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To find conditions for obtaining diffraction-quality crystals of a hammerhead RNA rapidly and reproducibly, we employed a "double screening" procedure in which we screened six different RNA synthetic constructs against 48 crystallization conditions using a newly devised sparse matrix. We obtained crystals immediately and diffraction-quality crystals of the sixth RNA construct within six months of initiating the screening of additional RNA sequences. The best crystals diffract to 2.9 A resolution when flash-cooled at synchrotron X-ray sources. Solid-support chemical synthesis combined with sparse matrix screening should allow rapid production of diffraction-quality crystals of a variety of small RNAs, reducing the time commitment for initiating such crystallography projects from several years to several months. The synthetic approach also makes introduction of modified bases to prevent self-cleavage and to generate isomorphous heavy-atom derivative crystals a rapid and straightforward process.

We have solved the crystal structure of an all-RNA hammerhead ribozyme having a single 2'-O-methyl cytosine incorporated at the active site to prevent cleavage. The conditions used differ from those in another recent solution in four significant ways: first, it is an all-RNA ribozyme rather than a DNA-RNA hybrid; second, the connectivity of the ribozyme backbone strands is different; third, the crystals were grown in the presence of a much lower concentration of salt; and fourth, the crystal packing scheme is very different. Nevertheless, the three-dimensional structure of the all-RNA hammerhead ribozyme is similar to the previous structure. Five potential Mg(II)-binding sites are identified, including one positioned near the ribozyme catalytic pocket. Upon this basis, as well as upon comparisons with the metal-binding sites in the structurally homologous uridine turn of tRNAPhe, we propose a mechanism for RNA catalytic cleavage.

Australian horticulture relies heavily on the introduced managed honey bee, Apis mellifera Linnaeus 1758 (Hymenoptera: Apidae), to pollinate crops. Given the risks associated with reliance upon a single species, it would be prudent to identify other taxa that could be managed to provide crop pollination services. We reviewed the literature relating to the distribution, efficiency and management potential of a number of flies (Diptera) known to visit pollinator-dependent crops in Australia and worldwide. Applying this information, we identified the taxa most suitable to play a greater role as managed pollinators in Australian crops. Of the taxa reviewed, flower visitation by representatives from the dipteran families Calliphoridae, Rhiniidae and Syrphidae was frequently reported in the literature. While data available are limited, there was clear evidence of pollination by these flies in a range of crops. A review of fly morphology, foraging behaviour and physiology revealed considerable potential for their development as managed pollinators, either alone or to augment honey bee services. Considering existing pollination evidence, along with the distribution, morphology, behaviour and life history traits of introduced and endemic species, 11 calliphorid, two rhiniid and seven syrphid species were identified as candidates with high potential for use in Australian managed pollination services. Research directions for the comprehensive assessment of the pollination abilities of the identified taxa to facilitate their development as a pollination service are described. This triage approach to identifying species with high potential to become significant managed pollinators at local or regional levels is clearly widely applicable to other countries and taxa.

Aaron Klug's group was one of the first to use a combination of X-ray diffraction and electron microscopy to study the structures of macromolecules. He helped to provide the intellectual framework for understanding the self-assembly of regular viruses and developed methods for analyzing their three-dimensional structures from electron microscope images, as well as the structures of helical polymers. He and his coworkers established the basic features of chromatin organization, including the structure of the repeating units (nucleosomes) and how they are stacked together. He studied a variety of molecules that interact with DNA or RNA, including disks of tobacco mosaic virus protein, a tRNA and a ribozyme, and also discovered the zinc-finger motif in nucleic acid-binding proteins. Thus, he has played a major part in developing the ideas and techniques that established structural molecular biology as an exciting new science during the second half of the twentieth century.

The WW domains are small proteins that contain a three-stranded, antiparallel beta-sheet. The 40-residue murine FBP28 WW domain rapidly formed twirling ribbon-like fibrils at physiological temperature and pH, with morphology typical of amyloid fibrils. These ribbons were unusually wide and well ordered, making them highly suitable for structural studies. Their x-ray and electron-diffraction patterns displayed the characteristic amyloid fiber 0.47-nm reflection of the cross-beta diffraction signature. Both conventional and electron cryomicroscopy showed clearly that the ribbons were composed of many 2.5-nm-wide subfilaments that ran parallel to the long axis of the fiber. There was a region of lower density along the center of each filament. Lateral association of these filaments generated twisted, often interlinked, sheets up to 40 nm wide and many microns in length. The pitch of the helix varied from 60 to 320 nm, depending on the width of the ribbon. The wild-type FBP28 fibers were formed under conditions in which multiexponential folding kinetics is observed in other studies and which was attributed to a change in the mechanism of folding. It is more likely that those phases result from initial events in the off-pathway aggregation observed here.

