Albert E. Dahlberg

Electrophoretic characterization of bacterial polyribosomes in agarose-acrylamide composite gels

Abstract Bacterial polyribosomes, ribosomes and ribosomal subunits were found to possess characteristic electrophoretic mobilities in polyacrylamide-agarose composite gels (containing 2 to 3% acrylamide). The mobilities of these particles were inversely proportional to their size. The smaller (30 s) subunit had the fastest mobility, and migrated at a rate similar to its constituent 16 s RNA. Polyribosomes containing eight ribosomes were the largest that could be resolved. Brief treatment with RNase converted the polyribosomes to faster migrating particles (70 s ribosomes). A unique feature of the electrophoretic analysis was the ability to identify, by a second electrophoresis, the type of RNA contained in ribosomal subunits, ribosomes and polyribosomes. Similarly, the subunit composition of ribosomes and polyribosomes could be established. The principal subunits observed were a faster (30 s) subunit containing 16 s RNA and a slower (50 s) subunit containing 23 s RNA. Polyribosomes resembled each other in their proportional content of these subunits. RNA isolated from polyribosome preparations was composed of three very similar but electrophoretically distinct 23 s RNAs; similarly the 16 s RNA was composed of two distinct RNAs. Another RNA migrating somewhat slower than the 16 s RNA (16 s–17 s) was observed. In addition to these multiple forms of RNA an unanticipated diversity of subunits was observed. One of the smaller subunits contained the slower 16 s–17 s RNA and may represent the 26 s precursor particle of the 30 s subunit. It is likely that some of the variations in subunit mobilities are related to differences in the size of the RNA which they contain. However, provided the protein is not removed, the electrophoretic mobilities of subunits and polyribosomes are not dependent on their containing intact RNA.

The Polypeptide Tunnel System in the Ribosome and Its Gating in Erythromycin Resistance Mutants of L4 and L22

Variations in the inner ribosomal landscape determining the topology of nascent protein transport have been studied by three-dimensional cryo-electron microscopy of erythromycin-resistant Escherichia coli 70S ribosomes. Significant differences in the mouth of the 50S subunit tunnel system visualized in the present study support a simple steric-hindrance explanation for the action of the drug. Examination of ribosomes in different functional states suggests that opening and closing of the main tunnel are dynamic features of the large subunit, possibly accompanied by changes in the L7/L12 stalk region. The existence and dynamic behavior of side tunnels suggest that ribosomal proteins L4 and L22 might be involved in the regulation of a multiple exit system facilitating cotranslational processing (or folding or directing) of nascent proteins.

Reading frame switch caused by base-pair formation between the 3' end of 16S rRNA and the mRNA during elongation of protein synthesis in Escherichia coli

Watson‐Crick base pairing is shown to occur between the mRNA and nucleotides near the 3′ end of 16S rRNA during the elongation phase of protein synthesis in Escherichia coli. This base‐pairing is similar to the mRNA‐rRNA interaction formed during initiation of protein synthesis between the Shine and Dalgarno (S‐D) nucleotides of ribosome binding sites and their complements in the 1540‐1535 region of 16S rRNA. mRNA‐rRNA hybrid formation during elongation had been postulated to explain the dependence of an efficient ribosomal frameshift on S‐D nucleotides precisely spaced 5′ on the mRNA from the frameshift site. Here we show that disruption of the postulated base pairs by single nucleotide substitutions, either in the S‐D sequence required for shifting or in nucleotide 1538 of 16S rRNA, decrease the amount of shifting, and that this defect is corrected by restoring complementary base pairing. This result implies that the 3′ end of 16S rRNA scans the mRNA very close to the decoding sites during elongation.

Analysis of mutations at residues A2451 and G2447 of 23S rRNA in the peptidyltransferase active site of the 50S ribosomal subunit

On the basis of the recent atomic-resolution x-ray structure of the 50S ribosomal subunit, residues A2451 and G2447 of 23S rRNA were proposed to participate directly in ribosome-catalyzed peptide bond formation. We have examined the peptidyltransferase and protein synthesis activities of ribosomes carrying mutations at these nucleotides. In Escherichia coli, pure mutant ribosome populations carrying either the G2447A or G2447C mutations maintained cell viability. In vitro, the G2447A ribosomes supported protein synthesis at a rate comparable to that of wild-type ribosomes. In single-turnover peptidyltransferase assays, G2447A ribosomes were shown to have essentially unimpaired peptidyltransferase activity at saturating substrate concentrations. All three base changes at the universally conserved A2451 conferred a dominant lethal phenotype when expressed in E. coli. Nonetheless, significant amounts of 2451 mutant ribosomes accumulated in polysomes, and all three 2451 mutations stimulated frameshifting and readthrough of stop codons in vivo. Furthermore, ribosomes carrying the A2451U transversion synthesized full-length β-lactamase chains in vitro. Pure mutant ribosome populations with changes at A2451 were generated by reconstituting Bacillus stearothermophilus 50S subunits from in vitro transcribed 23S rRNA. In single-turnover peptidyltransferase assays, the rate of peptide bond formation was diminished 3- to 14-fold by these mutations. Peptidyltransferase activity and in vitro β-lactamase synthesis by ribosomes with mutations at A2451 or G2447 were highly resistant to chloramphenicol. The significant levels of peptidyltransferase activity of ribosomes with mutations at A2451 and G2447 need to be reconciled with the roles proposed for these residues in catalysis.

