Parenting, Effortful Control, and Adolescents' Externalizing Problem Behavior: Moderation by Dopaminergic Genes.

Research shows that genetics and effortful control play an important role in the link between parenting and problem behavior. However, little is known about how these factors act simultaneously. This article used a moderated mediation model to examine whether effortful control mediated the link between parenting and externalizing problem behavior, and whether dopaminergic genes (i.e., polygenic index score including DAT1, DRD2, DRD4, COMT) moderated this link. Two three-wave studies were conducted on community samples (adolescents: Study 1: N = 457; Mage = 15.74; Study 2: N = 221; Mage = 12.84). There was no mediation by effortful control, but a moderation by dopaminergic reactivity was observed. Despite inconsistent evidence, this article indicates that the development of externalizing problem behavior is subject to genetic characteristics and parenting.

Lateralised motor behaviour leads to increased unevenness in front feet and asymmetry in athletic performance in young mature Warmblood horses.

REASON FOR PERFORMING STUDY: Foot stance in grazing significantly influences hoof conformation and development from foal to yearling age. OBJECTIVES: To conduct a longitudinal study to establish if the relationship between motor laterality and uneven front feet persisted in 3-year-old horses at the time of studbook selection and to investigate if such laterality and unevenness might influence the horses' ability to perform symmetrically while trotting, cantering and free jumping. METHODS: Seventeen clinically sound but untrained (with only minimal experience of handling) and sound Warmblood horses that had participated in a previous study were assessed as per the protocol reported. Laterality was tested in a preference test (PT) and z-values were calculated for analysis purposes. Laterality and hoof unevenness were related to both relative limb length and relative head size, while the ability to perform symmetrically was tested in free trot-canter transitions and free jumping exercises. Differences in performance between horses with and without a limb preference in the PT and those with 'uneven' and 'even' feet were tested for differences in performance metrics using Students' t test, while linearity was tested using a regression analysis (P<0.05). RESULTS: Significant laterality was still present in 24% of the 3-year-old horses and the relationship between laterality and uneven feet pairs was stronger than at foal and yearling stages. Horses with significant motor laterality had almost 4 times more unevenness, a smaller head and longer limbs and the relationship between body conformation and laterality was still present. There was a strong linear relation between unevenness, laterality and a bias or side preference for trot-canter transitions. However, this relationship was not significant during the free jumping exercise. CONCLUSION: Motor laterality and uneven feet pairs were still present and significantly related in the 3-year-old horses and both variables were also strongly related to sidedness in trot-canter transitions. POTENTIAL RELEVANCE: Warmblood studbooks should include quantitative data on laterality at the time of studbook admission as part of the selection criteria.

Myriapod haemocyanin: the first three-dimensional reconstruction of Scolopendra subspinipes and preliminary structural analysis of S. viridicornis.

Haemocyanins (Hcs) are copper-containing, respiratory proteins that occur in the haemolymph of many arthropod species. Here, we report the presence of Hcs in the chilopode Myriapoda, demonstrating that these proteins are more widespread among the Arthropoda than previously thought. The analysis of transcriptome of S. subspinipes subpinipes reveals the presence of two distinct subunits of Hc, where the signal peptide is present, and six of prophenoloxidase (PPO), where the signal peptide is absent, in the 75 kDa range. Size exclusion chromatography profiles indicate different quaternary organization for Hc of both species, which was corroborated by TEM analysis: S. viridicornis Hc is a 6 × 6-mer and S. subspinipes Hc is a 3 × 6-mer, which resembles the half-structure of the 6 × 6-mer but also includes the presence of phenoloxidases, since the 1 × 6-mer quaternary organization is commonly associated with hexamers of PPO. Studies with Chelicerata showed that PPO activity are exclusively associated with the Hcs. This study indicates that Scolopendra may have different proteins playing oxygen transport (Hc) and PO function, both following the hexameric oligomerization observed in Hcs.

Spectral diffusion and electron-phonon coupling of the B800 BChl a molecules in LH2 complexes from three different species of purple bacteria.

We have investigated the spectral diffusion and the electron-phonon coupling of B800 bacteriochlorophyll a molecules in the peripheral light-harvesting complex LH2 for three different species of purple bacteria, Rhodobacter sphaeroides, Rhodospirillum molischianum, and Rhodopseudomonas acidophila. We come to the conclusion that B800 binding pockets for Rhodobacter sphaeroides and Rhodopseudomonas acidophila are rather similar with respect to the polarity of the protein environment but that the packaging of the alphabeta-polypeptides seems to be less tight in Rb. sphaeroides with respect to the other two species.