Each sensory organ of the Drosophila peripheral nervous system is derived from a single sensory organ precursor cell (SOP). These originate in territories defined by expression of the proneural genes of the Achaete-Scute complex (AS-C). Formation of ectopic sensilla outside these regions is prevented by transcriptional repression of proneural genes. We demonstrate that the BTB/POZ-domain transcriptional repressor Tramtrack (Ttk) co-operates in this repression. Ttk is expressed ubiquitously, except in proneural clusters and SOPs. Ttk over-expression represses proneural genes and sensilla formation. Loss of Ttk enhances bristle-promoting mutants. Using neural repression as an assay, we dissected functional domains of Ttk, confirming the importance of the bric-à-brac-tramtrack-broad complex (BTB) motif. We show that the Ttk BTB domain is a protein-protein interaction motif mediating tetramer formation., (Copyright 2002 Elsevier Science Ireland Ltd.)

A study of papers on amyloid fibers suggested to us that cylindrical beta-sheets are the only structures consistent with some of the x-ray and electron microscope data. We then found that our own 7-year-old and hitherto enigmatic x-ray diagram of poly-L-glutamine fits a cylindrical sheet of 31 A diameter made of beta-strands with 20 residues per helical turn. Successive turns are linked by hydrogen bonds between both the main chain and side chain amides, and side chains point alternately into and out of the cylinder. Fibers of the exon-1 peptide of huntingtin and of the glutamine- and asparagine-rich region of the yeast prion Sup35 give the same underlying x-ray diagrams, which show that they have the same structure. Electron micrographs show that the 100-A-thick fibers of the Sup35 peptide are ropes made of three protofibrils a little over 30 A thick. They have a measured mass of 1,450 Da/A, compared with 1,426 Da/A for a calculated mass of three protofibrils each with 20 residues per helical turn wound around each other with a helical pitch of 510 A. Published x-ray diagrams and electron micrographs show that fibers of synuclein, the protein that forms the aggregates of Parkinson disease, consist of single cylindrical beta-sheets. Fibers of Alzheimer A beta fragments and variants are probably made of either two or three concentric cylindrical beta-sheets. Our structure of poly-L-glutamine fibers may explain why, in all but one of the neurodegenerative diseases resulting from extension of glutamine repeats, disease occurs when the number of repeats exceeds 37-40. A single helical turn with 20 residues would be unstable, because there is nothing to hold it in place, but two turns with 40 residues are stabilized by the hydrogen bonds between their amides and can act as nuclei for further helical growth. The A beta peptide of Alzheimer's disease contains 42 residues, the best number for nucleating further growth. All these structures are very stable; the best hope for therapies lies in preventing their growth.

The type 1 human immunodeficiency virus (HIV-1) contains a conical capsid comprising approximately 1,500 CA protein subunits, which organizes the viral RNA genome for uncoating and replication in a new host cell. In vitro, CA spontaneously assembles into helical tubes and cones that resemble authentic viral capsids. Here we describe electron cryo-microscopy and image reconstructions of CA tubes from six different helical families. In spite of their polymorphism, all tubes are composed of hexameric rings of CA arranged with approximate local p6 lattice symmetry. Crystal structures of the two CA domains were 'docked' into the reconstructed density, which showed that the amino-terminal domains form the hexameric rings and the carboxy-terminal dimerization domains connect each ring to six neighbours. We propose a molecular model for the HIV-1 capsid that follows the principles of a fullerene cone, in which the body of the cone is composed of curved hexagonal arrays of CA rings and the ends are closed by inclusion of 12 pentagonal 'defects'.

Aberrant protein processing with tissue deposition is associated with many common neurodegenerative disorders; however, the complex interplay of genetic and environmental factors has made it difficult to decipher the sequence of events linking protein aggregation with clinical disease. Substantial progress has been made toward understanding the pathophysiology of prototypical conformational diseases and protein polymerization in the superfamily of serine proteinase inhibitors (serpins). Here we describe a new disease, familial encephalopathy with neuroserpin inclusion bodies, characterized clinically as an autosomal dominantly inherited dementia, histologically by unique neuronal inclusion bodies and biochemically by polymers of the neuron-specific serpin, neuroserpin. We report the cosegregation of point mutations in the neuroserpin gene (PI12) with the disease in two families. The significance of one mutation, S49P, is evident from its homology to a previously described serpin mutations, whereas that of the other, S52R, is predicted by modelling of the serpin template. Our findings provide a molecular mechanism for a familial dementia and imply that inhibitors of protein polymerization may be effective therapies for this disorder and perhaps for other more common neurodegenerative diseases.

Importin-alpha is a cytosolic receptor that recognizes classical Nuclear Localization Signals (NLSs) and mediates import into the nucleus. We have used a number of methods to investigate the aggregation state of Xenopus importin-alpha both as a recombinant, purified protein and in cytosolic extracts. We have found that recombinant importin-alpha aggregates at a protein concentration similar to that estimated to be present in the Xenopus cytoplasm, and that the importin-alpha aggregation is relieved by NLS peptide binding, with the importin-alpha then binding the NLS as a monomer. We have also found that in HeLa cytosolic extracts, importin-alpha is present in an aggregated form. Similarly to the purified importin-alpha aggregation, NLS peptides relieve the aggregation of importin-alpha in the cytosol. These observations indicate that aggregation of importin-alpha in the cytosol may be an intrinsic property of the import receptor and may be functionally related to NLS binding.Our results suggest a novel mechanism for NLS recognition, whereby NLSs mediate disassembly of importin-alpha aggregates in the cytosol., (Copyright 1999 Academic Press.)

