The Excitement of Discovery: Selected Papers of Alexander Rich

The following sections are included:

• Foreword
• Introduction
• Contents

I was a postdoc with Alex and have built my career in DNA nanotechnology by benefitting from Alex’s pioneer work. Let’s talk first about hybridization: in their initial paper describing their proposed structure for DNA, Watson and Crick suggested that a double helical structure for RNA would not be possible, owing to the presence of the 2′-hydroxyl on the furanose ring of ribose. As is often the case, they were right in that RNA does not produce the B-form duplex that they proposed, but they ignored the possibility that a helix with other parameters (ultimately known as the A-form) could readily accommodate that extra group. The difference between the two helices can be understood if one takes a long strip of paper (such as the type that used to be found in adding machines), considers its midline to be the helix axis and the edges to be the backbone; a little verisimilitude can be added by drawing base pairs perpendicular to the midline. Twisting the paper about its midline results in a B-like helix, with the planes of the bases roughly perpendicular to the axis. However, if one wraps the strip around one’s arm, like the stripes on a barber pole with blue and red helical strips or a candy cane, one now has an A-like helix, the type of helix the RNA forms…

I had the good luck to start research at the dawn of molecular biology when it was possible to ask fundamental questions about the nature of the nucleic acids and how information is transferred in living systems. The search for answers led me into many different areas, often with the question of how molecular structure leads to biological function. Early work in this period provided some of the roots supporting the current explosive developments in life sciences. Here I give a brief account of my development, describe some contributions, and provide a hint of the exhilaration in discovering new things. Most of all, I had the good fortune to have inspiring teachers, stimulating colleagues, and excellent students.

When we think about scientific progress in the past, compared to progress in the future, there is a singular asymmetry. Many of the discoveries of the past look obvious. We ask, Why did it take people so long to uncover what today seems obvious? On the other hand, when we look to the future in research, it is unclear, clouded with many uncertainties. We cannot foresee which path will turn out to be correct. Here I look back and describe early research on RNA structure and protein biosynthesis, starting in an era before the term “molecular biology” was used…

A great deal of interest has been aroused by problems associated with the structure and function of ribonucleic acid. This substance is found widely distributed in various parts of the cell. Although some of it is in the nucleus, where it is near the deoxyribonucleic acid, most of the ribonucleic acid is in the cytoplasm, where it is either closely associated with protein to form the particles in the microsomal fraction or is free in the supernatant. Because it constitutes almost half the microsomal particles in which protein synthesis is carried out, it is widely believed that it takes a fundamental role in this process, such as helping to determine the sequence of amino-acids in the newly synthesized protein. It has also been demonstrated that ribonucleic acid can function as a carrier of genetic information. If ribonucleic acid is isolated from the tobacco mosaic virus, it is capable of infecting the tobacco leaf and thereby producing a large number of now virus particles which carry the genetic markers of the original virus…

One of the central problems in molecular biology today is the mechanism whereby genetic information which is stored in the deoxyribosenucleic acid (DNA) is transferred to other molecular species. It is quite clear that genetic information is contained in the DNA molecules. This was initially demonstrated by work on the bacterial transforming factor and more recently by the analysis of the mechanism of bacteriophage infection. It is equally clear at the present time that the major mode of expressing a genetic potentiality is by governing protein synthesis. Protein synthesis is carried out in the microsomal particles. However, the information bearing elements in protein synthesis are believed to be found in the ribosenucleic acid (RNA), which constitutes over one half of the microsomal particle. The other component of these particles is protein, and it is unlikely that this has an important role in ordering the sequence of amino acids because the microsomal protein component seems to be common to all particles even though they are synthesizing widely different proteins…

We have a great deal of information today which strongly but indirectly suggests that there is some relation between the sequence of nucleotides which are found in the two major types of nucleic acid in the cell Since we assume that one nucleic acid sequence copies from another, we describe this process as a “transfer of information…”

Fifty-two years ago I was venturing to the basement of Cal Tech chemistry with some regularity, looking at nucleic-acid diffraction data using the school’s admittedly primitive fiber X-ray facilities. My postdoctoral advisor at the time, Linus Pauling, had been interested in finding the structure of DNA, but Watson and Crick had largely eclipsed that effort. Now, collaborating with Jim Watson, who had returned from Cambridge, I was taken with a challenge put forth in his famed double-helix manuscript…

The sodium salt of the dinucleoside phosphate adenosyl-3′, 5′-uridine phosphate crystallizes in the form of a right handed antiparallel double helix with Watson-Crick hydrogen bonding between uracil and adenine. A sodium ion is located in the minor groove of the helix complexed to both uracil rings.

Adenylyl-3′, 5′-uridine and 9-aminoacridine cocrystallise in a heavily hydrated monoclinic lattice. The adenine residues are hydrogen bonded with uracil residues and the base pairs are stacked parallel to each other in the crystal with 9-aminoacridine sandwiched directly between them.

