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Many viruses reprogram ribosomes to produce two different proteins from two different reading frames. So-called −1 frameshifting often involves pairwise alignment of two adjacent tRNAs at a ‘slippery' sequence in the ribosomal A and P sites such that an overlapping codon is shifted upstream by one base relative to the zero frame. In the majority of cases, an RNA pseudoknot located downstream stimulates this type of frameshift. Crystal structures of the frameshifting RNA pseudoknot from Beet Western Yellow Virus (BWYV) have provided a detailed picture of the tertiary interactions stabilizing this folding motif, including a minor-groove triplex and quadruple-base interactions. The structure determined at atomic resolution revealed the locations of several magnesium ions and provided insights into the role of structured water stabilizing the RNA. Systematic in vitro and in vivo mutational analyses based on the structural results revealed specific tertiary interactions and regions in the pseudoknot that drastically change frameshifting efficiency. Here, we summarize recent advances in our understanding of pseudoknot-mediated ribosomal frameshifting on the basis of the insights gained from structural and functional studies of the RNA pseudoknot from BWYV.

The first crystal structure of a protein, the Zα high affinity binding domain of the RNA editing enzyme ADAR1, bound to left-handed Z-DNA was recently described. The essential set of residues determined from this structure to be critical for Z-DNA recognition was used to search the database for other proteins with the potential for Z-DNA binding. We found that the tumor-associated protein DLM-1 contains a domain with remarkable sequence similarities to ZαADAR. Here we report the crystal structure of this DLM-1 domain bound to left-handed Z-DNA at 1.85 Å resolution. Comparison of Z-DNA binding by DLM-1 and ADAR1 reveals a common structure-specific recognition core within the binding domain. However, the domains differ in certain residues peripheral to the protein–DNA interface. These structures reveal a general mechanism of Z-DNA recognition, suggesting the existence of a family of winged-helix proteins sharing a common Z-DNA binding motif.

The Zα domain of the human RNA editing enzyme double‐stranded RNA deaminase I (ADAR1) binds to left‐handed Z‐DNA with high affinity. We found by analytical ultracentrifugation and CD spectroscopy that two Zα domains bind to one d(CG)3T4(CG)3hairpin which contains a stem of six base pairs in the Z‐DNA conformation. Both wild‐type Zα and a C125S mutant show a mean dissociation constant of 30 nM as measured by surface plasmon resonance and analytical ultracentrifugation. Our data suggest that short (≥6 bp) segments of Z‐DNA within a gene are able to recruit two ADAR1 enzymes to that particular site.

Direct read-out of information from DNA into RNA allows the genome to be faithfully reproduced in RNA. This outcome occurs in what may be called "hard-wired" organisms. On the other hand, in what we refer to as "soft-wired" organisms, RNA is processed extensively, allowing a number of different messages to be produced from the same gene. As a consequence, the nucleotide sequences present in RNA (referred to here as the ribotype) differ from those present in DNA (the genotype). In soft-wired organisms, RNA processing can be thought of as a series of steps, one or more of which have two mutually exclusive outcomes: a "default" outcome and an "alternative" outcome. In the presence of appropriate regulatory signals, the RNA is processed using the alternative pathway, while the default pathway is used in their absence. The setup is functionally equivalent to that found in binary "logic gates." In both cases, "logical operations" are implemented by using regulatory signals to establish a conditional relationship between input and output and can be described using the Boolean operators AND, OR, and NOT. In the case of RNA processing events, the outcomes can be used either to directly regulate cellular responses or to control other RNA processing events. In the latter case, "networks" are established that make processing of one RNA contingent on another. Such networks allow cells to respond to their surroundings by changing the connectivity between different RNA processing events, using RNA as a substrate to compute an appropriate response. As such logical operations impact phenotype, they are subject to natural selection. Through reverse transcription, successful outcomes can be incorporated into the genome.