The genome of the human immunodeficiency virus (HIV) is packaged within an unusual conical core particle located at the center of the infectious virion. The core is composed of a complex of the NC (nucleocapsid) protein and genomic RNA, surrounded by a shell of the CA (capsid) protein. A method was developed for assembling cones in vitro using pure recombinant HIV-1 CA-NC fusion proteins and RNA templates. These synthetic cores are capped at both ends and appear similar in size and morphology to authentic viral cores. It is proposed that both viral and synthetic cores are organized on conical hexagonal lattices, which by Euler's theorem requires quantization of their cone angles. Electron microscopic analyses revealed that the cone angles of synthetic cores were indeed quantized into the five allowed angles. The viral core and most synthetic cones exhibited cone angles of approximately 19 degrees (the narrowest of the allowed angles). These observations suggest that the core of HIV is organized on the principles of a fullerene cone, in analogy to structures recently observed for elemental carbon.

Possible stereochemical determinants of the orientation of TBP on the TATA box are discussed using the crystal coordinates of TBP-TATA complexes, which have been determined by other groups. The C-terminal half of the TBP beta-sheet interacts with the TATA site of the DNA, and the N-terminal half with the A-rich site, so that the two sites with distinct curvatures produce a unique fit. Although chemical contacts take place between one side of the beta-sheet and the DNA minor groove, the interaction seems to be facilitated indirectly by the characteristics of the other side of the beta-sheet and the DNA major groove. Thus, Ala71, Leu162 and Pro190 differentiate the curvature of the beta-sheet in the N- and C-halves. The methyl positions in the DNA major groove modulate the bendability of the two DNA sites by using differences in the rolling capacity of TA and AT compared with PyT, and in the shifting capacity of AT compared with TT. The deformations of the first steps (TA and PyT) in the two sites are the largest and thus are important for the overall bending of the DNA. The differences between the two DNA sites are greatest at the second steps (AT and TT) and so these are important for determining the orientation of TBP.

Sequence-specific conformational differences between dinucleotide steps are characterised using published crystal coordinates with special attention to steric hindrance of the methyl group of a T base to the neighbouring base, and, more importantly, to the sugar-phosphate backbone. The TT step is inflexible and B-like, as it has two methyl groups which interlock with each other and with the sugar-phosphate backbones. AT slides, or overtwists, so that the methyl groups move away from the backbones, both lead the step towards the A-conformation. TA is most flexible as it does not have such restriction. These characteristics are observed with other pyrimidine-pyrimidine, pyrimidine-purine, purine-pyrimidine steps, respectively, but to less extent, depending on the number of non-A:T basepairs in the steps.

The Z (Glu342-->Lys) and Siiyama (Ser53-->Phe) deficiency variants of alpha 1-antitrypsin result in the retention of protein in the endoplasmic reticulum of the hepatocyte by loop-sheet polymerization in which the reactive center loop of one molecule is inserted into a beta-pleated sheet of a second. We show here that antitrypsin Mmalton (Phe52-deleted), which is associated with the same liver inclusions, is also retained at an endoglycosidase H-sensitive stage of processing in the Xenopus oocyte and spontaneously forms polymers in vivo. These polymers, obtained from the plasma of an Mmalton/QO (null) bolton heterozygote, were much shorter than other antitrypsin polymers and contained a reactive center loop-cleaved species. Monomeric mutant antitrypsin was also isolated from the plasma. The monomeric component had a normal unfolding transition on transverse urea gradient gel electrophoresis and formed polymers in vitro more readily than M, but less readily than Z, antitrypsin. The A beta-sheet accommodated a reactive center loop peptide much less readily than Z antitrypsin, which in turn was less receptive than native M antitrypsin. The nonreceptive conformation of the A sheet in antitrypsin Mmalton had little effect on kinetic parameters, the formation of SDS-stable complexes, the S to R transition, and the formation of the latent conformation. Comparison of the results with similar findings of short chain polymers associated with the antithrombin variant Rouen VI (Bruce, D., Perry, D., Borg, J.-Y., Carrell, R. W., and Wardell, M. R. (1994) J. Clin. Invest. 94, 2265-2274) suggests that polymerization is more complicated than the mechanism proposed earlier. The Z, Siiyama, and Mmalton mutations favor a conformational change in the antitrypsin molecule to an intermediate between the native and latent forms. This would involve a partial overinsertion of the reactive loop into the A sheet with displacement of strand 1C and consequent loop-C sheet polymerization.

We have solved the crystal structure of an all-RNA hammerhead ribozyme by isomorphous replacement which contains a single 2'-O-methyl-cytosine at the active site to prevent cleavage. We describe the hammerhead RNA structure from a point of view which shows how the structural elements are disposed to bring the active site nucleotide into the catalytic pocket of the ribozyme. Five potential Mg(II) binding sites can be identified in the hammerhead RNA electron density maps. Of these, one is a newly identified metal site positioned near the ribozyme catalytic pocket, and another site corresponds to a Mn(II) site identified in the previous hammerhead RNA structure. We propose a mechanism for RNA catalytic cleavage on the basis of this new metal-binding site, as well as upon comparisons between the catalytic pocket of the hammerhead RNA and the metal-binding sites in the structurally homologous uridine turn of the anticodon loop in tRNAPhe.

