Structure of a mispaired RNA double helix at 1.6-A resolution and implications for the prediction of RNA secondary structure.

Abstract

The nonamer r(GCUUCGGC)dBrU, where dBrU is 5-bromo-2'-deoxyuridine, contains the tetraloop sequence UUCG. It crystallizes in the presence of Rh(NH3)6CI3. In solution the oligomer is expected to form a hairpin loop but the x-ray structure analysis, to a resolution of 1.6 A, indicates an eight-base-pair A-RNA duplex containing a central block of two GNU and two C U pairs. Self-pairs which approximate to Watson-Crick geometry are also formed in the extended crystal structure between symmetry-related BrU residues and are part of infinite double-helical stacks. The G U pair is a wobble base pair analogous to the GOT pair found in DNA fragments. The CU mismatch involves one hydrogen-bonded contact between the bases and a bridging water molecule which ensures a good fit ofthe base pair in the RNA helix. The BrU BrU pair is held by two hydrogen bonds in an orientation which is compatible with duplex geometry. The structure observed within the crystal has some parallels with the structure of globular RNAs, and the presence of stable, noncanonical base pairs has implications for the prediction of RNA secondary structure.Tertiary structure is essential to the biological function of many single-stranded RNAs. Base pairing between complementary segments in a sequence generates a series of doublehelical stems that connect unpaired regions or loops. These can fold further into compact assemblies stabilized by additional hydrogen bonds and by hydrophobic and electrostatic interactions (1). The various levels of structural order are most completely characterized in tRNAs, for which a number of crystal structures have been determined (2-4). In the case of larger and more complex RNA species, including ribosomal components and RNase P, present understanding of secondary structure relies heavily on assumptions about the relative stabilities of possible base-pairing arrangements (5). These follow the specificities of adenine for uracil and guanine for cytosine observed in both ribonucleotides and deoxyribonucleotides during replication and transcription and are also mirrored in the experimentally measured stabilities of short oligonucleotide duplexes. Although the double helix is a central feature of these systems, none is a conspicuously direct model of the environment within a globular RNA structure.The present analysis of an RNA double helix is the second to be reported for an oligonucleotide containing a "tetraloop" sequence. Tetraloops are common in natural singlestranded RNA and, although the stems of such structural elements are variable, the unpaired regions are found to be tetranucleotides of mainly two types: a group with the general sequence r(GNRA) and the specific r(UUCG). Examples of both classes have been examined extensively in solution, where they exist as monomeric, thermally stable, looped species which parallel the behavior of the parent sequences in RNA of higher molecular weight (6,7).In crystals of both the present sequence and a dodecamer studied by Holbrook et al. (8), where r(UUCG) is embedded in se...

RNA hydration: three nanoseconds of multiple molecular dynamics simulations of the solvated tRNA Asp anticodon hairpin 1 1Edited by J. Karn

“…Moreover, in three out of six simulations, a long-lived water bridge between atoms (G30)H 21 , (U40)O 2 and (C31)O 2 is observed ( Figure 6). Our simulations, in agreement with crystal structures Betzel et al, 1994;Cruse et al, 1994;Baeyens et al, 1995;Lietzke et al, 1996), suggest that several hydration patterns around G-U base-pairs may be possible. However, in the simulations, the hydroxyl group of the uridine does not participate in long-lived hydrogen bond interactions, as proposed on the basis of crystallographic results Cruse et al, 1994;Lietzke et al, 1996).…”

“…Concerning the G30-U40 base-pair, a long-lived water molecule linking the (G30)H 21 , (U40)O 2 and (C31)O 2 atoms is found in simulations S 2 , S 5 and S 6 (Table 5 and Figure 6). Such a water molecule may be involved in the stabilization of the G-U base-pair as it ®ts in a hydration pocket observed in several crystal structures Betzel et al, 1994;Cruse et al, 1994). However, it is absent in three simulations out of six and, when present, does not make a long-lived hydrogen bond contact with the U40 hydroxyl group, although it remains in its vicinity (Figure 6).…”

“…In a duplex containing two G-U base-pairs (Lietzke et al, 1996), one of the basepairs contributes to crystal packing interactions through an intermolecular hydrogen bond between a uridine O 2 H group and a cytosine O 2 atom of an adjacent duplex, as reported also by Baeyens et al (1995) in another structure. For the second G-U base-pair, water presumably mediates an interaction between the 2 H -hydroxyl group and the guanine N 2 amino group, as described by Cruse et al (1994) for another structure. Such bridges were previously observed in tRNAs .…”

1.76 Å Structure of a Pyrimidine Start Alternating A-RNA Hexamer r(CGUAC)dG

“…During the past decade other RNA crystal structures have been unravelled: the hammerhead ribozyme (Pley et al, 1994;Scott et al, 1995Scott et al, , 1996, the P4±P6 domain of group I intron (Cate et al, 1996) after the model of the entire intron was proposed by Michel & Westhof (1990), complexes of RNA with proteins (Rould et al, 1989;Ruff et al, 1991;Biou et al, 1994;Arnez & Steitz, 1994;Oubridge et al, 1994;Valegard et al, 1994) and duplex RNA (Dock-Bregeon et al, 1988Holbrook et al, 1991;Leonard et al, 1994;Betzel et al, 1994;Cruse et al, 1994;Portmann et al, 1995;Schindelin et al, 1995;Baeyens et al, 1995;. Complex RNA's, like tRNA, rRNA, ribozyme, have double helical stems, loops and other tertiary structures, while DNA's are commonly composed of duplexes.…”

Crystals of the chemically synthesized acceptor stem of tRNAAlafromEscherichia colidiffracting to high resolution

“…Moreover, only a few short RNA molecules were analyzed by X-ray crystallography (Baeyens, De Bondt & Holbrook, 1995;Betzel et al, 1994;Cruse et al, 1994;Leonard et al, 1994;Holbrook, Cheong, Tinoco & Kim, 1991;Dock-Bregeon et al, 1988;Schindelin et al, 1995;Portmann, Usman & Egli, 1995). One reason for few structural investigations of short RNA's was the lack of a suitable method to produce large amounts of RNA tbr a long time.…”