Collagen is the most abundant protein in the human body and its importance in health and disease continues to be the subject of many investigations. Alex intuitively understood that when he made it the subject of his research early in his career. In 1961, along with Francis Crick, Alex proposed a seminal model (Rich et al., 1955; Rich et al., 1961) of the triple-helical collagen chain, based on fiber diffraction studies, in which the three chains were connected by one hydrogen bond per repeating Gly-X-Y tripeptide. This model was later shown to be correct following various studies of the structures of collagen-like polypeptides…

Very recently Bamford and his colleagues have described their work on a second form of polyglycine, which they call polyglycine II. The purpose of this communication is to suggest a structure for this substance…

Very recently, Ramachandran and Kartha have made an important contribution by proposing a coiled-coil structure for collagen. We believe this idea to be basically correct but the actual structure suggested by them to be wrong…

Extract from an interview with Alex Rich on the history of the discovery of the structure of collagen. Having been invited over to England by Francis Crick, initially to work on polyadenylic acid, Alex arrived with his RNA fibers in July of 1955 and found himself staying with the Crick’s in Cambridge…

The second installment of an interview with Alex Rich on the modeling of the structure of collagen.

“What passed for frontier structural biology in those days was to deduce the structure you were interested in (using clues, intuition and whatever wit you had) and then to see whether it agreed with the X-ray diffraction data (or whatever other data were available). It was a different animal entirely from the kind of work done today. For one thing, in today’s structure determinations you have redundancy — you have more data points than you have unknowns — and thus when you get a solution you know it is almost certainly correct. In those days you had more unknowns, that is, the coordinates, than data points…

Having ‘solved’ the structure of polyglycine II in August of 1955, Alex Rich and Francis Crick realize, a month later, that they are now in a position to build a model of the structure of collagen. In the final segment of an interview with Alex Rich, he tells of how they completed the model and how, almost forty years later, it was shown to be correct…

An important element in Alex’s lab during the early 1960s was the presence of Howard Dintzis, who, together with Mike Naughton, was doing the “Dintzis experiment,” proving that proteins, globin in this case, were made in the N-to-C direction. Paul Knopf was Howard’s graduate student, repeating the “Dintzis experiment” in vitro, using rabbit reticulocyte extracts. I was Alex’s first graduate student; he had me studying in vitro translation with E. coli extracts, in particular trying to translate poliovirus RNA, not very successfully yet at the time. Paul and I thought it might make more sense to translate an animal virus in an animal cell system, and added poliovirus RNA to Paul’s extracts. Since we had ³²P-labeled poliovirus RNA, courtesy of Jim Darnell, we asked if the RNA would associate with the reticulocyte ribosomes. But everything ended up in the pellet of the sucrose gradient! We stepped back a little and asked what reticulocyte ribosomes looked like in a sucrose gradient, using a pulse label with ¹⁴C amino acids to identify the active reticulocyte ribosomes. The result is shown in Fig. 1. After a few control experiments, we were sure that the active ribosomes were in a multiple-ribosome structure, resistant to detergents, ionic strength and DNAse, but exquisitely sensitive to RNAse and to the shear forces used in most preparations of ribosomes at the time. As soon as Alex saw the result he realized what it meant, namely, these were several ribosomes travelling along a single mRNA. Our fellow graduate student, Henry Slayter, doing his PhD with Cecil Hall, took some EM pictures, a few of which have now been republished countless times. These confirmed our suggestion, based on the sucrose gradient analysis, that the predominant form was five ribosomes on the globin mRNA of ∼150 codons. We wrote up the paper, and Alex sent it to John Edsall for submission to PNAS. There was a bit of difficulty with reviewers from the other side of Cambridge, but the paper was soon published.1 Fortunately, Alex had sent a preprint to RB Roberts at the Carnegie Institute, who pointed out that our original term, “multisome,” was a mixture of Roman and Greek roots, so in the proofs the name was changed to “polyribosome,” or “polysome” for short, which has stuck ever since. It soon became apparent that protein synthesis in all organisms takes place on polysomes, which represent a naturally efficient use of mRNA…

Ribosomes are RNA-protein particles which have a diameter of approximately 230 A in the electron microscope. For many years it has been believed that they are the site of protein synthesis. However, in studies with rabbit reticulocytes, we have been able to show that the site of hemoglobin synthesis in vivo is not the single ribosome but rather a cluster of ribosomal particles, which we have called a “polyribosome” or simply a polysome (1). Somewhat similar observations have also been made by Gierer (2). In this paper we describe electron-microscopic studies of this protein-synthesizing structure…

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.