Four inherited neurodegenerative diseases are linked to abnormally expanded repeats of glutamine residues in the affected proteins. Molecular modeling followed by optical, electron, and x-ray diffraction studies of a synthetic poly(L-glutamine) shows that it forms beta-sheets strongly held together by hydrogen bonds. Glutamine repeats may function as polar zippers, for example, by joining specific transcription factors bound to separate DNA segments. Their extension may cause disease either by increased, nonspecific affinity between such factors or by gradual precipitation of the affected proteins in neurons.

The Cys2-His2 zinc-finger is the most widely occurring DNA-binding motif. The first structure of a zinc-finger/DNA complex revealed a fairly simple mechanism for DNA recognition suggesting that the zinc-finger might represent a candidate template for designing proteins to recognize DNA. Residues at three key positions in an alpha-helical 'reading head' play a dominant role in base-recognition and have been targets for mutagenesis experiments aimed at deriving a recognition code. Here we report the structure of a two zinc-finger DNA-binding domain from the protein Tramtrack complexed with DNA. The amino-terminal zinc-finger and its interaction with DNA illustrate several novel features. These include the use of a serine residue, which is semi-conserved and located outside the three key positions, to make a base contact. Its role in base-recognition correlates with a large, local, protein-induced deformation of the DNA helix at a flexible A-T-A sequence and may give insight into previous mutagenesis experiments. It is apparent from this structure that zinc-finger/DNA recognition is more complex than was originally perceived.

Monoclonal antibodies against the 110-kD component of the yeast spindle pole body (SPB) were used to clone the corresponding gene SPC110. SPC110 is identical to NUF1 (Mirzayan, C., C. S. Copeland, and M. Synder. 1992. J. Cell Biol. 116:1319-1332). SPC110/NUF1 has an MluI cell cycle box consensus sequence in its putative promoter region, and we found that the transcript was cell cycle regulated in a similar way to other MluI-regulated transcripts. Spc110p/Nuflp has a long central region with a predicted coiled-coil structure. We expressed this region in Escherichia coli and showed by rotary shadowing that rods of the predicted length were present. The 110-kD component is localized in the SPB to the gap between the central plaque and the sealed ends of the nuclear microtubules near the inner plaque (Rout, M., and J. V. Kilmartin. 1990. J. Cell Biol. 111:1913-1927). We found that rodlike structures bridge this gap. When truncations of SPC110 with deletions in the coiled-coil region of the protein replaced the wild-type gene, the gap between the central plaque and the ends of the microtubules decreased in proportion to the size of the deletion. This suggests that Spc110p connects these two parts of the SPB together and that the coiled-coil domain acts as a spacer element.

Background: The steroid/nuclear hormone receptors are a large family of conserved ligand-activated transcription factors that regulate gene expression through binding to response elements upstream of their target genes. Most members of this family bind to DNA as homodimers or heterodimers and recognize the sequence, spacing and orientation of the two half-sites of their response elements. The recognition and discrimination of the sequence and arrangements of these half-sites are mediated primarily by a highly conserved DNA-binding domain., Results: Here we describe the DNA-binding properties of the isolated DNA-binding domain of the oestrogen receptor, the ERDBD, and its refined NMR structure. This domain is monomeric in solution, but two molecules bind cooperatively to specific DNA sequences; this cooperativity determines the arrangement of half-sites that is recognized by the ERDBD. The 10 carboxy-terminal residues and a region of 15 residues within the domain are disordered in the solution structure, yet are important for DNA binding., Conclusion: The cooperative nature of ERDBD binding to DNA is important. The previously-determined X-ray structure of the ERDBD dimer bound to DNA shows that the 15 internal residues disordered in solution make contact both with DNA and with the corresponding region of the other monomer. These results suggest that these residues become ordered during the process of binding to DNA, forming the dimer interface and thus contributing to the cooperative interaction between monomers.

The nuclear hormone receptors are a superfamily of ligand-activated DNA-binding transcription factors. We have determined the crystal structure (at 2.4 A) of the fully specific complex between the DNA-binding domain from the estrogen receptor and DNA. The protein binds as a symmetrical dimer to its palindromic binding site consisting of two 6 bp consensus half sites with three intervening base pairs. This structure reveals how the protein recognizes its own half site sequence rather than that of the related glucocorticoid receptor, which differs by only two base pairs. Since all nuclear hormone receptors recognize one or the other of these two consensus half site sequences, this recognition mechanism applies generally to the whole receptor family.