The use of force plate measurements to titrate the dosage of a new COX-2 inhibitor in lame horses.

REASONS FOR PERFORMING STUDY: Lameness is a highly prevalent condition in horses and the principal cause of removal from athletic activity. In clinical studies to evaluate nonsteroidal anti-inflammatory drug therapies, force plates are commonly used to assess improvement of lameness objectively. HYPOTHESIS: To use a force plate to determine the optimal dose of a new COX-2 inhibitor (firocoxib) that will reduce lameness, when administered orally to horses once daily. METHODS: Sixty-four horses that exhibited chronic lameness presumed due to osteoarthritis, including navicular disease, in at least one of the frontlimbs and at a stable level of severity, were included. Horses were treated per os s.i.d. for 7 days as follows: vehicle control, firocoxib at 0.05, 0.1 or 0.25 mg/kg bwt. Force plate analysis of each horse was done for the selected (most) lame frontlimb at trot. Once between Days -19 and -4 (initial examination), and again on Day -2 or -1 (baseline), pretreatment force plate assessments were performed, and thereafter horses were assessed on Days 0, 2 and 6, approximately 10 h post treatment each time. Peak vertical force (PVF) and lameness grades at initial examination and at baseline, and their change from baseline in the 4 different treatment groups were analysed statistically at a significance level of P < 0.05. RESULTS: The PVF results were found to be superior to vehicle control already at Day 0 for 0.25 mg/kg bwt and at Days 2 and 6 for 0.1 and 0.25 mg/kg bwt (P < 0.05). Mean clinical lameness for both concentrations decreased > 1 grade at Day 6. CONCLUSIONS AND CLINICAL RELEVANCE: With the dosage of 0.25 mg/kg bwt lameness did not improve more than with 0.1 mg/kg bwt. Thus, 0.1 mg/kg bwt s.i.d. was considered to be the effective dose at reducing chronic lameness in horses presumed due to osteoarthritis, including navicular disease.

Unveiling ribosomal structures: the final phases.

Unprecedented insights into the structure of the ribosome have been gained recently: X-ray crystallographic studies have yielded 5-9 A resolution structures and cryo-electron microscopy has elucidated the structure of the Escherichia coli ribosome in different functional states. A 7.5 A cryo-electron microscopy structure of the large subunit indicates that this technique is still in the race to determine the ribosome structure.

The 80S rat liver ribosome at 25 A resolution by electron cryomicroscopy and angular reconstitution.

BACKGROUND: The ribosome is central to protein synthesis in all living organisms. Single-particle electron cryomicroscopy has recently led to the determination of three-dimensional structures of bacterial ribosomes to approximately 20 A, which have since revolutionised our understanding of ribosomal function. The structure we present here of the 80S rat liver ribosome leads the way to similar progress for mammalian ribosomes. RESULTS: Among the new details revealed by our 25 A structure of the 80S rat liver ribosome are channels within the subunits, a large 'flat ribosomal surface' (FRS) on the outer surface of the large subunit and structural extensions of the mammalian compared to the bacterial ribosome. The main large subunit channel in both the bacterial and the mammalian species starts at the peptidyl transferase centre, below the central protuberance, and ends in the FRS, at the lower back of the large subunit. Structurally, the channels of both species can be directly superimposed. CONCLUSIONS: The mammalian structural extensions--none of which trespass the FRS--can be interpreted in terms of rRNA inserts and additional protein content over that of bacterial ribosomes. The main large subunit channel, which ends at the FRS, is the best candidate for the exit channel for proteins targeted for the endoplasmic reticulum.

The 70S Escherichia coli ribosome at 23 A resolution: fitting the ribosomal RNA.

BACKGROUND: The ribosome--essential for protein synthesis in all organisms--has been an evasive target for structural studies. The best available structures for the 70S Escherichia coli ribosome or its 30S and 50S subunits are based on electron microscopical tilt experiments and are limited in resolution to 28-55 A. The angular reconstitution approach, which exploits the random orientations of particles within a vitreous ice matrix, can be used in conjunction with cryo-electron microscopy to yield a higher-resolution structure. RESULTS: Our 23 A resolution map of the 70S ribosome elucidates many structural details, such as an extensive system of channels within the 50S subunit and an intersubunit gap ideally shaped to accommodate two transfer RNA molecules. The resolution achieved is sufficient to allow the preliminary fitting of double-helical regions of an earlier three-dimensional ribosomal RNA model. CONCLUSIONS: Although we are still a long way from attaining an atomic-resolution structure of the ribosome, cryo-electron microscopy, in combination with angular reconstitution, is likely to yield three-dimensional maps with gradually increasing resolution. As exemplified by our current 23 A reconstruction, these maps will lead to progressive refinement of models of the ribosomal RNA.

