Staphylococcus aureus domain V functions in Escherichia coli ribosomes provided a conserved interaction with domain IV is restored.

Domain V of Escherichia coli 23 S rRNA (residues 2023-2630) was replaced by that from Staphylococcus aureus, thereby introducing 132 changes in the rRNA sequence. The resulting ribosomal mutant was unable to support cell growth. The mutant was rescued, however, by restoring an interaction between domains IV and V (residues 1782 and 2586). Although the importance of this interaction, U/U in E. coli, C/C in S. aureus, is therefore demonstrated, it cannot be the only tertiary interaction important for ribosomal function as the rescued hybrid grew more slowly than the wild type. Additionally, although the single-site mutations U1782C and U2586C in E. coli are viable, the double mutant is lethal.

Pseudoknot in the central domain of small subunit ribosomal RNA is essential for translation.

Phylogenetic comparison of rRNA sequences has suggested that a pseudoknot structure exists in the central domain of small-subunit rRNA. In Escherichia coli 16S rRNA, this pseudoknot would form when positions 570 and 571 pair with positions 865 and 866. Mutations were introduced into this pseudoknot at the phylogenetically invariant nucleotides U571 and A865. Single mutations of U to A at 571 or A to U at 865 dramatically altered the structural stability of the 30S subunit and also impaired the function of the subunit in translation. When the mutations were combined to create a compensatory pairing, the normal structure of the 30S subunit was restored, and the function of the mutant subunit in translation returned to wild-type levels. These results demonstrate the existence of a higher order structure in rRNA that directly affects the folding of the 30S subunit. Given the position of this structure in the three-dimensional model of the small subunit and the additional interactions that are likely to form in the same rRNA region, the central domain pseudoknot appears to contribute to a complex structure of rRNA that controls the conformational state of the ribosome.

Evidence for a conformational change in the exit site of the Escherichia coli ribosome upon tRNA binding.

The exit (E) site of the Escherichia coli ribosome was investigated using oligodeoxyribonucleotides complementary to single-stranded regions of ribosomal RNA suggested to be involved in tRNA binding in the E site [Moazed, D., & Noller, H. (1989) Cell 57, 585-597]. Radiolabeled DNA oligomers (probes) were hybridized in situ to complementary sites on the ribosomal RNA of ribosomes or ribosomal subunits, and the effects of simultaneous tRNA or antibiotic binding on probe binding were measured using a nitrocellulose filtration binding assay. Site specificity of probe binding was assured using ribonuclease H to cleave the ribosomal RNA at the site of probe binding. When 50S subunits were hybridized with a probe spanning bases 2109-2119 and deacylated tRNA was added incrementally, probe binding decreased, suggesting that the probe and tRNA competed for the same binding site or that tRNA was allosterically affecting the probe binding site. When 70S ribosomes were substituted for 50S subunits, probe binding to this site initially increased and then decreased at higher concentrations of deacylated tRNA. Titrating probe-ribosome complexes with acylated tRNA, N-acetyl-acylated tRNA, tetracycline, or chloramphenicol had no effect on probe binding. The data presented provide evidence for tRNA/rRNA interaction at or near the E site of the E. coli ribosome and suggest that a conformational change occurs in the E site when deacylated tRNA is bound to the P site. The data suggest that deacylated tRNA in the P site serves as a translocational trigger by causing the E site to change conformations, making it more available for tRNA (and probe) binding and therefore promoting translocation.(ABSTRACT TRUNCATED AT 250 WORDS)

Single-base mutations at position 2661 of Escherichia coli 23S rRNA increase efficiency of translational proofreading.