Z-DNA is a high energy conformer of B-DNA that forms in vivoduring transcription as a result of torsional strain generated by a moving polymerase. An understanding of the biological role of Z-DNA has advanced with the discovery that the RNA editing enzyme double-stranded RNA adenosine deaminase type I (ADAR1) has motifs specific for the Z-DNA conformation. Editing by ADAR1 requires a double-stranded RNA substrate. In the cases known, the substrate is formed by folding an intron back onto the exon that is targeted for modification. The use of introns to direct processing of exons requires that editing occurs before splicing. Recognition of Z-DNA by ADAR1 may allow editing of nascent transcripts to be initiated immediately after transcription, ensuring that editing and splicing are performed in the correct sequence. Structural characterization of the Z-DNA binding domain indicates that it belongs to the winged helix-turn-helix class of proteins and is similar to the globular domain of histone-H5.

On each of two unmanned Viking spacecraft, a number of investigations are planned to be conducted on the surface of Mars this summer. Included in the science payload are instruments designed to determine the state of chemical evolution, Including the possible presence of living organisms, on that planet.

The DNA fragment d(CG) 3was co-crystallized with N-(2-aminoethyl)-1,4-diaminobutane (PA(24)), a chemically synthesized polyamine. The complex crystal contained one polyamine, 3 magnesium cations and one sodium cation per duplex of d(CG) 3, and well diffracted the X-ray intensities up to 1.0 Å resolution. The d(CG) 3took a left-handed Z-DNA conformation, and the PA(24) molecule electrostatically interacted with the phosphate groups of the d(CG) 3duplex.

The crystal structure of Escherichia coli tRNAfMetan initiator transfer RNA, has been determined. While grossly similar to that of the chain-elongating yeast tRNAPhe, there are three major differences. One involves the folding of the anticodon loop; in particular, the position of the constant uridine, U33. This difference was unexpected and may be of functional significance

The role of problem-solving self-appraisal and negative life stress in hopelessness among college students was examined. Subjects (n =186) completed the Problem-Solving Inventory, the Life Experiences Survey, the Self-Rating Depression Scale, and the Hopelessness Scale. The results of a hierarchical multiple regression analysis showed that problem-solving self-appraisal and its interaction with negative life stress are independent predictors of hopelessness beyond depressed mood. Results are discussed in terms of the implications for future research and treatment of hopelessness and suicidal behavior.

The Zα domain of the human RNA editing enzyme double-stranded RNA deaminase I (ADAR1) binds to left-handed Z-DNA with high affinity. We found by analytical ultracentrifugation and CD spectroscopy that two Zα domains bind to one d(CG) 3T 4(CG) 3hairpin which contains a stem of six base pairs in the Z-DNA conformation. Both wild-type Zα and a C125S mutant show a mean dissociation constant of 30 nM as measured by surface plasmon resonance and analytical ultracentrifugation. Our data suggest that short (≥6 bp) segments of Z-DNA within a gene are able to recruit two ADAR1 enzymes to that particular site.

In an attempt to evaluate the self-regulation model of test anxiety, the effects of outcome expectancy, self-focused attention, and test anxiety were examined with regard to the performance of college student subjects. Forty high-test-anxious and 40 low-test-anxious subjects received prior success (positive expectancy) or failure (negative expectancy) feedback on a word association task that was presented as predictive of performance on verbal achievement tasks. Half the subjects then completed the subsequent achievement tasks in the presence of a mirror to induce self-focused attention. A triple interaction was found on two of the three dependent measures: among test-anxious subjects with induced self-focused attention, significant performance facilitation occurred under conditions of positive expectancy and significant performance decrements occurred under conditions of negative expectancy. Failure appeared to induce a reactance effect among low-test-anxious subjects. The results are consistent with those recently reported in the self-esteem and depression literatures.

Within a stress-cognitive vulnerability paradigm, the present study tested an interactional "state of mind" model of hopelessness among correctional inmates. One hundred forty-six male inmates volunteered to complete measures of problem-solving appraisal, perceived social alienation, rigid/irrational beliefs, reasons for living, hopelessness, and depression. It was hypothesized that each of the cognitive factors would interact with jail stress to account for hopelessness, independent of mood. The results of hierarchical regression analyses supported this hypothesis. Jail stress was related to hopelessness through interaction with the cognitive mediators. Depression, low appraised problem-solving effectiveness, and few reasons for living also had main effects in predicting hopelessness. Results are discusssed in terms of a state of mind model of hopelessness.