Polyribosomes or polysomes are clusters of ribosomes which are held together by RNA. They can be seen in the electron microscope as linear arrays of ribosomes which are linked by thin strands, 10-15 Å in diameter. Very low concentrations of ribonuclease convert these ribosomal clusters into single ribosomal units. It has been shown that the polysome is the site of protein synthesis in investigations with reticulocytes, liver, HeLa cells, and slime moulds…

IT has been shown in several animal, microbial and plant systems that the assembly of polypeptide chains during protein synthesis occurs on clusters of ribosomes called ‘polyribosomes’ or simply ‘polysomes’. These polysomes appear to be held together by messenger RNA. They are usually identified by their absorbancy at 260 mμ and their protein synthetic function is recognized by the incorporation of isotopically labelled amino-acids into the nascent proteins attached to the ribosomes. In cells which are synthesizing many different proteins, all the proteins are labelled. However, the formation of individual globular proteins has been studied in the case of hæmoglobin and of β-galactosidase. Experiments are reported hero which were performed to examine the formation of collagen, a triple-stranded fibrous protein. It is particularly well suited to such an investigation due to its characteristic content of hydroxyprolino, which is derived metabolically from proline and which can be regarded as a label specific for collagen. Lowther et al. and Prockop et al. have shown collagen to be associated with the microsomal fraction during synthesis. Our results indicate that collagen is synthesized on very largo polysomes…

A typical mammalian cell contains instructions for making many thousands of different proteins and has the capacity to turn out thousands of protein molecules every minute. To a very large extent the living cell is an expression of the particular kinds of proteins it manufactures. It has been known for several years that the site of protein synthesis within the cell is the particle called the ribosome. Visible only in the electron microscope, ribosomes are approximately spherical and can be seen throughout the substance of all living cells. Although the internal structure of these particles is obscure, it has been established that they are composed of protein and ribonucleic acid (RNA) in about equal amounts…

Peptidyl transferase, the ribosomal enzyme which synthesizes peptide bonds, can catalyse the formation of ester linkages between α-hydroxyacyl-tRNA and aminoacyl-tRNA when ribosomes carry out a specific translation of phage R17 RNA in vitro.

Alex was the first person who opened my sense of excitement of scientific discovery which has been driving my scientific curiosity and career ever since. But my encounter with him happened by an “unexpected and chance circumstance.” In 1966, I was in my last year in the graduate program on crystallographic studies of carbohydrate molecules at the University of Pittsburgh under the guidance of George A. (“Jeff”) Jeffrey, and I decided to learn about “New Biology” (later called Molecular Biology) for my postdoctoral training. One day, Jeff asked me whether I would be interested in doing my postdoctoral research with one of his friends at MIT. When he told me that his friend worked in the Department of Biology, I said “YES” immediately, because I saw an opportunity for learning New Biology, which was my “intention.” At that point I did not know anything about the accomplishments or international reputation of Alex since my graduate study subject was very different from that of Alex’s field. At my first encounter with Alex at MIT, I immediately liked him, especially his friendly openness, relaxed and down-to-earth manner and unpretentiousness, all the attributes that were totally foreign to a young Asian who did his undergraduate and master’s studies in South Korea…

An orthorhombic form of crystalline formylmethionine transfer RNA has been obtained which contains one molecule as the asymmetric unit of the unit cell. Three-dimensional x-ray diffraction data have been collected up to a resolution of 12 angstroms, and from this a Patterson function has been calculated. The junction contains an elongated ridge of interatomic vectors parallel to the c-axis of the crystal. Analysis of the function suggests that the molecules are elongated and dimerized in an overlapping antiparallel fashion along the c-axis. The dimer has a length near 109 angstroms and a width of 35 angstroms in one direction. The individual molecular length is approximately 80 angstroms with an irregular cross section measuring 25 by 35 angstroms.

Yeast phenylalanyl transfer RNA crystallizes in a simple orthorhombic unit cell (a = 33.2, b = 56.1, c = 161 Å), and the crystal yields an x-ray diffraction pattern with a resolution of 2.3 Å. From an analysis of the packing in the unit cell it is concluded that the molecular dimensions are approximately 80 by 33 by 28 Å. The diffraction pattern viewed along the a-axis has a distribution characteristic of double-helical nucleic acids. However, this distribution is not found when the pattern is viewed along the b-axis. This has been interpreted as indicating that the double-helical portions of the transfer RNA molecule are approximately half a helical turn in length, and therefore can contain 4-7 base pairs. These results are consistent with the cloverleaf formulation of transfer RNA secondary structure…

The 3-angstrom electron density map of crystalline yeast phenylalanine transfer RNA has provided us with a complete three-dimensional model which defines the positions of all of the nucleotide residues in the molecule. The overall features of the molecule are virtually the same as those seen at a resolution of 4 angstroms except that many additional details of tertiary structure are now visualized. Ten types of hydrogen bonding are identified which define the specificity of tertiary interactions. The molecule is also stabilized by considerable stacking of the planar purines and pyrimidines. This tertiary structure explains, in a simple and direct fashion, chemical modification studies of transfer RNA. Since most of the tertiary interactions involve nucleotides which are common to all transfer RNA’s, it is likely that this three-dimensional structure provides a basic pattern of folding which may help to clarify the three-dimensional structure of all transfer RNA’s.