Elucidating the medium-resolution structure of ribosomal particles: an interplay between electron cryo-microscopy and X-ray crystallograhy.

BACKGROUND: Ribosomes are the universal cellular organelles that accomplish the translation of the genetic code into proteins. Electron cryo-microscopy (cryo-EM) has yielded fairly detailed three-dimensional reconstructions of ribosomes. These were used to assist in the determination of higher resolution structures by X-ray crystallography. RESULTS: Molecular replacement studies using cryo-EM reconstructions provided feasible packing schemes for crystals of ribosomes and their two subunits from Thermus thermophilus, and of the large subunits from Haloarcula marismortui. For the large subunits, these studies also confirmed the major heavy-atom sites obtained by single isomorphous replacement combined with anomalous diffraction (SIRAS) and by multiple isomorphous replacement combined with anomalous diffraction (MIRAS) at approximately 10 A. Although adequate starting phases could not be obtained for the small subunits, the crystals of which diffract to 3.0 A, cryo-EM reconstructions were indispensable for analyzing their 7.2 A multiple isomorphous replacement (MIR) map. This work indicated that the conformation of the crystallized small subunits resembles that seen within the 70S ribosomes. Subsequently, crystals of particles trapped in their functionally active state were grown. CONCLUSIONS: Single-particle cryo-EM can contribute to the progress of crystallography of non-symmetrical, large and flexible macromolecular assemblies. Besides confirming heavy-atom sites, obtained from flat or overcrowded difference Patterson maps, the cryo-EM reconstructions assisted in elucidating packing arrangements. They also provided tools for the identification of the conformation within the crystals and for the estimation of the level of inherent non-isomorphism.

The Escherichia coli large ribosomal subunit at 7.5 A resolution.

BACKGROUND: In recent years, the three-dimensional structure of the ribosome has been visualised in different functional states by single-particle cryo-electron microscopy (cryo-EM) at 13-25 A resolution. Even more recently, X-ray crystallography has achieved resolution levels better than 10 A for the ribosomal structures of thermophilic and halophilic organisms. We present here the 7.5 A solution structure of the 50S large subunit of the Escherichia coli ribosome, as determined by cryo-EM and angular reconstitution. RESULTS: The reconstruction reveals a host of new details including the long alpha helix connecting the N- and C-terminal domains of the L9 protein, which is found wrapped like a collar around the base of the L1 stalk. A second L7/L12 dimer is now visible below the classical L7/L12 'stalk', thus revealing the position of the entire L8 complex. Extensive conformational changes occur in the 50S subunit upon 30S binding; for example, the L9 protein moves by some 50 A. Various rRNA stem-loops are found to be involved in subunit binding: helix h38, located in the A-site finger; h69, on the rim of the peptidyl transferase centre cleft; and h34, in the principal interface protrusion. CONCLUSIONS: Single-particle cryo-EM is rapidly evolving towards the resolution levels required for the direct atomic interpretation of the structure of the ribosome. Structural details such as the minor and major grooves in rRNA double helices and alpha helices of the ribosomal proteins can already be visualised directly in cryo-EM reconstructions of ribosomes frozen in different functional states.

Shoeing sound warmblood horses with a rolled toe optimises hoof-unrollment and lowers peak loading during breakover.

REASONS FOR PERFORMING STUDY: Overload injuries in sport horses commonly occur; shoeing techniques are believed to be important in prevention of these injuries, but there is a paucity of scientific information identifying the potential connection. OBJECTIVES: To test a horseshoe with a modified rolled toe designed to ease the process of breakover and decrease loading of lesion-prone structures of the distal limb. METHODS: Twenty clinically sound Warmblood horses trotted over a track containing a pressure/force measuring system and 6 infrared cameras. The horses were measured with 2 types of shoes, standard flat shoes and shoes with a rolled toe. The shoeing procedure was randomised and horses had 2 days between measurements to adapt to the shoes. RESULTS: Limb placement and timing characteristics, e.g. breakover duration, did not change significantly. There was an improvement in the ease of movement to roll over the toe in the shoes with a rolled toe, due mainly to a smoother hoof-unrollment pattern. The peak indicative moment decreased substantially at the onset of breakover in the shoe with the rolled toe. CONCLUSIONS: With a rolled toe the process of hoof-unrollment is smoother, which improves the coordination of this process, and lowers peak loading of the distal limb during breakover. POTENTIAL RELEVANCE: This study stresses the importance of proper shoeing in sound horses, showing that shoe modifications can optimise the loading characteristics of the distal limb and therefore might be a means to prevent sport horses from overload injuries.