Antitrypsin Siiyama is a rare example of the deficiency variants of antitrypsin that accumulate in the endoplasmic reticulum of the hepatocyte. The common example is Z antitrypsin, which has a mutation (Glu342-->Lys) at the junction of the head of the fifth strand of the A sheet and the base of the reactive center loop. It was previously shown that Z antitrypsin spontaneously polymerizes due to the insertion of the reactive center loop of one molecule into the A sheet of a second. The mutation in antitrypsin Siiyama (Ser53-->Phe) affects a residue that provides a ridge for the sliding movement that opens the A sheet, and it had been predicted that this would result in the same type of loop-sheet polymerization observed with the Z variant. We confirm this here and show that virtually all the plasma antitrypsin in a homozygote for the Siiyama variant was polymerized due to non-covalent bonding with a loss of accessibility of the reactive center loop. The common basis of the polymerization of Z and Siiyama antitrypsin is supported by identical findings on electron microscopy. Taken together these results confirm that loop-sheet polymerization is a general mechanism and as such is likely to be responsible for the intracellular inclusions associated with liver pathology.

The structure of GH5, the globular domain of the linker histone H5, has been solved to 2.5 A resolution by multiwavelength anomalous diffraction on crystals of the selenomethionyl protein. The structure shows a striking similarity to the DNA-binding domain of the catabolite gene activator protein CAP, thereby providing a possible model for the binding of GH5 to DNA.

The three-dimensional structure has been determined of a complex of the dye Congo Red, a specific stain for amyloid deposits, bound to the amyloid protein insulin. One dye molecule intercalates between two globular insulin molecules at an interface formed by a pair of anti-parallel beta-strands. This result, together with analysis of the primary sequences of other amyloidogenic proteins and peptides suggests that this mode of dye-binding to amyloid could be general. Moreover, the structure of this dye-binding interface between protein molecules provides an insight into the polymerization of amyloidogenic proteins into amyloid fibres. Thus the detailed characterization, at a resolution of 2.5 A, of the dye binding site in insulin could form a basis for the design of agents targeted against a variety of amyloid deposits.

We describe here the purification and partial characterization of a 200 kDa keratan sulphate proteoglycan found in the pericellular matrix of human embryonal carcinoma cells. Previously we have shown that this molecule is recognized by a monoclonal antibody (GCTM-2). The antigen was isolated using ion-exchange chromatography and gel filtration, purification being monitored by e.l.i.s.a. using GCTM-2. Metabolic labelling of GCT 27 C-4 embryonal carcinoma cells with sodium [35S]sulphate resulted in the incorporation of [35S]sulphate into the purified molecule. Throughout the purification procedure, the peaks of 35S radioactivity were coincident with the peaks of immunoreactivity, and this label was released both by digestion with keratanase and chondroitinase, confirming the proteoglycan nature of the antigen. The intact molecule ran as a single broad band of 200 kDa, which has been identified by silver staining and immunoblotting following gel electrophoresis. Amino acid analysis of the purified antigen indicated a high content of serine, glycine and aspartic acid/asparagine residues. Visualization by rotary-shadowing electron microscopy suggests that the purified material forms large aggregates, even under denaturing conditions. Deglycosylation of this preparation with trifluoromethanesulphonic acid yielded a major band of 55 kDa and a minor band of 48 kDa. The biochemical nature of the molecule described here, along with tissue distribution studies using GCTM-2, indicates that the antigen is not related to previously described keratan sulphate proteoglycans.

Most northern Europeans have only the normal M form of the plasma protease inhibitor alpha 1-antitrypsin, but some 4% are heterozygotes for the Z deficiency variant. For reasons that have not been well-understood, the Z mutation results in a blockage in the final stage of processing of antitrypsin in the liver such that in the Z homozygote only 15% of the protein is secreted into the plasma. The 85% of the alpha 1-antitrypsin that is not secreted accumulates in the endoplasmic reticulum of the hepatocyte; much of it is degraded but the remainder aggregates to form insoluble intracellular inclusions. These inclusions are associated with hepatocellular damage, and 10% of newborn Z homozygotes develop liver disease which often leads to a fatal childhood cirrhosis. Here we demonstrate the molecular pathology underlying this accumulation and describe how the Z mutation in antitrypsin results in a unique molecular interaction between the reactive centre loop of one molecule and the gap in the A-sheet of another. This loop-sheet polymerization of Z antitrypsin occurs spontaneously at 37 degrees C and is completely blocked by the insertion of a specific peptide into the A-sheet of the antitrypsin molecule. Z antitrypsin polymerized in vitro has identical properties and ultrastructure to the inclusions isolated from hepatocytes of a Z homozygote. The concentration and temperature dependence of this loop-sheet polymerization has implications for the management of the liver disease of the newborn Z homozygote.