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.

It is now widely known that the instructions for the assembly and organization of a living system are embodied in the DNA molecules contained within the living cell. The sequence of nucleotide bases along the linear chain of the DNA molecule specifies the structure of the thousands of proteins that are the construction materials of the cell and the catalysts of its intricate biochemical reactions. By itself. however. a DNA molecule is rather like a strip of magnetic recording tape: the information embodied in its structure cannot be expressed without a decoding mechanism…

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.

Four different crystals of d(CpGpCpGpCpG) have been solved by x-ray diffraction analysis and all form similar left-handed double helical Z-DNA molecules in the crystal lattice. Two different conformations are observed for the phosphates in the GpC sequences, as the phosphates are found either facing the helical groove or rotated away from it. The latter conformation is often found when hydrated magnesium ions are complexed to a phosphate oxygen atom. These different conformations may be used when right-handed B-DNA joins left-handed Z-DNA. Atomic coordinates and torsion angles are presented for both types of Z-DNA.

The carcinogen aflatoxin B1 was reacted with a polymer of alternating deoxyguanine and deoxycytosine residues to determine the effect that adduct formation has on the conversion of this polymer from the right-handed B-DNA form found at low salt concentrations to the left-handed Z-DNA form found at high salt concentrations. Reaction with aflatoxin strongly inhibited the salt-induced conversion of this polymer from B-DNA to Z-DNA. This inhibition could be detected even at relatively low binding levels.

In the equilibrium between B-DNA and Z-DNA in poly(dC-dG), the [Co(NH₃)₆]³⁺ ion stabilizes the Z form 4 orders of magnitude more effectively than the Mg²⁺ ion. The structural basis of this difference is revealed in Z-DNA crystal structures of d(CpGpCpGpCpG) stabilized by either Na⁺/Mg²⁺ or Na⁺/Mg²⁺ plus [Co(NH₃)₆]³⁺. The crystals diffract X-rays to high resolution, and the structures were refined at 1.25 Å. The [Co(NH₃)₆]³⁺ ion forms five hydrogen bonds onto the surface of Z-DNA, bonding to a guanine 06 and N7 as well as to a phosphate group in the Z_II conformation. The Mg²⁺ ion binds through its hydration shell with up to three hydrogen bonds to guanine N7 and 06. Higher charge, specific fitting of more hydrogen bonds, and a more stable complex all contribute to the great effectiveness of [Co(NH₃)₆]³⁺ in stabilizing Z-DNA.

The boundary between two segments of Z-DNA that differ in the phase of their syn-anti alternation about the glycosidic bond is termed a z-z junction. Using chemical probes and two-dimensional gel electrophoresis, we examined a z-z junction consisting of the sequence d[(CG)₈(CG)₈] inserted into a plasmid and used energy minimization techniques to devise a three-dimensional model that is consistent with the available data. We show that both alternating CG segments undergo the B-Z transiton together to form a z-z junction. The junction is very compact, displaying a distinctive reactivity signature at the two base pairs at the junction. In particular, the 5’ cytosine of the CC dinucleotide at the junction is hyperreactive toward hydroxylamine, and the two guanines of the GG dinucleotide on the complementary strand are less reactive toward diethyl pyrocarbonate than are the surrounding Z-DNA guanines. Statistical mechanical treatment of the 2-D gel data yields a ΔG for forming the Z-Z junction equal to 3.5 kcal, significantly less than the cost of a B-Z junction and approximately equal to the cost of a base out of alternation (i.e., a Z-DNA pyrimidine in the syn conformation). The computer-generated model shows little distortion of the Z helix outside of the central two base pairs, and the energy of the structure and the steric accessibility of the reactive groups are consistent with the data.

Left-handed Z-DNA is a higher-energy form of the double helix, stabilized by negative supercoiling generated by transcription or unwrapping nucleosomes. Regions near the transcription start site frequently contain sequence motifs favourable for forming Z-DNA, and formation of Z-DNA near the promoter region stimulates transcription. Z-DNA is also stabilized by specific protein binding; several proteins have been identified with low nanomolar binding constants. Z-DNA occurs in a dynamic state, forming as a result of physiological processes then relaxing to the right-handed B-DNA. Each time a DNA segment turns into Z-DNA, two B–Z junctions form. These have been examined extensively, but their structure was unknown. Here we describe the structure of a B–Z junction as revealed by X-ray crystallography at 2.6Å resolution. A 15-base-pair segment of DNA is stabilized at one end in the Z conformation by Z-DNA binding proteins, while the other end remains B-DNA. Continuous stacking of bases between B-DNA and Z-DNA segments is found, with the breaking of one base pair at the junction and extrusion of the bases on each side (Fig. 1). These extruded bases may be sites for DNA modification…

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.