Developmental aspects of distal limb conformation in the horse: the potential consequences of uneven feet in foals.

REASONS FOR PERFORMING STUDY: Distal limb conformation is generally accepted to be an important item with respect to performance and soundness in mature horses, but little is known about the developmental aspects. OBJECTIVES: To gain insight into the development of distal limb conformation and to assess the possible consequences of uneven feet in foals. METHODS: Conformation of the distal front limbs of 23 Warmblood foals was scored visually and measured using radiographs, at ages 27 and 55 weeks. At the same ages, pressure measurements were made under both front feet. RESULTS: At both ages the hoof-pastern axis was broken-backwards on radiographs, but only occasionally recognised as such, when scored by eye. Over time, the hoof angle decreased, while both the angles of the dorsal and solar surfaces of the distal phalanx (P3) increased and the parallelism between hoof wall and P3 improved. The foals with uneven feet at age 27 weeks showed a significant difference in distal limb loading that persisted until age 55 weeks. CONCLUSIONS: The alignment of the distal limb in the sagittal plane increased in a 6 month period. Visual assessment was not sensitive enough to appreciate this. The growth processes in the distal limb could not compensate for existing unevenness and ensuing asymmetrical limb loading. POTENTIAL RELEVANCE: Foals have a different conformation of the distal limb from mature horses, which should be taken into account when interpreting radiographs. Unevenness of the feet resulted in asymmetrical loading of the proximal and distal interphalangeal joint, which might lead to increased susceptibility to overload injuries and decreased performance at mature age.

Hoof growth between two shoeing sessions leads to a substantial increase of the moment about the distal, but not the proximal, interphalangeal joint.

REASONS FOR PERFORMING STUDY: There is little insight into the effects of routine farriery on the internal structures of the distal limb in sound horses. OBJECTIVES: To measure the effect of change in hoof conformation during a shoeing interval on the moments about the proximal and distal interphalangeal joints (PIPJ, DIPJ) and to determine whether and how the horse compensates for this change in hoof conformation. METHODS: Both front feet of 9 sound Warmblood horses were measured while standing on a pressure-force measuring system and radiographed in a lateromedial direction shortly after shoeing and 8 weeks later. From these data, ground reaction forces (GRF) and lever arms were measured in order to calculate joint moments. RESULTS: After 8 weeks, the moment about the PIPJ did not increase significantly, but the moment about the DIPJ did so, indicating a compensatory mechanism for a change in hoof conformation in the DIPJ. CONCLUSIONS: Standing horses compensate for hoof conformation change during an 8-week shoeing interval, which leads to increased DIPJ extension and consequently an increased loading of the deep digital flexor tendon. POTENTIAL RELEVANCE: This study quantifies the effect of a shoeing interval on the internal structures of the foot and helps to determine an appropriate shoeing interval for individual horses in which the hoof with the lowest hoof angle is the best indicator. The exact determination of an optimal individual shoeing interval requires further study.

Uneven feet in a foal may develop as a consequence of lateral grazing behaviour induced by conformational traits.

REASONS FOR PERFORMING STUDY: Conformational traits are important in breeding, since they may be indicative for performance ability and susceptibility to injuries. OBJECTIVES: To study whether certain desired conformational traits of foals are related to lateralised behaviour while foraging and to the development of uneven feet. METHODS: Twenty-four Warmblood foals, born and raised at the same location, were studied for a year. Foraging behaviour was observed by means of weekly 10 min scan-sampling for 8 h. A preference test (PT) was developed to serve as a standardised tool to determine laterality. The foals were evaluated at age 3, 15, 27 and 55 weeks. The PT and distal limb conformation were used to study the relation between overall body conformation, laterality and the development of uneven feet. Pressure measurements were used to determine the loading patterns under the feet. RESULTS: About 50% of the foals developed a significant preference to protract the same limb systematically while grazing, which resulted in uneven feet and subsequently uneven loading patterns. Foals with relatively long limbs and small heads were predisposed to develop laterality and, consequently unevenness. CONCLUSIONS: Conformational traits may stimulate the development of laterality and therefore indirectly cause uneven feet.

