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The "ice" structure for the E. coli ribosome clearly illustrates how the ribosome could work and it is used as the basis of a detailed account of ribosome function. There have been objections to this structure as it does not fit with current beliefs about the position of the T-site, for the arriving tRNA, or the Exit-site: these beliefs are wrong. The elongation factor EF-Tu is required at both the T-site and the E-site, and these two attachment areas were combined into the false site. The E-site has been placed at the L1-ridge because deacylated tRNAs attach to protein L1, but none of these experiments used a cognate codon to get a positive identification of this as the E-site. The reason L1 attaches to deacylated tRNAs is that it is at the E-site in polysomes, which contain closely stacked, or overlapping, ribosomes with the L1-ridge at the interface between ribosomes. The revised data allows the 1989 ribosome model to be reconciled with the most contradictory results and it is then used to develop a more detailed picture of the ribosome.

An in vitro system developed for the site-specific mutagenesis of 16S RNA of Escherichia coli ribosomes (Krzyzosiak et al., Biochemistry 26, 2353-2364, 1987) was used to make 10 single base changes around C1400, the residue known to be at the decoding site. C1400 was replaced by U, A, or G, 5 single base deletions at and to either side of C1400 were made, and C or U was inserted next to C1400. Another mutant possessed 7 additional nucleotides at the 3' end of the 16S RNA such that a stem and loop involving the anti-Shine-Dalgarno sequence could form. Another series of 8 mutants tested hypothetical base-pairing between C1404, G1405 and C1496, G1497. The activity of these 19 mutants in A and P site binding, and in initiation-dependent and initiation-independent peptide synthesis was determined. None of the base substitutions of C1400 were strongly inhibitory. The insertions and deletions completely blocked initiation-dependent peptide synthesis but markedly stimulated the initiation-independent reaction. The effects of tRNA binding were variable. The only alteration to block all ribosomal function was the deletion of G1401. The extra stem and loop at the 3'-end blocked initiation-dependent peptide synthesis, but all other assays were normal. The mutants which break and reform the hypothetical base-pairs had a functional pattern that suggest the contiguous base-pairs do exist and are functionally important.

In the presence of up to 6 microM chloramphenicol, balanced exponential growth of Escherichia coli occurred at a reduced rate after an adjustment period. The inhibition of ribosome function by chloramphenicol within growing cells was inferred from measurements of growth rate and cell composition during balanced growth and of pulse-labeling of cells by radioactive proline after a 10-min exposure to chloramphenicol. In each case the results were consistent with simple noncompetitive inhibition of protein synthesis, with 50% inhibition occurring at 2 microM chloramphenicol, the concentration that gave 50% binding of chloramphenicol to purified ribosomes in vitro. The differences between these results and those obtained with cell-free protein synthesizing systems were shown to be in part due to slow binding of chloramphenicol and in part due to the slow rate and lack of saturation of the cell-free protein-synthesizing systems now available. During balanced growth in concentrations of chloramphenicol 1 microM or higher, the net rate of maturation of ribosomal ribonucleic acid was also inhibited (50% at 2.8 microM). The specific growth rate during balanced growth was inhibited by 50% at 1.8 microM chloramphenicol, but at higher concentrations inhibition was greater than expected from the simple noncompetitive dose-response observed for inhibition of polypeptide synthesis. However, the inhibition of maturation of ribosomal ribonucleic acid plus the inhibition of protein synthesis quantitatively accounted for the observed inhibition of growth. Thus, we have presented for the first time an essentially complete account of the effects of partially inhibitory concentrations of an antibiotic on the growth physiology of a bacterium.

The RecA proteins of the unusually strong protease-constitutive mutants recA1202 and recA1211 can use RNA in addition to single-stranded DNA (ssDNA) as a cofactor in the cleavage of the LexA repressor in vitro. In the presence of rRNA or tRNA, the effectiveness of these proteins decreased in the order RecA1202 greater than RecA1211 much greater than RecA+, which is also the order of their in vivo constitutive protease activities. The effectiveness of rRNA was comparable to that of ssDNA in the cleavage of the LexA repressor by either mutant protease. Although all the common nucleoside triphosphates can act as positive effectors for LexA cleavage by the two mutant proteins in the presence of ssDNA (W. B. Wang, M. Sassanfar, I. Tessman, J. W. Roberts, and E. S. Tessman, J. Bacteriol. 170:4816-4822, 1988), only dATP, ATP, and ATP-gamma-S were effective in the presence of RNA. Our results explain more fully why certain recA mutants have high constitutive protease activities in vivo.

The role of 5 S RNA within the large ribosomal subunit of the extremely thermophilic archaebacterium Sulfolobus solfataricus has been analysed by means of in vitro reconstitution procedures. It is shown that Sulfolobus 50 S subunits reconstituted in the absence of 5 S RNA are inactive in protein synthesis and lack 2-3 ribosomal proteins. Furthermore, it has been determined that in the course of the in vitro assembly process Sulfolobus 5 S RNA can be replaced by the correspondent RNA species of E.coli; Sulfolobus reconstituted particles containing the eubacterial 5 S molecule are stable and active in polypeptide synthesis at high temperatures.

Plasmid pPM114 carries the Escherichia coli 16S ribosomal RNA gene under the control of a T7 promoter. It can generate in vitro transcribed 16S rRNA that can be assembled into functional 30S ribosomal subunits. Two deletion mutants were derived from pPM114, by partial or total deletion of the conserved 900 stem/loop region of the 16S rRNA. These mutants, pMG delta 10 and pMG delta 23, respectively lack bases 895 to 904 and 889 to 911 of the 16S rRNA. The amputated 16S rRNA transcripts synthesized from these mutated plasmids were assembled into 30S subunits which were as active under the direction of an artificial or a natural messenger as subunits reconstructed with the full-length 16S rRNA transcript. They also responded as well to the stimulation of misreading by streptomycin, although the deleted region is proximal to the streptomycin binding domain. However, when we attempted to delete the 895-904 or 889-911 region from the 16S rRNA gene in plasmid pKK3535 which carries the rrnB operon, no transformants harbouring plasmids with one of these deletions could be recovered. These observations suggest that the 900 stem/loop region of the 16S rRNA is not required for the ribosomal function but is probably essential for important cell regulatory functions.

An Escherichia coli K12 strain, carrying the promotor and proximal portion of the 16-S rRNA gene from rrnB cloned in the high-copy-number plasmid psF2124, has been examined for abnormalities in ribosome biogenesis. Both ribosomal RNA accumulation and ribosome content are depressed in this strain as compared to the control strain carrying the plasmid vector alone. The rate of total protein synthesis, however, appears to be normal. In contrast, the rate of ribosomal protein synthesis, relative to total protein synthesis, is elevated. The rates of synthesis of individual ribosomal proteins were determined and found to vary greatly, ranging from severe under-synthesis (displayed especially by proteins L7/L12) to massive over-synthesis (displayed particularly in the case of protein S7). Analysis of the rates of synthesis of other proteins coded for by the S12 operon revealed that protein S12 was moderately over-produced, but elongation factors EF-G and EF-Tu appear to be synthesized at the same rate as EF-Ts, all three being moderately under-synthesized relative to total soluble proteins.