When human U937 cells are placed in agarose microbeads and treated with a detergent, the cytoplasmic membrane is lysed and the nuclear membrane is permeabilized. However, the nuclei remain intact and maintain both replication and transcription. Biotin labeled monoclonal antibodies against Z-DNA have been diffused into this system and used to measure the amount of Z-DNA present in the nuclei. It has previously been shown that the amount of Z-DNA present decreases due to relaxation by topoisomerase I and increases as the level of transcription increases. Here we measure the formation of Z-DNA in the c-myc gene by crosslinking the antibodies to DNA using laser radiation at 266 nm for 10 ns. The crosslinked DNA is isolated by restriction digestion, separation of antibody labeled fractions through the biotin residue, and subsequent proteolysis to remove the crosslinked antibody. Three AluI restriction fragments of the c-myc gene are shown to form Z-DNA when the cell is transcribing c-myc. The Z-DNA forming segments are near the promoter regions of the gene. However, when U937 cells start to differentiate and transcription of the c-myc gene is down-regulated, the Z-DNA content goes to undetectable levels within 30–60 min.

Editing of RNA changes the read-out of information from DNA by altering the nucleotide sequence of a transcript. One type of RNA editing found in all metazoans uses double-stranded RNA (dsRNA) as a substrate and results in the deamination of adenosine to give inosine, which is translated as guanosine. Editing thus allows variant proteins to be produced from a single pre-mRNA. A mechanism by which dsRNA substrates form is through pairing of intronic and exonic sequences before the removal of noncoding sequences by splicing. Here we report that the RNA editing enzyme, human dsRNA adenosine deaminase (DRADA1, or ADAR1) contains a domain (Zα) that binds specifically to the left-handed Z-DNA conformation with high affinity (K_D = 4 nM). As formation of Z-DNA in vivo occurs 5′ to, or behind, a moving RNA polymerase during transcription, recognition of Z-DNA by DRADA1 provides a plausible mechanism by which DRADA1 can be targeted to a nascent RNA so that editing occurs before splicing. Analysis of sequences related to Zα has allowed identification of motifs common to this class of nucleic acid binding domain.

The editing enzyme double-stranded RNA adenosine deaminase includes a DNA binding domain, Zα, which is specific for left-handed Z-DNA. The 2.1 angstrom crystal structure of Zα complexed to DNA reveals that the substrate is in the left-handed Z conformation. The contacts between Zα and Z-DNA are made primarily with the “zigzag” sugar-phosphate backbone, which provides a basis for the specificity for the Z conformation. A single base contact is observed to guanine in the syn conformation, characteristic of Z-DNA. Intriguingly, the helix-turn-helix motif, frequently used to recognize B-DNA, is used by Zα to contact Z-DNA.

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 Zα_ADAR. 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 E3L gene product found in all poxviruses is required for the lethality of mice in vaccinia virus infection. Both the C-terminal region, consisting of a double-stranded RNA-binding motif, and the N-terminal region (vZ_E3L), which is similar to the Zα family of Z-DNA-binding proteins, are required for infection. It has recently been demonstrated that the function of the N-terminal domain depends on its ability to bind Z-DNA; Z-DNA-binding domains from unrelated mammalian proteins fully complement an N-terminal deletion of E3L. Mutations that decrease affinity for Z-DNA have similar effects in decreasing pathogenicity. Compounds that block the Z-DNA-binding activity of E3L may also limit infection by the poxvirus. Here we show both an in vitro and an in vivo assay with the potential to be used in screening for such compounds. Using a conformation-specific yeast one-hybrid assay, we compared the results for Z-DNA binding of vZE3L with those for human Zβ_ADAR1, a peptide that has similarity to the Zα motif but does not bind Z-DNA, and with a mutant of hZβ_ADAR1, which binds Z-DNA. The results suggest that this system can be used for high-throughput screening.

The Zα domains represent a growing subfamily of the winged helix-turnhelix (HTH) domain family whose members share a remarkable ability to bind specifically to Z-DNA and/or Z-RNA. They have been found exclusively in proteins involved in interferon response and, while their importance in determining pox viral pathogenicity has been demonstrated, their actual target and biological role remain obscure. Cellular proteins containing Zα domains bear a second homologous domain termed Zβ, which appears to lack the ability to bind left-handed nucleic acids. Here, we present the crystal structure of the Zβ domain from the human double-stranded RNA adenosine deaminase ADAR1 at 0.97 Å, determined by single isomorphous replacement including anomalous scattering. Zβ maintains a winged-HTH fold with the addition of a C-terminal helix. Mapping of the Zβ conservation profile on the Zβ surface reveals a new conserved surface formed partly by the terminal helix 4, involved in metal binding and dimerization and absent from Zα domains. Our results show how two domains similar in fold may have evolved into different functional entities even in the context of the same protein.