Molecular shape of Lumbricus terrestris erythrocruorin studied by electron microscopy and image analysis.

The molecular structure of erythrocruorin (hemoglobin) from Lumbricus terrestris has been studied by electron microscopy of negatively stained particles. Over 1000 molecular projections were selected from a number of electron micrographs and were then classified by multivariate statistical image-processing techniques. The two main groups of top and side views were each subdivided into smaller classes with significantly different features. About half of the top-view projections exhibit perfect hexagonal symmetry at the current resolution of about 2.0 nm, while the other top views lack this symmetry, probably as a result of tilting of the molecules relative to the carbon support film. The side views were separated into two 'families', each associated with the two different stable side-view positions the molecules can take. From these narrow stable side-views, the two families of projections are, again, generated by tilting. The symmetry properties of the three non-tilted projections show that Lumbricus erythrocruorin has a pointgroup D6 (622) symmetry rather than D3 (32).

Three-dimensional structure of bovine NADH:ubiquinone oxidoreductase of the mitochondrial respiratory chain.

We have studied the structure of bovine heart mitochondrial NADH:ubiquinone (Q) oxidoreductase (EC 1.6.99.3) by image analysis of electron micrographs. A three-dimensional reconstruction was calculated from a tilt-series of a two-dimensional crystal of the molecule. Our interpretation of the position of the molecule in the unit cell of the crystal is supported by additional (low-resolution) analysis of images of single molecules. The three-dimensional reconstruction was calculated with the aid of an iterative real-space reconstruction algorithm. The various projections used as input to the algorithm were obtained by averaging the images of the tilted crystal through a Fourier-space peak-filtering procedure. The reconstructed unit cell measures 15.2 X 15.2 nm in the plane of the two-dimensional crystal and has a height of 10-11 nm. The unit cell contains one molecule consisting of four large subunits. At the present resolution of about 1.3 nm in the untilted projection, these four monomers are seen as two dimers related by a two-fold axis. Two views of the single particles have been recognized; they are the top and side view of the building block of the crystal. After computer image alignment and correspondence analysis, clusters of similar particles have been averaged. In the averages an uneven stain distribution is seen around the molecules, which may result from preferential staining of hydrophilic parts of the molecule. The molecular mass of the whole molecule was determined from scanning transmission electron microscopy measurements as (1.6 +/- 0.2) X 10(6) daltons.

Structure of mitochondrial F1-ATPase studied by electron microscopy and image processing.

The structure of soluble F1-ATPase (EC 3.6.1.3) has been investigated by computer analysis of individual molecular images extracted from electron micrographs of negatively stained particles. A total of 1241 images was interactively selected from several digitized micrographs and these images were subsequently aligned relative to different reference images. They were then submitted to a multivariate statistical classification procedure. We have focussed our attention on the main 'hexagonal' view which represents some 40% of our population of images. In this view, six masses are located on the outer region of the projection which are associated with the alpha and the beta subunits of the protein. A seventh mass is located close to the centre of the hexagon, but slightly off its exact midpoint. It has the shape of the letter V and its two legs point to two of the outer protein masses, or one alpha-beta subunit pair. The corner of the V has a density as high as those of the large subunits. Possible subunit arrangements and their consequences for the mechanism of ATP synthesis are discussed.

A new family of powerful multivariate statistical sequence analysis techniques.

A novel multivariate statistical approach is presented for extracting and exploiting intrinsic information present in our ever-growing sequence data banks. The information extraction from the sequences avoids the pitfalls of intersequence alignment by analyzing secondary invariant functions derived from the sequences in the data bank rather than the sequences themselves. Such typical invariant function is a 20 x 20 histogram of occurrences of amino acid pairs in a given sequence or fragment thereof. To illustrate the potential of the approach an analysis of 10,000 protein sequences from the National Biomedical Research Foundation Protein Identification Resource is presented, whose analysis already reveals great biological detail. For example, zeta-hemoglobin is found to lie close to amphibian and fish chi-hemoglobin which, in turn, is an important clue to the physiological function of this mammalian early embryonic hemoglobin. The multivariate statistical framework presented unifies such apparently unrelated issues as phylogenetic comparisons between a set of sequences and distance matrices between the constituents of the biological sequences. The Multivariate Statistical Sequence Analysis (MSSA) principles can be used for a wide spectrum of sequence analysis problems such as: assignment of family memberships to new sequences, validation of new incoming sequences to be entered into the database, prediction of structure from sequence, discrimination of coding from non-coding DNA regions, and automatic generation of an atlas of protein or DNA sequences. The MSSA techniques represent a self-contained approach to learning continuously and automatically from the growing stream of new sequences. The MSSA approach is particularly likely to play a significant role in major sequencing efforts such as the human genome project.

