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...
The crystal structure of Fab R19.9, derived from an anti-p-azobenzenearsonate monoclonal antibody, has been determined and refined to 2.8-A resolution by x-ray crystallographic techniques. Monoclonal antibody R19.9 (IgG2bK) shares some idiotopes with a major idiotype (CRIA) associated with A/J anti-p-azobenzenearsonate antibodies.The amino acid sequences of the variable (V) parts of the heavy (VH) and light (VL) polypeptide chains of monoclonal antibody R19.9 were determined through nucleotide sequencing of their mRNAs. The VL region is very similar to that of CRIA-positive anti-p-azobenzenearsonate antibodies as is VH, except for its third complementarity-determining region, which is three amino acids longer; it makes a loop, unique to R19.9, that protrudes into the solvent. A large number of tyrosine residues in the complementarity-determining region of VH and VL, with their side chains pointing towards the solvent, may have an important function in antigen binding. Murine antibodies to model antigens have provided valuable experimental systems to study the molecular bases of the specificity, diversity, and genetic control of immune responses. The hapten, p-azobenzenearsonate (Ar), has been used in several laboratories as a suitable probe for such studies (1-5), which have been facilitated by the presence of an intrastrain cross-reactive idiotype, designated CRIA, among the anti-Ar antibodies of A/J mice or ofclosely related strains. The expression of CRIA is linked to genetic loci encoding heavy (H) chains (6) and light (L) chains (7). On the average, about half of the anti-Ar antibodies induced by keyhole limpet hemocyanin-Ar in A/J mice share this idiotype. The variable (V) regions, VH and VL, of CRIA antibodies appear to be encoded by single germ-line genes (8,9), and the diversity (D) region is encoded by a variant of the DFL16.1 gene (10). CRIA molecules also utilize the VK10, K chain joining (J) 1, and, almost invariably, JH2 gene segments (4, 5, 11). Idiotypeexpressing antibodies from hyperimmunized mice display somatic variants ofamino acid sequences in each ofthese gene segments (4, 5, 12), whereas the antibodies from an early primary response reflect few if any mutations (5). The VH region appears to be somewhat more susceptible to somatic variation than VL (4). A disproportionate number of mutations in VH and VL occurs in their complementarity-determining regions (CDR); this probably reflects selection by antigen of variants with higher affinity (5).Among the serum anti-Ar antibodies of immunized A/J mice are molecules that carry some but not all ofthe idiotopes associated with CRIA (13,14). Such antibodies are bound by anti-CRIA antibodies, but they are unable to completely displace labeled CRI+ antibodies from such anti-idiotype antibodies. Antibodies of this type were designated "minor idiotypes" (13,14). The subject of the present investigation, monoclonal antibody (mAb) R19.9 (IgG2bK), has these serological properties and is thus a member of a minor idiotypic anti-Ar family. The ...
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