Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: purification, crystallization and structure determination.

We describe the crystallization and structure determination of the 30 S ribosomal subunit from Thermus thermophilus. Previous reports of crystals that diffracted to 10 A resolution were used as a starting point to improve the quality of the diffraction. Eventually, ideas such as the addition of substrates or factors to eliminate conformational heterogeneity proved less important than attention to detail in yielding crystals that diffracted beyond 3 A resolution. Despite improvements in technology and methodology in the last decade, the structure determination of the 30 S subunit presented some very challenging technical problems because of the size of the asymmetric unit, crystal variability and sensitivity to radiation damage. Some steps that were useful for determination of the atomic structure were: the use of anomalous scattering from the LIII edges of osmium and lutetium to obtain the necessary phasing signal; the use of tunable, third-generation synchrotron sources to obtain data of reasonable quality at high resolution; collection of derivative data precisely about a mirror plane to preserve small anomalous differences between Bijvoet mates despite extensive radiation damage and multi-crystal scaling; the pre-screening of crystals to ensure quality, isomorphism and the efficient use of scarce third-generation synchrotron time; pre-incubation of crystals in cobalt hexaammine to ensure isomorphism with other derivatives; and finally, the placement of proteins whose structures had been previously solved in isolation, in conjunction with biochemical data on protein-RNA interactions, to map out the architecture of the 30 S subunit prior to the construction of a detailed atomic-resolution model.

Structure of a bacterial 30S ribosomal subunit at 5.5 A resolution.

The 30S ribosomal subunit binds messenger RNA and the anticodon stem-loop of transfer RNA during protein synthesis. A crystallographic analysis of the structure of the subunit from the bacterium Thermus thermophilus is presented. At a resolution of 5.5 A, the phosphate backbone of the ribosomal RNA is visible, as are the alpha-helices of the ribosomal proteins, enabling double-helical regions of RNA to be identified throughout the subunit, all seven of the small-subunit proteins of known crystal structure to be positioned in the electron density map, and the fold of the entire central domain of the small-subunit ribosomal RNA to be determined.

A detailed view of a ribosomal active site: the structure of the L11-RNA complex.

We report the crystal structure of a 58 nucleotide fragment of 23S ribosomal RNA bound to ribosomal protein L11. This highly conserved ribonucleoprotein domain is the target for the thiostrepton family of antibiotics that disrupt elongation factor function. The highly compact RNA has both familiar and novel structural motifs. While the C-terminal domain of L11 binds RNA tightly, the N-terminal domain makes only limited contacts with RNA and is proposed to function as a switch that reversibly associates with an adjacent region of RNA. The sites of mutations conferring resistance to thiostrepton and micrococcin line a narrow cleft between the RNA and the N-terminal domain. These antibiotics are proposed to bind in this cleft, locking the putative switch and interfering with the function of elongation factors.

Location of translational initiation factor IF3 on the small ribosomal subunit.

The location of translational initiation factor IF3 bound to the 30S subunit of the Thermus thermophilus ribosome has been determined by cryoelectron microscopy. Both the 30S.IF3 complex and control 30S subunit structures were determined to 27-A resolution. The difference map calculated from the two reconstructions reveals three prominent lobes of positive density. The previously solved crystal structure of IF3 fits very well into two of these lobes, whereas the third lobe probably arises from conformational changes induced in the 30S subunit as a result of IF3 binding. Our placement of IF3 on the 30S subunit allows an understanding in structural terms of the biochemical functions of this initiation factor, namely its ability to dissociate 70S ribosomes into 30S and 50S subunits and the preferential selection of initiator tRNA by IF3 during initiation.

Structure of the carboxyl-terminal dimerization domain of the HIV-1 capsid protein.

The carboxyl-terminal domain, residues 146 to 231, of the human immunodeficiency virus-1 (HIV-1) capsid protein [CA(146-231)] is required for capsid dimerization and viral assembly. This domain contains a stretch of 20 residues, called the major homology region (MHR), which is conserved across retroviruses and is essential for viral assembly, maturation, and infectivity. The crystal structures of CA(146-231) and CA(151-231) reveal that the globular domain is composed of four helices and an extended amino-terminal strand. CA(146-231) dimerizes through parallel packing of helix 2 across a dyad. The MHR is distinct from the dimer interface and instead forms an intricate hydrogen-bonding network that interconnects strand 1 and helices 1 and 2. Alignment of the CA(146-231) dimer with the crystal structure of the capsid amino-terminal domain provides a model for the intact protein and extends models for assembly of the central conical core of HIV-1.