Correlation of the expansion segments in mammalian rRNA with the fine structure of the 80 S ribosome; a cryoelectron microscopic reconstruction of the rabbit reticulocyte ribosome at 21 A resolution.

Samples of 80 S ribosomes from rabbit reticulocytes were subjected to electron cryomicroscopy combined with angular reconstitution. A three-dimensional reconstruction at 21 A resolution was obtained, which was compared with the corresponding (previously published) reconstruction of Escherichia coli 70 S ribosomes carrying tRNAs at the A and P sites. In the region of the intersubunit cavity, the principal features observed in the 70 S ribosome (such as the L1 protuberance, the central protuberance and A site finger in the large subunit) could all be clearly identified in the 80 S particle. On the other hand, significant additional features were observed in the 80 S ribosomes on the solvent sides and lower regions of both subunits. In the case of the small (40 S) subunit, the most prominent additions are two extensions at the base of the particle. By comparing the secondary structure of the rabbit 18 S rRNA with our model for the three-dimensional arrangement of E. coli 16 S rRNA, these two extensions could be correlated with the rabbit expansion segments (each totalling ca 170 bases) in the regions of helix 21, and of helices 8, 9 and 44, respectively. A similar comparison of the secondary structures of mammalian 28 S rRNA and E. coli 23 S rRNA, combined with preliminary modelling studies on the 23 S rRNA within the 50 S subunit, enabled the additional features in the 60 S subunit to be sub-divided into five groups. The first (corresponding to a total of ca 335 extra bases in helices 45, 98 and 101) is located on the solvent side of the 60 S subunit, close to the L7/L12 area. The second (820 bases in helices 25 and 38) is centrally placed on the solvent side of the subunit, whereas the third group (totaling 225 bases in helices 18/19, 27/29, 52 and 54) lies towards the L1 side of the subunit. The fourth feature (80 bases in helices 78 and 79) lies within or close to the L1 protuberance itself, and the fifth (560 bases in helix 63) is located underneath the L1 protuberance on the interface side of the 60 S subunit.

A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA. III. The topography of the functional centre.

We describe the locations of sites within the 3D model for the 16 S rRNA (described in two accompanying papers) that are implicated in ribosomal function. The relevant experimental data originate from many laboratories and include sites of foot-printing, cross-linking or mutagenesis for various functional ligands. A number of the sites were themselves used as constraints in building the 16 S model. (1) The foot-print sites for A site tRNA are all clustered around the anticodon stem-loop of the tRNA; there is no "allosteric" site. (2) The foot-print sites for P site tRNA that are essential for P site binding are similarly clustered around the P site anticodon stem-loop. The foot-print sites in 16 S rRNA helices 23 and 24 are, however, remote from the P site tRNA. (3) Cross-link sites from specific nucleotides within the anticodon loops of A or P site-bound tRNA are mostly in agreement with the model, whereas those from nucleotides in the elbow region of the tRNA (which also exhibit extensive cross-linking to the 50 S subunit) are more widely spread. Again, cross-links to helix 23 are remote from the tRNAs. (4) The corresponding cross-links from E site tRNA are predominantly in helix 23, and these agree with the model. Electron microscopy data are presented, suggestive of substantial conformational changes in this region of the ribosome. (5) Foot-prints for IF-3 in helices 23 and 24 are at a position with close contact to the 50 S subunit. (6) Foot-prints from IF-1 form a cluster around the anticodon stem-loop of A site tRNA, as do also the sites on 16 S rRNA that have been implicated in termination. (7) Foot-print sites and mutations relating to streptomycin form a compact group on one side of the A site anticodon loop, with the corresponding sites for spectinomycin on the other side. (8) Site-specific cross-links from mRNA (which were instrumental in constructing the 16 S model) fit well both in the upstream and downstream regions of the mRNA, and indicate that the incoming mRNA passes through the well-defined "hole" at the head-body junction of the 30 S subunit.