Biologists were puzzled by the discovery of left-handed Z-DNA because it seemed unnecessary. Z-DNA was stabilized by the negative supercoiling generated by transcription, which indicated a transient localized conformational change. Few laboratories worked on the biology of Z-DNA. However, the discovery that certain classes of proteins bound to Z-DNA with high affinity and great specificity indicated a biological role. The most recent data show that some of these proteins participate in the pathology of poxviruses…

The sequence d(GGGGTTTTGGGG) from the 3′ overhang of the Oxytricha telomere has been crystallized and its three-dimensional structure solved to 2.5 A resolution. The oligonucleotide forms hairpins, two of which join to make a four-stranded helical structure with the loops containing four thymine residues at either end. The guanine residues are held together by cyclic hydrogen bonding and an ion is located in the centre. The four guanine residues in each segment have a glycosyl conformation that alternates between anti and syn. There are two four-stranded molecules in the asymmetric unit showing that the structure has some intrinsic flexibility.

The crystal structure of d(CCCAAT), refined at 2.0 Å resolution, shows a four stranded molecule in which two parallel duplexes intercalate with opposite polarity, using cytosine•protonated cytosine base pairs. The intercalation motif in this structure is extended by adenine•adenine base pairs. Two topologically distinct broad grooves are found in the lath-like central part of the molecule with the phosphate groups on one side bent over towards each other, stabilized by bridging water molecules. At the 3’ ends, two arrangements of intermolecular A•A•T base triplets are found, involving both asymmetric and symmetric A•A base pairs joined to thymine residues by Watson-Crick and reverse Hoogsteen base pairing, respectively.

In DNA replication, Okazaki fragments are formed as double-stranded Intermediates during synthesis of the lagging strand. They are composed of the growing DNA strand primed by RNA and the template strand. The DNA oligonucleotide d(GGGTATACGC) and the chimeric RNA-DNA oligonucleotide r(GCG)d(TATACCC) were combined to form a synthetic Okazaki fragment and its three-dimensional structure was determined by x-ray crystallography. The fragment adopts an overall A-type conformation with 11 residues per turn. Although the base-pair geometry, particularly in the central TATA part, is distorted, there is no evidence for a transition from the A- to the B-type (Oil(ormation at the junction between RNA·DNA hybrid and DNA duplex. The RNA trimer may, therefore, lock the complete fragment in an A-type conformation.

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.

Forty years ago, we learned of the major double helical structure adopted by DNA. It combined both elegance and simplicity in its design. Since then we have learned that DNA can also adopt other conformations. We now know that it can exist in a variety of triple-stranded and quadruple-stranded forms, as well as forms that are left-handed. The list of alternative conformations that can be adopted by this molecule is still growing. These conformations represent a major biological challenge to understand their role in biological systems. This type of work represents an active frontier in molecular biology today.

DNA and RNA are acids contain bases, form salts and are held together by sugars. In DNA, the sugar is 2′-deoxyribose and in RNA it is ribose. By the time I joined Alex’s lab in 1989, the important role of the sugar in determining the shape and function of the nucleic acids had long been recognized. As recounted in a short paper titled “The double helix: a tale of two puckers” (Rich, 2003), Alex made a string of discoveries that paved the way to a deeper understanding of how the sugar influences the conformation of DNA and RNA…

Many pathogenic viruses use programmed –1 ribosomal frameshifting to regulate translation of their structural and enzymatic proteins from polycistronic mRNAs. Frameshifting is commonly stimulated by a pseudoknot located downstream from a slippery sequence, the latter positioned at the ribosomal A and P sites. We report here the structures of two crystal forms of the frameshifting RNA pseudoknot from beet western yellow virus at resolutions of 1.25 and 2.85 Å. Because of the very high resolution of 1.25 Å, ten mono- and divalent metal ions per asymmetric unit could be identified, giving insight into potential roles of metal ions in stabilizing the pseudoknot. A magnesium ion located at the junction of the two pseudoknot stems appears to play a crucial role in stabilizing the structure. Because the two crystal forms exhibit mostly unrelated packing interactions and local crystallographic disorder in the high-resolution form was resolvable, the two structures offer the most detailed view yet of the conformational preference and flexibility of an RNA pseudoknot.

The era of the double helix began 50 years ago with publication of the Watson-Crick formulation and the fiber X-ray diffraction patterns from groups led by Maurice Wilkins2 and Rosalind Franklin3. Analysis of the diffraction pattern, especially the fibers of the hydrated B-form, could be immediately interpreted as consistent with a double helix: the weakness of the first-layer line and the virtual absence of the fourth-layer line clearly were consistent with two chains wrapping around each other with the phosphate groups on the outside…

Interleukin 1 (IL-l) is a protein with several biological activities regulating host defense and immune responses. We report here the Isolation or human IL-l eDNA. It encodes a precursor polypeptide or 269 amino acids (30,747 M_r). mRNA Isolated by hybridization to this cDNA was translated in a reticulocyte cell-free system, yielding immunoprecipitable IL-l. Furthermore, this hybrid-selected mRNA was injected into Xenopus laevis oocytes, which subsequently secreted biologically active IL-l. The cDNA nucleotide sequence suggests that IL-l is initially translated as a precursor molecule that is subsequently processed into the 15,000-20,000 M_r protein usually associated with IL-l activity.