We have employed the rapid-freeze technique to prepare specimens for electron microscopy of a coat protein solution of tobacco mosaic virus at equilibrium at pH 7.0 and 6.8, ionic strength 0.1 M and 20 degrees C. The former are the conditions for the most rapid assembly of the virus from its isolated protein and RNA. At both pH values, the equilibrium mixture contains approximately 80% of a "20 S" aggregate and 20% of a "4 S" aggregate (the so-called A-protein). The specimens were prepared either totally unstained or positively stained with methyl mercury nitrate, which binds to an amino acid residue (Cys27) internally located within the subunit, which we show not to affect the virus assembly. The images in the electron microscope are compatible only with the major structure for the "20 S" aggregate at pH 7.0 containing two rings of subunits and these aggregates display the same binding contacts as those seen between the aggregate that forms the asymmetric unit in the crystal, which has been shown by X-ray crystallography to be a disk containing two rings, each of 17 subunits, oriented in the same direction. In contrast, the images from specimens prepared at pH 6.8 show the major structure to be a proto-helix at this slightly lower pH, demonstrating that the technique of cryo-electron microscopy is capable of distinguishing between these aggregates of tobacco mosaic virus coat protein. The main structure in solution at pH 7.0 must therefore be very similar to that in the crystal, although slight differences could occur and there are probably other, minor, components in a mixture of species sedimenting around 20 S under these conditions. The equilibrium between aggregates is extremely sensitive to conditions, with a drop of 0.2 pH unit tipping the disk to proto-helix ratio from approximately 10:1 at pH 7.0 to 1:10 at pH 6.8. This direct determination of the structure of the "20 S" aggregate in solution, under conditions for virus assembly, contradicts some recent speculation that it must be helical, and establishes that, at pH 7.0, it is in fact predominantly a two-layer disk as it had been modelled before.

We have grown single crystals of long DNA molecules, 54 and 65 base pairs in length which encompass the binding site of the Xenopus protein transcription factor IIIA (TFIIIA). These molecules are considerably longer than those previously crystallised. X-ray diffraction shows that the crystals are ordered to a resolution of 9 A. The DNA molecules are arranged side to side in layers which are orientated at 120 degrees with respect to adjacent layers. The phosphate backbones of molecules in adjacent layers are interdigitated in both the major and minor grooves. The intensity distribution indicates that the average structure is B-like, although CD spectra in solution are more consistent with the structure, intermediate between A- and B-, found in crystals of a nonamer fragment of the binding site. The B-character in our crystals may therefore be impressed by the tight packing between layers.

In view of the likely role of H1-H1 interactions in the stabilization of chromatin higher order structure, we have asked whether interactions can occur between the globular domains of the histone molecules. We have studied the properties of the isolated globular domains of H1 and the variant H5 (GH1 and GH5) and we have shown (by sedimentation analysis, electron microscopy, chemical cross-linking and nucleoprotein gel electrophoresis) that although GH1 shows no, and GH5 little if any, tendency to self-associate in dilute solution, they bind highly cooperatively to DNA. The resulting complexes appear to contain essentially continuous arrays of globular domains bridging 'tramlines' of DNA, similar to those formed with intact H1, presumably reflecting the ability of the globular domain to bind more than one DNA segment, as it is likely to do in the nucleosome. Additional (thicker) complexes are also formed with GH5, probably resulting from association of the primary complexes, possibly with binding of additional GH5. The highly cooperative nature of the binding, in close apposition, of GH1 and GH5 to DNA is fully compatible with the involvement of interactions between the globular domains of H1 and its variants in chromatin folding.

Ovalbumin, the major protein in avian egg-white, is a non-inhibitory member of the serine protease inhibitor (serpin) superfamily. The crystal structure of uncleaved, hen ovalbumin was solved by the molecular replacement method using the structure of plakalbumin, a proteolytically cleaved form of ovalbumin, as a starting model. The final refined model, including four ovalbumin molecules, 678 water molecules and a single metal ion, has a crystallographic R-factor of 17.4% for all reflections between 6.0 and 1.95 A resolution. The root-mean-square deviation from ideal values in bond lengths is 0.02 A and in bond angles is 2.9 degrees. This is the first crystal structure of a member of the serpin family in an uncleaved form. Surprisingly, the peptide that is homologous to the reactive centre of inhibitory serpins adopts an alpha-helical conformation. The implications for the mechanism of inhibition of the inhibitory members of the family is discussed.

The human immunodeficiency virus type 1 rev protein binds with high affinity (Kd less than 1-3 nM) to a purine-rich "bubble" containing bulged GG and GUA residues on either side of a double-helical RNA stem-loop located toward the 5' end of rev-response element RNA. High-affinity rev binding is maintained when the bubble is placed in heterologous stem-loop structures, but rev binding is reduced when either the bulged residues or flanking base pairs in the stem are altered. Rev binding to the purine-rich bubble nucleates assembly of long filamentous ribonucleoprotein structures containing polymers of rev bound to flanking RNA sequences. It is proposed that rev regulates human immunodeficiency virus RNA expression by selectively packaging viral transcripts carrying the rev-response element sequence into rod-like nucleoprotein complexes that block splicing of the packaged mRNAs.

The sea urchin sperm-specific histones H1 and H2B are multiply phosphorylated in spermatids, dephosphorylated in the final stages of spermatogenesis to give mature sperm, and rephosphorylated upon fertilization. Phosphorylation in spermatids, and probably at fertilization, occurs at repeated -Ser-Pro-X-Basic-motifs in the distinctive N-terminal basic domains of both histones and at the end of the much longer C-terminal domain of H1. Here we identify the consequences of multiple phosphorylation through comparison of some physical and biochemical properties of spermatid (phosphorylated) and sperm (dephosphorylated) chromatin and histones. Study of the DNA binding properties of the intact histones and isolated basic domains suggests that phosphorylation at three dispersed sites in the C-terminal tail of H1 has little effect on its overall DNA binding affinity, whereas, strikingly, binding of the N-terminal domains of H2B and H1 is abolished by phosphorylation at four or six tandemly repeated sites respectively. Together with the relative timing of events in vivo, this suggests that phosphorylation/dephosphorylation of the N-terminal (and distal end of the C-terminal) tail of H1, and/or the N-terminal tail of H2B, effectively controls intermolecular interactions between adjacent chromatin filaments, and hence chromatin packing in the sperm nucleus.

