interactions; for example, the hammerhead ribozyme structure (Pley et al+, 1994) provided a tetraloop-minor groove interaction that is similar to the now commonly observed tetraloop-tetraloop receptor motif (Costa & Michel, 1995;Cate et al+, 1996)+ Implications for pre-mRNA splicing and self-splicing Group II RNAs…”

NMR and XAS reveal an inner-sphere metal binding site in the P4 helix of the metallo-ribozyme ribonuclease P

“…This work represents the successful combination of NMR and XAS for studying metal coordination by RNA, and should be applicable to the study of other metal-requiring ribozymes. Furthermore, the study of the inner-sphere metal-binding site identified in helix P4 not only provides a molecular level characterization of the environment of a catalytic and/or cocatalytic metal ion in RNase P, but also suggests that tandem guanosines adjacent to a bulged nucleotide could be a general metal ion binding motif in RNA (22,23).…”