Condensation of single molecules from solution into crystals represents a transition between distinct energetic states. In solution, the atomic interactions within the molecule dominate. In the crystalline state, however, a set of additional interactions are formed between molecules in close contact in the lattice—these are the packing interactions. The crystal structures of d(CCCT), d(TAACCC), d(CCCAAT), and d(AACCCC) have in common a four-stranded intercalated cytosine segment, built by stacked layers of cytosine · cytosine⁺ (C · C⁺) base pairs coming from two parallel duplexes that intercalate into each other with opposite polarity. The intercalated cytosine segments in these structures are similar in their geometry, even though the sequences crystallized in different space groups. In the crystals, adenine and thymine residues of the sequences are used to build the three-dimensional crystal lattice by elaborately interacting with symmetry-related molecules. The packing elements observed provide novel insight about the copious ways in which nucleic acid molecules can interact with each other—for example, when folded in more complicated higher order structures, such as mRNA and chromatin. © 1998 John Wiley & Sons, Inc. Biopoly 44: 257–267, 1997

The RNA-specific adenosine deaminase (ADAR1, herein referred to as ADAR) is an interferon-inducible RNA-editing enzyme. ADAR catalyzes the C-6 deamination of adenosine in double-stranded (ds) structures present in viral RNAs and cellular pre-mRNAs as well as synthetic dsRNA substrates. ADAR possesses three functionally distinct copies of the highly conserved double-stranded RNA binding R motif (RI, RII, RIII) implicated in the recognition of dsRNA structures within the substrate RNAs. ADAR is also a Z-DNA-binding protein. Two Z-DNA binding motifs (Zα and Zβ) present in ADAR correspond to repeated regions homologous to the N-terminal region of the vaccinia virus E3L protein. Here we describe assay methods for measurement of ADAR enzymatic activity, dsRNA binding activity, and Z-DNA binding activity.

A study was conducted over a 6-wk. period to investigate factors associated with the development of depression among college students. Subjects were 134 first-semester freshmen conscripted from the general psychology subject pool. Scores were obtained from self-report questionnaires involving measures of life events, social support, and cognitive appraisal at the beginning of the semester and again later. The result of stepwise multiple regression analyses of Time 1 and Time 2 administrations indicated that the variables most strongly and consistently associated with depression were loneliness, interpersonal mistrust, and neuroticism. Two-panel cross-lagged correlational analyses indicated that loneliness preceded depression. Moreover, the data suggested that interpersonal mistrust and neuroticism were related to depression throughthe variable of loneliness.

Antibodies which are specific to the Z–DNA conformation have been purified and characterized on the basis of their binding to three different DNA polymers which can form this left-handed helix. These antibodies bind specifically to polytene chromosomes of Drosophila melanogaster as visualized by fluorescent staining. The staining is found in the interband regions and its intensity varies among different interbands in a reproducible manner. This is the first identification of the Z-DNA conformation in material of biological origin.

The crystal structure of a complex containing deoxy CpG and a terpyridine platinum compound (TPH) shows a DNA double helical fragment with TPH intercalated between two Watson–Crick GC base pairs. The DNA unwinding angle is 23° and the pucker of the deoxyribose rings differ at the 3′ and 5′ ends.

An 8-15S RNA fraction from calf parathyroid glands stimulated the incorporation of radioactive lysine and methionine into protein by 15- to 30-fold in a wheat germ extract. The major product, representing 25% of the total protein synthesized, could be bound to an antiserum to parathyroid hormone and binding was inhibited by parathyroid hormone. The chromatographic mobilities of the two tryptic peptides of the cell-free product that contained methionine were identical to the corresponding peptides of parathyroid hormone. Upon electrophoresis in acidic or sodium dodecyl sulfate-acrylamide gels, the cell-free product migrated more slowly than either parathyroid hormone or its biosynthetic precursor, proparathyroid hormone. Analysis of cyanogen bromide products indicated that the cell-free product contained an additional sequence of amino acids at the amino-terminal end. A protein corresponding to the cell-free product could not be detected in intact cells even during incubations with [3H]leucine as short as 2 min, which suggests the protein may be a transient precursor to proparathyroid hormone.