HIV-1 tat protein binds specifically to HIV-1 TAR RNA. A Scatchard analysis of tat binding has shown that the purified protein forms a one-to-one complex with HIV-1 TAR RNA with a dissociation constant of Kd = 12 nM. Tat binding in vitro is dependent upon the presence of 3 non-base paired U residues which produce a 'bulge' in the TAR RNA stem-loop structure. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6 to 8-fold lower affinity than wild-type TAR. By contrast, mutations that alter the sequence of the 6 nucleotide-long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson-Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs encoding TAR sequences known to bind tat weakly, are not stimulated efficiently by tat in vivo. HIV-1 regulator of virion expression (rev) protein binds specifically to RNA transcripts containing the 223 nucleotide-long RRE sequence with an apparent dissociation constant of 1-3 nM. The minimum binding site for rev is a 'bubble' containing 2 G residues on one side and the sequence AGU on the other. Rev is able to bind efficiently to this restricted site in the context of the RRE sequence as well as in the context of a stable RNA duplex with a sequence unrelated to that found in the RRE.(ABSTRACT TRUNCATED AT 250 WORDS)

The serpins are a widely distributed family of proteins with diverse functions; they include the key serine protease inhibitors of human plasma as well as noninhibitory homologues such as hormone-binding globulins, angiotensinogen and egg-white ovalbumin. Sequence alignment based on the crystal structure. On the cleaved form of the archetypal serpin, alpha 1-antitrypsin, indicates that the serpins share a common highly ordered structure. On cleavage of the reactive centre peptide bond, they characteristically undergo a remarkable conformational change, the newly generated C terminus moving some 70 A to the opposite pole of the molecule. The structure of this post-cleavage form is known, but the conformation of the intact serpins and in particular that of their reactive centre is not. Wright et al.'s structure of plakalbumin (ovalbumin cleaved by subtilisin) has provided evidence for the conformational change that results from cleavage. We have now determined the structure of native ovalbumin to 1.95 A resolution and have found that the intact peptide loop forming the analogue to the reactive centre of the inhibitory serpins takes the unexpected form of a protruding, isolated helix. This model of the intact structures of the serpins suggests how they may interact with their target proteases.

Defined lengths of chromatin were prepared by brief digestion with micrococcal nuclease and fractionation in a sucrose gradient. A length containing a given number of 200 base pair repeating units appeared as the same number of 100 A beads in the electron microscope. The distance between beads within a length was small, usually less than about 20 A.

Image reconstruction to 22 A resolution of the histone octamer (H3)2(H4)2(H2A)2(H2B)2 shows it to have a 2-fold axis of symmetry, and the overall shape of the left-handed helical spool on which to wind about two turns of a flat superhelix of DNA in the nucleosome. From this structure and the results of various cross-linking studies, we have deduced the arrangement of the individual histones. We propose that the (H3)2(H4)2 tetramer forms a dislocated disk which defines the central turn of DNA, while the two H2A-H2B dimers lie one on each face, each associated with about one half a turn.

The pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was treated with chymotrypsin at pH 7 and 0 degrees C. Loss of the overall catalytic activity lagged behind the rapid cleavage of the lipoate acetyltransferase polypeptide chains, whose apparent Mr fell from 57 000 to 45 000 as judged by sodium dodecylsulphate/polyacrylamide gel electrophoresis. The inactive chymotrypsin-treated enzyme had lost the lipoic-acid-containing regions of the lipoate acetyltransferase chains, yet remained a highly assembled structure. Treatment of this chymotryptic core complex with trypsin at pH 7.0 and 0 degrees C caused a further shortening of the lipoate acetyltransferase polypeptide chains to an apparent Mr of 28 000 and was accompanied by disassembly of the complex. The lipoic-acid-containing regions are therefore likely to be physically exposed in the intact complex, protruding from the structural core formed by the lipoate acetyltransferase component between the subunits of the other component enzymes. Proton nuclear magnetic resonance spectroscopy demonstrated that the enzyme complex contains large regions of polypeptide chain with remarkable intramolecular mobility, most of which were retained after excision of the lipoic-acid-containing regions with chymotrypsin. It is likely that the highly mobile regions are in the lipoate acetyltransferase component and facilitate movement of the lipoic acid residues. Such polypeptide chain mobility provides the molecular basis of a novel system of active-site coupling in the 2-oxo acid dehydrogenase multienzyme complexes.