Bovine parathyroid tissue incubated in vitro secretes a protein that is distinct from both parathyroid hormone and proparathyroid hormone and comprises about 50 percent of the total secreted protein. This protein appears to be an aggregate consisting of two or more subunits of molecular weight 70,000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Although the function of this protein is unknown, the secretion rates of both the protein and parathyroid hormone respond in parallel to changes in the concentration of calcium in the medium.

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.

Evidence acquired by molecular biologists during the past 25 years points to the interaction of protein molecules with DNA and RNA as a primary source of genetic and metabolic control in living organisms. Just as the description at an atomic level of the DNA molecule had enormous implications for the storage and transfer of genetic information, it seems probable that a detailed explanation of the means by which proteins interact with the control points on the DNA will suggest the mechanisms by which the flow of information is mediated. Such an explanation does not seem possible unless the molecular components and distribution of chemical bonds can be directly visualized. The only available means for obtaining such an image is through the application of the X-ray diffraction technique to single crystals of DNA-binding proteins complexed with fragments of the nucleic acid…

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 stale of chemical evolution, including the possible presence of living organisms, on that planet.

Three different types of biological experiments on samples of martian surface material (“soil”) were conducted inside the Viking lander. In the carbon assimilation or pyrolytic release experiment, ¹⁴CO₂ and ¹⁴CO were exposed to soil in the presence of light. A small amount of gas was found to be converted into organic material. Heat treatment of a duplicate sample prevented such conversion. In the gas exchange experiment, soil was first humidified (exposed to water vapor) for 6 sols and then wet with a complex aqueous solution of metabolites. The gas above the soil was monitored by gas chromatography. A substantial amount of O₂ was detected in the first chromatogram taken 2.8 hours after humidification. Subsequent analyses revealed that significant increases in CO₂ and only small changes in N₂ had also occurred. In the labeled release experiment, soil was moistened with a solution containing several ¹⁴C-labeled organic compounds. A substantial evolution of radioactive gas was registered, but did not occur with a duplicate heat-treated sample. Alternative chemical and biological interpretations are possible for these preliminary data. The experiments are still in process, and these results so far do not allow a decision regarding the existence of life on the planet Mars.

A 16-residue peptide [(AJa-Glu-Ala-Glu-AJaLys-AJa-Lys)z) bas a characteristic β-sheet circular dichroism spectrum in water. Upon the addition of salt, the peptide spontaneously assembles to form a macroscopic membrane. The membrane does not dissolve in heat or in acidic or alkaline solutions, nor does it dissolve upon addition of guanidine hydrochloride, SDS/urea, or a variety of proteolytic enzymes. Scanning EM reveals a network of Interwoven filaments ≈10-20 nm In diameter. An important component of the stability is probably due to formation of complementary ionic bonds between glutamic and lysine side chains. This phenomenon may be a model for studying the insoluble peptides found in certain neurological disorders. It may also have implications for biomaterials and origin-of-life research.

A 16-amino acid oligopeptide forms a stable β-sheet structure in water. In physiological solutions it is able to self-assemble to forma macroscopic matrix that stains with Congo red. On raising the temperature of the aqueous solution above 70°C, an abrupt structural transition occurs in the CD spectra from a β-sheet to a stable α-helix without a detectable random-coil intermediate. With cooling, it retained the α-helical form and took several weeks at room temperature to partially return to the β-sheet form. Slow formation of the stable β-sheet structure thus shows kinetic irreversibility. Such a formation of very stable β-sheet structures is found in the amyloid of a number of neurological diseases. This oligopeptide could be a model system for studying the protein conformational changes that occurs in scrapie or Alzheimer disease. The abrupt and direct conversion from a β-sheet to an α-helix may also be found in other processes, such as protein folding and protein–protein interaction. Furthermore, such drastic structure changes may also be exploited in biomaterials designed as sensors to detect environmental changes.

A new type of self-assembling peptide (sapeptide) scaffolds that serve as substrates for neurite outgrowth and synapse formation is described. These peptide-based scaffolds are amenable to molecular design by using chemical or biotechnological syntheses. They can be tailored to a variety of applications. The sapeptide scaffolds are formed through the spontaneous assembly of ionic self-complementary β-sheet oligopeptides under physiological conditions, producing a hydrogel material. The scaffolds can support neuronal cell attachment and differentiation as well as extensive neurite outgrowth. Furthermore, they are permissive substrates for functional synapse formation between the attached neurons. That primary rat neurons form active synapses on such scaffold surfaces in situ suggests these scaffolds could be useful for tissue engineering applications. The buoyant sapeptide scaffolds with attached cells in culture can be transported readily from one environment to another. Furthermore, these peptides did not elicit a measurable immune response or tissue inflammation when introduced into animals. These biological materials created through molecular design and self assembly may be developed as a biologically compatible scaffold for tissue repair and tissue engineering.

