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The ribosome is able to monitor the structure of the nascent peptide and can stall in response to specific peptide sequences. Such programmed stalling is used for the regulation of gene expression. The molecular mechanisms of the nascent-peptide recognition and ribosome stalling are unknown. We identified the conserved and posttranscriptionally modified 23S rRNA nucleotide m(2)A2503 located at the entrance of the ribosome exit tunnel as a key component of the ribosomal response mechanism. A2503 mutations abolish nascent-peptide-dependent stalling at the leader cistrons of several inducible antibiotic resistance genes and at the secM regulatory gene. Remarkably, lack of the C2 methylation of A2503 significantly function induction of expression of the ermC gene, indicating that the functional role of posttranscriptional modification is to fine-tune ribosome-nascent peptide interactions. Structural and biochemical evidence suggest that m(2)A2503 may act in concert with the previously identified nascent-peptide sensor, A2062, in the ribosome exit tunnel to relay the stalling signal to the peptidyl transferase centre.

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.

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.