This paper deals with the crystal structure of d(CG)3‐spermidine complex. The DNA fragment, d(CG)3, was crystallized with N‐(2‐amino‐propyl)‐1,4‐diamino‐butane, PA(34), spermidine. The results of its X‐ray crystallographic analysis showed many intermolecular contacts between d(CG)3and spermidine, but the binding mode of spermidine to the d(CG)3molecule is different from that of the d(CG)3and N‐(2‐amino‐ethyl)‐1,4‐diamino‐butane [PA(24)]complex: a spermidine molecule bound to the d(CG)3and its symmetrically related neighboring d(CG)3molecules through the water molecules with hydrogen bonds, while one PA(24) molecule connected directly to one d(CG)3molecule, but not to its neighboring d(CD)3molecule. In the crystal, the d(CG)3molecule was the left‐handed Z‐form, and three magnesium cations and a sodium cation were observed around the d(CG)3moiety with different binding modes from the case of the d(CG)3‐PA(24) complex.

In vitrolinked transcription-translation of chloroplast DNA has been used to show that the large subunit of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] is encoded by Zea mayschloroplast DNA. A BamHI-generated chloroplast DNA sequence cloned in Escherichia coliis shown to direct the in vitrosynthesis of this protein identified as large subunit by its size, serological properties, and limited proteolytic digestion products.

The role of loneliness, irrational beliefs, and deficient reasons for living in predicting vulnerability to hopelessness under conditions of negative life stress was examined. Subjects (N 178) completed the UCLA Loneliness Scale, Rational Beliefs Inventory, and the Reasons for Living Inventory at the beginning of the semester. Then, at midterm, measures of negative life stress, depression, and hopelessness were obtained from the same subjects. It was hypothesized that the vulnerability factors would interact with negative life stress to predict hopelessness, independent of depressed mood. The results of multiple regression analyses supported this hypothesis. Implications for research, prevention, and treatment are noted.

The DNA fragment d(CG)3was co‐crystallized with N‐(2‐aminoethyl)‐1,4‐diaminobutane (PA(24)), a chemically synthesized polyamine. The complex crystal contained one polyamine, 3 magnesium cations and one sodium cation per duplex of d(CG)3, and well diffracted the X‐ray intensities up to 1.0 Å resolution. The d(CG)3took a left‐handed Z‐DNA conformation, and the PA(24) molecule electrostatically interacted with the phosphate groups of the d(CG)3duplex.

Three 8-base pair (bp) segments of alternating purine-pyrimidine from the simian virus 40 enhancer region form Z-DNA on negative supercoiling; minichromosome DNase I-hypersensitive sites determined by others bracket these three segments. A survey of transcriptional enhancer sequences reveals a pattern of potential Z-DNA-forming regions which occur in pairs 50–80 bp apart. This may influence local chromatin structure and may be related to transcriptional activation.

The DNA fragment d(CpGpCpGpCpG) crystallises as a left-handed double helical molecule with Watson–Crick base pairs and an antiparallel organisation of the sugar phosphate chains. The helix has two nucleotides in the asymmetric unit and contains twelve base pairs per turn. It differs significantly from right-handed B-DNA.

DNA complementary in sequence to the messenger RNA for pre-proparathyroid hormone was synthesised using reverse transcriptase. In a linked transcription–translation system using RNA polymerase and cell-free extract from wheat germ, the DNA directed the synthesis of a protein identified as pre-proparathyroid hormone by N-terminal Sequencing and by electrophoretic and immunologic criteria.