Two single-base substitutions were constructed in the 2660 loop of Escherichia coli 23S rRNA (G2661-->C or U) and were introduced into the rrnB operon cloned in plasmid pKK3535. Ribosomes were isolated from bacteria transformed with the mutated plasmids and assayed in vitro in a poly(U)-directed system for their response to the misreading effect of streptomycin, neomycin, and gentamicin, three aminoglycoside antibiotics known to impair the proofreading control of translational accuracy. Both mutations decreased the stimulation of misreading by these drugs, but neither interfered with their binding to the ribosome. The response of the mutant ribosomes to these drugs suggests that the 2660 loop, which belongs to the elongation factor Tu binding site, is involved in the proofreading step of the accuracy control. In vivo, both mutations reduced read-through of nonsense codons and frameshifting, which can also be related to the increased efficiency in proofreading control which they confer to ribosomes.

A single base mutation at position 2661 in E. coli 23S ribosomal RNA affects the binding of ternary complex to the ribosome.

A single base substitution mutation from guanine to cytosine was constructed at position 2661 of Escherichia coli 23S rRNA and cloned into the rrnB operon of the multi-copy plasmid pKK3535. The mutant plasmid was transformed into E.coli to determine the effect of the mutation on cell growth as well as the structural and functional properties of the mutant ribosomes in vivo and in vitro. The results show that the mutant ribosomes have a slower elongation rate and an altered affinity for EF-Tu-tRNA-GTP ternary complex. This supports previous findings which indicated that position 2661 is part of a region of 23S rRNA that forms a recognition site for binding of the ternary complex in the ribosomal A site. Combinations of the 2661 mutation with various mutations in ribosomal protein S12 also demonstrate that elements of both ribosomal subunits work in concert to form this binding site.

Interaction of tRNA with domain II of 23S rRNA.

The interaction of tRNA with domain II of 23S rRNA in E. coli ribosomes has been probed using short, complementary DNA oligodeoxyribonucleotides. Specifically, cDNA oligomers to the region 801-811 of the 23S rRNA were used to ascertain the interaction of this region with tRNA. It was found that when tRNA was bound to the P site, considerable competition occurred between tRNA and the cDNA oligomers which base paired with the nucleotides 807-811. However, A-site bound tRNA neither displaced, nor was displaced, by cDNA oligomers to this region. Additionally, the binding of tRNA lacking the CACCA nucleotides on the 3' terminus was unaffected by the presence a cDNA oligomer complementary to nucleotides 803-811, indicating that the cDNA-tRNA competition was dependent on the 3' terminal nucleotides of tRNA.

Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis.

A single base was mutated from guanine to adenine at position 791 in 16S rRNA in the Escherichia coli rrnB operon on the multicopy plasmid pKK3535. The plasmid-coded rRNA was processed and assembled into 30S ribosomal subunits in E. coli and caused a retardation of cell growth. The mutation affected crucial functional roles of the 30S subunit in the initiation of protein synthesis. The affinity of the mutant 30S subunits for 50S subunits was reduced and the association equilibrium constant for initiation factor 3 was decreased by a factor of 10 compared to wild-type 30S subunits. The interrelationship among the region of residue 790 in 16S rRNA, subunit association, and initiation factor 3 binding during initiation complex formation, as revealed by this study, offers insights into the functional role of rRNA in protein synthesis.

Involvement of bases 787-795 of Escherichia coli 16S ribosomal RNA in ribosomal subunit association.

A nine-base DNA oligomer [d(GTATCTAAT)] was used to probe the accessibility and function of bases in the region 787-795 of Escherichia coli 16S rRNA. Hybridization of the cDNA [d(GTATCTAAT)] to 16S rRNA in situ was carried out by binding the probe to intact 30S ribosomal subunits. Nitrocellulose filter binding showed that cDNA hybridization saturated with increasing probe concentration, suggesting that the probe was binding to a discrete site or sites. RNase H digestion of the rRNA under the DNA . rRNA hybrid and sequencing of the resultant RNA fragments verified that the cDNA probe bound specifically to the 787-795 region. Hybridization experiments using the cDNA probe showed that bases in the 787-795 region of 16S rRNA are exposed on the surface of 30S subunits. The functional role of bases 787-795 was then tested by assaying various ribosomal activities with the cDNA in place. Results of these functional assays demonstrate that this 16S rRNA region is directly involved in the association of 30S and 50S subunits.