Probing the structure of the Escherichia coli 10Sa RNA (tmRNA).

The conformation of the Escherichia coli 10Sa RNA (tmRNA) in solution was investigated using chemical and enzymatic probes. Single- and double-stranded domains were identified by hydrolysis of tmRNA in imidazole buffer and by lead(II)-induced cleavages. Ribonucleases T1 and S1 were used to map unpaired nucleotides and ribonuclease V1 was used to identify paired bases or stacked nucleotides. Specific atomic positions of bases were probed with dimethylsulfate, a carbodiimide, and diethylpyrocarbonate. Covariations, identified by sequence alignment with nine other tmRNA sequences, suggest the presence of several tertiary interactions, including pseudoknots. Temperature-gradient gel electrophoresis experiments showed structural transitions of tmRNA starting around 40 degrees C, and enzymatic probing performed at selected temperatures revealed the progressive melting of several predicted interactions. Based on these data, a secondary structure is proposed, containing two stems, four stem-loops, four pseudoknots, and an unstable structural domain, some connected by single-stranded A-rich sequence stretches. A tRNA-like domain, including an already reported acceptor branch, is supported by the probing data. A second structural domain encompasses the coding sequence, which extends from the top of one stem-loop to the top of another, with a 7-nt single-stranded stretch between. A third structural module containing pseudoknots connects and probably orients the tRNA-like domain and the coding sequence. Several discrepancies between the probing data and the phylogeny suggest that E. coli tmRNA undergoes a conformational change.

Integration of hepatitis B vaccination into rural African primary health care programmes.

OBJECTIVE: To determine the efficacy of hepatitis B vaccine when added to the routine expanded programme on immunisation under field conditions in rural Africa. DESIGN: Infants were immunised according to two schedules--an early schedule at birth, 3 months, and 6 months and a later schedule to correspond with routine vaccination in the expanded programme on immunisation at 3 months, 4 1/2 months, and 6 months. SETTING: Venda, northern Transvaal, South Africa, a self governing region of 7460 square kilometers varying from rural villages to small towns. SUBJECTS: The 1989 birth cohort of Venda. MAIN OUTCOME MEASURES: Coverage for hepatitis B vaccine at first, second, and third doses; serological assessment of vaccine efficacy by prevalence of antibodies to hepatitis B surface antigen in infants who had completed the three dose course of immunisation; antibodies to hepatitis B core antigen to determine if natural infection occurred. RESULTS: Vaccine coverage for hepatitis B dropped sharply from 99% to 53% to 39% for the first, second, and third dose respectively. In contrast, vaccine coverage was maintained at 97-99% for the three doses of poliomyelitis vaccine. Serological evaluation of vaccine efficacy showed that only 3.5% of recipients of all three doses failed to develop antibodies to hepatitis B surface antigen. Only 6.6% of vaccine recipients were vaccinated according to either the early or later schedules whereas 93.4% received their doses of vaccine at intervals beyond the limits of either of the planned schedules. There was, however, no significant difference in seroconversion to the surface antigen between the "unscheduled" or scheduled groups of those who were vaccinated according to the early or late schedules. The pattern of prevalence of antibodies to hepatitis B core antigen, which showed a sharp fall in children aged over 7 months, suggested that the antibodies were acquired passively rather than by active infection. CONCLUSIONS: Supplementation of the present expanded programme on immunisation with hepatitis B vaccine in rural Africa is fraught with difficulties. However, the vaccine was effective within a fairly wide spacing of dosage. Adding hepatitis B vaccine to diphtheria, tetanus, and pertussis as a tetravalent vaccine is proposed as a means of effectively integrating it into the expanded programme on immunisation in Third World settings.

Analysis of an immunisation programme in a rural area.

An immunisation programme in a rural area of southern Africa is analysed. The combination of fixed and mobile immunisation points is discussed with reference to staffing levels, patient convenience, vaccine handling, costs and efficiency. A system of community involvement in the programme is assessed. Mobile immunisation teams to complement fixed clinic services, and active community involvement are suggested for immunisation programmes in rural areas.