The molecular structure of monoclinic crystals of yeast phenylalanine tRNA is analyzed by comparing it to the orthorhombic crystals of the same material whose structure has been determined. Changing the packing of the molecule from the head-to-head, tail-to-tail arrangement in the orthorhombic lattice to a head-to-tail packing makes it possible to generate a proposed structure for the monoclinic unit cell. The structure factors for the proposed arrangement have been calculated and compared with those experimentally observed from monoclinic crystals. The residuals from this comparison are low enough to conclude that at 4-Å resolution, the three-dimensional structure of the molecule in the monoclinic crystal is essentially the same as that in the orthorhombic crystal. In addition, a correlation coefficient calculated from intensities based on a skeletal model of the molecule also confirmed the structure in the monoclinic cell. Electron density difference maps, as well as the presence of close contacts in the anticodon loop region of the monoclinic crystal, suggest that the anticodon loop may have a slightly different conformation than that observed in the orthorhombic crystals.

The three-dimensional structure of yeast phenylalanine tRNA serves as a useful basis for understanding the tertiary structure of all tRNAs. A large number of tRNA sequences have been surveyed and some general conclusions are drawn. There are only a few regions in the molecule in which there are differences in the number of nucleotides; and the structure of yeast phenylalanine tRNA can accommodate these differences by forming or enlarging protuberances on the surface of the basic framework molecule. The nature and distribution of the differences in number of nucleotides are surveyed and possible hydrogen bonding interactions are discussed for a number of tRNA classes. The two most significant features of the molecule are the large number of stacking interactions which are seen to include most of the nucleotides in the molecule and the system of specific hydrogen bonding interactions. It is likely that these stabilizing elements are preserved in all tRNA structures.

The DNA fragment, d(CG)3, was co‐crystallized with N‐(3‐amino‐propyl)‐N‐(5‐aminopropyl)‐1,4‐diaminobutane (thermospermine; PA(334)), a polyamine metabolized from the nucleic acid. By using a good crystal with dimensions of 0.5 × 0.5 × 0.5 mm3, X‐ray intensity data were collected up to 1.0 Å resolution. Two thermospermine molecules and a magnesium cation were bound to the left‐handed double‐helical d(CG)3molecule. The d(CG)3molecule adopted the left‐handed Z‐conformation and two thermospermine molecules and a magnesium cation neutralized the negative charges of the phosphate groups of the d(CG)3molecule. Furthermore, the binding modes between d(CG)3and thermospermine were different from those of d(CG)3complexes with PA(24), spermidine and spermine. This is the first case in which it was determined by X‐ray crystallographic analysis that one of two thermospermine molecules bound three d(CG)3duplexes which were symmetrically related to each other, and the other formed two hydrogen bonds at the N(5) and N(9) atoms with two adjacent nucleotide phosphate groups of a single d(CG)3strand at the minor groove. Furthermore, no direct coordination bond was found between the d(CG)3molecule and the magnesium cation.

Many viruses regulate translation of polycistronic mRNA using a -1 ribosomal frameshift induced by an RNA pseudoknot. A pseudoknot has two stems that form a quasi-continuous helix and two connecting loops. A 1.6 Å crystal structure of the beet western yellow virus (BWYV) pseudoknot reveals rotation and a bend at the junction of the two stems. A loop base is inserted in the major groove of one stem with quadruple-base interactions. The second loop forms a new minor-groove triplex motif with the other stem, involving 2'-OH and triple-base interactions, as well as sodium ion coordination. Overall, the number of hydrogen bonds stabilizing the tertiary interactions exceeds the number involved in Watson–Crick base pairs. This structure will aid mechanistic analyses of ribosomal frameshifting.

Z‐DNA, the left‐handed conformer of DNA, is stabilized by the negative supercoiling generated during the movement of an RNA polymerase through a gene. Recently, we have shown that the editing enzyme ADAR1 (double‐stranded RNA adenosine deaminase, type 1) has two Z‐DNA binding motifs, Zα and Zβ, the function of which is currently unknown. Here we show that a peptide containing the Zα motif binds with high affinity to Z‐DNA as a dimer, that the binding site is no larger than 6 bp and that the Zα domain can flip a range of sequences, including d(TA)3, into the Z‐DNAconformation. Evidence is also presented to show that Zα and Zβ interact to form a functional DNA binding site. Studies with atomic force microscopy reveal that binding of Zα to supercoiled plasmids is associated with relaxation of the plasmid. Pronounced kinking of DNA is observed, and appears to be induced by binding of Zα. The results reported here support a model where the Z‐DNA binding motifs target ADAR1 to regions of negative supercoiling in actively transcribing genes. In this situation, binding by Zα would be dependent upon the local level of negative superhelicity rather than the presence of any particular sequence.