Clathrin triskelions form polyhedral cages with hexagonal and pentagonal faces when dialyzed against suitable assembly buffers. However, when the buffer is made 12% saturated in ammonium sulfate and the dialysis is performed at 4 degrees C, clathrin polymerizes into cubes. The cube is constructed from eight triskelions with one at each corner. The edge length of the cube is approximately 45 nm, equivalent to the length of the leg of a triskelion. Thus, each edge of the cube is composed of two antiparallel legs overlapping over their whole length. The interactions between the legs in the cube are a subset of those postulated to occur in cages. Indeed, the cube can be derived from a pentagonal dodecahedron by removing 12 of the 20 triskelions with only slight adjustment of the legs of the remaining triskelions. The cube forms regular arrays and appears to be a favorable species for crystallization of clathrin.

The higher-order structure of chromatin isolated from sea urchin sperm, which has a long nucleosomal DNA repeat length (approximately 240 bp), has been studied by electron microscopy and X-ray diffraction. Electron micrographs show that this chromatin forms 300 A filaments which are indistinguishable from those of chicken erythrocytes (approximately 212 bp repeat); X-ray diffraction patterns from partially oriented samples show that the edge-to-edge packing of nucleosomes in the direction of the 300 A filament axis, and the radial disposition of nucleosomes around it, are both similar to those of the chicken erythrocyte 300 A filament, which is described by the solenoid model. The invariance of the structure with increased linker DNA length is inconsistent with many other models proposed for the 300 A filament and, furthermore, means that the linker DNA must be bent. The low-angle X-ray scattering in the 300-400 A region both in vitro and in vivo differs from that of chicken erythrocyte chromatin. The nature of the difference suggests that 300 A filaments in sea urchin sperm in vivo are packed so tightly together that electron-density contrast between individual filaments is lost; this is consistent with electron micrographs of the chromatin in vitro.

The bases of yeast tRNA(Phe) which react with carbodiimide and methoxyamine have been determined and this information has been combined with chemical modification studies of other workers to produce a composite picture of base accessibility in this tRNA. The results are compared with the three-dimensional structure which we have recently determined. The bases which react chemically lie in exposed positions in the three-dimensional model and those which do not are either in the double helical stem regions or else are involved in maintaining the tertiary structure through pairing or stacking interactions.

Chromatin prepared by brief digestion of nuclei with micrococcal nuclease, and extracted in 0.2 mM EDTA, appears in the electron microscope as filaments of about 100 A diameter which coil loosely. In 0.2 mM Mg++ these "nucleofilaments" condense into a supercoil or solenoidal structure of pitch about 110 A corresponding to the diameter of a nucleofilament. It is proposed that the x-ray reflections at orders of 110 A observed in chromatin originate in the spacing between turns of the solenoid rather than that between nucleosomes along the nucleofilament. The solenoidal structure appears to need histone H1 for its stabilization. Under certain conditions, isolated nucleosomes can also aggregate into a similar structure. The solenoidal structure can be correlated with the "thread" of diameter about 300 A observed by other workers in nuclei.

A rapid and reproducible procedure for the resolution of 'native' and 'activated' forms of properdin (a component of the alternative activation pathway of complement), by gel filtration on the polyvinyl matrix Fractogel TSK HW-55(S), is reported. This fractionation permitted effective screening of samples for conditions that cause activation. Only 'native' properdin was detected in serum, even after activation of the alternative pathway by yeast cell walls. Transformation of 'native' into 'activated' properdin in vitro was produced by freeze-thawing of the protein, but not upon binding to and dissociation from the C3 convertase, C3bBb. Electron microscopy showed that only the 'native' population contained the discrete cyclic structures described previously by Smith, Pangburn, Vogel & Müller-Eberhard [(1984) J. Biol. Chem. 259, 4582-4588]. 'Activated' properdin, which was eluted from the gel-filtration column close to the breakthrough peak, was mainly composed of large amorphous aggregates. We therefore conclude that properdin 'activation' is not a physiological event that occurs in serum on complement activation, but is an artifact of isolation. Fractionation of properdin on Fractogel TSK HW-55(S) has, however, enabled detailed analysis of functional heterogeneity within the 'native' population.

The spontaneous self-assembly mechanism of tobacco mosaic virus (TMV) led us to predict that contiguous foreign RNA sequences of unlimited length would be fully encapsidated into ribonuclease-resistant, virus-like particles ("pseudovirions"), provided the cognate origin-of-assembly sequence from TMV RNA was present. We describe the construction of SP6-transcription plasmids and in vitro synthesis of recombinant RNAs bearing a correctly oriented, 3' terminal, origin-of-assembly from TMV RNA together with 5'-coding regions for chicken egg white prelysozyme, calf preprochymosin or Xenopus borealis rRNA. In the presence of a "disk" preparation of TMV coat protein, complete assembly of the prelysozyme coding sequence into ribonuclease-resistant, rod-shaped pseudovirions was directed specifically by the 3'-TMV RNA sequence. Helical particles of the predicted length were visible in the electron microscope and could be separated by sucrose gradient centrifugation. This in vitro RNA packaging system may offer a more efficient, protective route to introduce foreign mRNAs into whole plants or protoplasts by "pseudoinfection", particularly when studying the transient expression of mRNAs incapable of replicating or spreading from cell to cell. However, we have detected some constraints in the assembly elongation process, especially with highly structured transcripts such as rRNA. The significance of these observations for current views on the cooperative self-assembly mechanism of TMV is discussed.