Linus Pauling died on 19 August at his home in California at the age of 93. He was widely regarded as the greatest chemist of the twentieth century — such was the depth of his intuitive understanding of the subject that his co-workers felt he sometimes knew the answer to a question before developing a theory to explain it…

Alexander Rich and Charles F. Stevens, respectively an early collaborator of Crick’s and a long-standing colleague at the Salk Institute, describe the life and work of one of the great thinkers of twentieth-century biology.

After entering Harvard College as a freshman in the fall of 1942, I experienced the usual confusion of a young student without a clear goal in mind. It took me almost 2 years to decide that I wanted to study biochemical sciences after having flirted with philosophy and physics. My first contact with John Edsall was probably in the spring of 1944 when I began taking a biochemistry course, and John was the head of the biochemical sciences tutorial program. Of course, this was in the middle of World War II, and I had already enlisted in the Navy V12 Program and remained at Harvard in that program. Like many others, however, I left college in mid-1944 to undertake a more active form of military duty and remained away from Harvard until the beginning of the Spring Term of 1946. At that time, I returned to complete undergraduate training and do an undergraduate thesis in biochemical sciences. John Edsall agreed to supervise my thesis, and this led to my introduction to Raman and infrared spectroscopy with some focus on the amino acids that John had been studying for many years.

I have been given the virtually impossible job of summarizing molecular biology in thirty minutes. Thus my colleagues should excuse me for Simplifying the work excessively…

Developments are now taking place in our study of the molecular basis of genetics which suggest that within the next decade or two we will have fairly complete information about the molecular structure and, more specifically, the molecular sequence information in the human genome. This is a significant development in science and one which will profoundly broaden and deepen our understanding of biological problems in general, and of Homo sapiens, ourselves. This development is the outgrowth of an astonishingly rapid rate in the growth of molecular biology, i.e., molecular analysis applied to biological phenomena. The central area in this effort has been the understanding that the nucleic acids provide the molecular basis of inheritance, and that DNA contains the genes which are inherited from parents and passed on to progeny. DNA was virtually unknown to the general public three or four decades ago., but it has now been incorporated into the vocabulary of the educated public.

The following sections are included:

• Education
• Positions
• Honors
• Membership on Editorial Boards
• Membership on Committees
• Society Memberships
• Publications
• Books
• SCIENTIFIC PUBLICATIONS OF ALEXANDER RICH

The following sections are included:

• Captain Alex
• Alex Rich
• People, Parties, and Zigzag Stitching with Alex
• Papa Alex
• My Uncle Alex
• Alex and Jane; Early Days Together
• What If We Had Been Caught? A Memoir of My Life with Alex
• To Z or Not to Z, That Was the Question
• Master and Apprentice
• A Very Good Friend of Half a Century: Alexander Rich
• Alex and the Iron Curtain
• My Years with Alex
• I Wish I’d Listened to Alex More Often
• Alexander Rich’s 90th Birthday Symposium
• So Many Ideas, So Little Time
• Making Discovery Seem Easy, or At Least Possible
• An Undergraduate in Wonderland: A Formative and Nurturing Experience at MIT
• Lessons from the Rich Lab: Focus on the Important Projects
• Alexander Rich, the Importance of RNA and the Development of Nucleic Acid Hybridization
• Lessons Learned in Alex’s lab
• Alex Rich’s Lab in Early 1960s
• Alex Rich: Friend, Teacher, Mentor and Role Model
• My Walks with Alex Rich
• Alex in Wonderland II — Ideas
• What Would Alex Do?
• Enthusiasm Substituting for Experience and Energy for Insight
• My Rich Time with Alex
• Night Lessons in Applied Joyce: From Poincaré to Alex Rich
• The Night Alex Stole My Fork
• My Years of Friendship with Alex
• Blue Sky Ideas (Ode to Alex Rich)
• Five Years with Alex Rich (1972–1977)
• Alex Rich
• Alex as Career Councilor
• Adventures with Z-DNA
• Impact of Alex on My Life
• Alex Rich: An Appreciation
• True and Genuine Story of the Life and Actions of Alexander Rich: Not Made Up of Fiction and Fable
• A Rich Lab Experience for an Undergraduate
• The Great Crystallization Caper
• The Alex Rich Lab, 1962: Reminiscences of an Antioch College Co-op
• A Tribute to Alex a Great Sailor
• Cover of Newsweek
• Science and the Kitchen
• Science, Art and Many Friends: My Time with Alex Rich
• Alex Lab: A Kaleidoscope of People, RNA- and DNA Molecules
• Sage Advice from Alex Rich

This chapter contains a photo collection depicting Alexander Rich’s life.

The following section is included:

• Corpus Acknowledgments