The molecule r(GCG)d(TATACGC) is self-complementary and forms two DNA–RNA hybrid segments surrounding a central region of double helical DNA its molecular structure has been solved by X-ray analysis. All three parts of the molecule adopt a conformation which is close to that seen in the 11-fold RNA double helix. The conformation of the ribonucleotides is partly determined by water molecules bridging between the ribose O2′ hydroxyl group and cytosine O2. The hybrid–DNA duplex junction contains no structural discontinuities. However, the central DNA TATA sequence has some structural irregularities.

A tRNATrpwas purified from rabbit reticulocytes which suppresses the UGA termination codon of β-haemoglobin mRNA. Evidence is presented that the β-haemoglobin readthrough protein is found in reticulocyte translations and intact cells. Some natural readthrough proteins perform essential functions; they are synthesised through suppression of UGA or UAG but not UAA termination codons.

At present we have some understanding about the mechanisms of prebiotic synthesis and polymerization. Experiments are also being carried out on the prebiotic replication of the nucleic acids. However, the greatest gap in our knowledge is the prebiotic mechanism for the polynucleotide directed synthesis of polypeptides. Some insight into potential mechanisms of this type is afforded by the recent determination of the three-dimensional structure of yeast phenylalanine rRNA. The poly-nucleotide chain id found to be highly folded and the molecule is found in an L shaped configuration. One end of the L shaped molecule contains the anticodon loop; that arm of the L is made of the double helical anticodon and dihydro U stems. The other end of the L shaped molecule contains the 3'OH adenosine to which the amino acid is added during aminoacylation. That arm of the L is built of the double helical CCA and T?C stems. The corner of the L is formed by the close approximation of the T?C loop and dihydro U loop. The distance between the anticodon and the 3'OH adenosine end of the molecule is 76 Å. Comments are made about the manner in which the tRNA molecule may have evolved and in addition, some suggestions are presented about the prebiotic interactions of primitive tRNA polynucleotides and messenger RNA strands.

HeLa cells normally contain a distribution of polysome sizes, and the largest polysomes contain over 40 ribosomes. After infection with polio virus and actinomycin-D treatment, a new class of polio-induced polysomes are found, some of which contain up to 60 ribosomes. Examinaton of these polysomes suggests a mechanism for protein synthesis with this polycistronic RNA.

If an initiation complex is formed with E. coli ribosomes, viral messenger RNA and formylmethionyl-tRNA and the unprotected messenger RNA is then digested with ribonuclease, the ribosome is still able to synthesize a pentapeptide fragment. The point of entry for messenger RNA on the ribosome therefore seems to be four codons removed from the initiator site. The same distance was found for the peptidyl site.

Lowering the temperature of E. coli to 8° C results in an accumulation of ribosomal subunits arising from a block in initiating protein synthesis. It is likely that this explains the fact that cell division in E. coli does not occur at temperatures below 8° C.

The sodium salt of guanylyl-3′,5′-cytidine crystallizes in a monoclinic unit cell with one molecule in the asymmetric unit. Each molecule is related to another molecule by a 2-fold rotation axis which results in the formation of an antiparallel, right-handed double helix with complementary hydrogen bonding between the guanine and cytosine residues. The crystal is heavily hydrated with 36 water molecules in the unit cell. The geometry of this crystalline double helix is very similar to those which have been derived from studies of fiber x-ray diffraction patterns of double-stranded RNA, even though the latter do not yield data at atomic resolution.

At 3Å resolution, the electron density map of crystalline tRNA shows the polynucleotide chain as an alternating series of ribose and phosphate peaks. Bases are seen, especially in the double helical stem regions. A complete three-dimensional model of the L-shaped molecule has been built.