Mutations in 16S ribosomal RNA disrupt antibiotic--RNA interactions.

Two of six mutations at a base‐paired site in Escherichia coli 16S rRNA confer resistance to nine different aminoglycoside antibiotics in vivo. Chemical probing of mutant and wild‐type ribosomes in the presence of paromomycin indicates that interactions between the antibiotic and 16S rRNA in mutant ribosomes are disrupted. The altered interactions measured in vitro correlate precisely with resistance seen in vivo and may be attributable to specific structural changes observed in the mutant rRNA.

A conformational change in the ribosomal peptidyl transferase center upon active/inactive transition

The ribosome is a dynamic particle that undergoes many structural changes during translation. We show through chemical probing with dimethyl sulfate (DMS) that conformational changes occur at several nucleotides in the peptidyl transferase center upon alterations in pH, temperature, and monovalent ion concentration, consistent with observations made by Elson and coworkers over 30 years ago. Moreover, we have found that the pH-dependent DMS reactivity of A2451 in the center of the 23S rRNA peptidyl transferase region, ascribed to a perturbed pKa of this base, occurs only in inactive 50S and 70S ribosomes. The degree of DMS reactivity of this base in the inactive ribosomes depends on both the identity and amount of monovalent ion present. Furthermore, G2447, a residue proposed to be critical for the hypothesized pKa perturbation, is not essential for the conditional DMS reactivity at A2451. Given that the pH-dependent change in DMS reactivity at A2451 occurs only in inactive ribosomes, and that this DMS reactivity can increase with increasing salt (independently of pH), we conclude that this observation cannot be used as supporting evidence for a recently proposed model of acid/base catalyzed ribosomal transpeptidation.

Major rearrangements in the 70S ribosomal 3D structure caused by a conformational switch in 16S ribosomal RNA.

Dynamic changes in secondary structure of the 16S rRNA during the decoding of mRNA are visualized by three‐dimensional cryo‐electron microscopy of the 70S ribosome. Thermodynamically unstable base pairing of the 912–910 (CUC) nucleotides of the 16S RNA with two adjacent complementary regions at nucleotides 885–887 (GGG) and 888–890 (GAG) was stabilized in either of the two states by point mutations at positions 912 (C912G) and 885 (G885U). A wave of rearrangements can be traced arising from the switch in the three base pairs and involving functionally important regions in both subunits of the ribosome. This significantly affects the topography of the A‐site tRNA‐binding region on the 30S subunit and thereby explains changes in tRNA affinity for the ribosome and fidelity of decoding mRNA.

Effects of mutagenesis of a conserved base-paired site near the decoding region of Escherichia coli 16 S ribosomal RNA☆

Eleven of 15 possible single and double mutations were constructed in a cloned copy of Escherichia coli 16 S rDNA at a base-paired site, 1409-1491. Expression of any of these mutations was detrimental to the growth of E. coli. Mutations that substituted unpaired purine bases were lethal in the system described. Otherwise, the degree of detrimental effect on growth-rate was not directly correlated with specific rRNA primary or secondary structures. Using reverse transcription of rRNA isolated from subunits or 70 S ribosomes, we were able to determine the amount of mutant rRNA used in translation. From these experiments, we found that the lethal mutations appeared to be selectively excluded from the pool of 70 S ribosomes following expression from a repressible plasmid. In contrast, a non-lethal mutation was present in subunits, ribosomes and polysomes in approximately equal amounts. Mutations that disrupted base-pairing were found to confer varying levels of resistance to nine aminoglycosides, including four neomycins, two kanamycins, gentamicin, apramycin and hygromycin. A high frequency of reversion from resistant and slow-growth phenotypes due to a host mutation was observed.

Initiation factor IF2, thiostrepton and micrococcin prevent the binding of elongation factor G to the Escherichia coli ribosome.

The bacterial translational GTPases (initiation factor IF2, elongation factors EF-G and EF-Tu and release factor RF3) are involved in all stages of translation, and evidence indicates that they bind to overlapping sites on the ribosome, whereupon GTP hydrolysis is triggered. We provide evidence for a common ribosomal binding site for EF-G and IF2. IF2 prevents the binding of EF-G to the ribosome, as shown by Western blot analysis and fusidic acid-stabilized EF-G.GDP.ribosome complex formation. Additionally, IF2 inhibits EF-G-dependent GTP hydrolysis on 70 S ribosomes. The antibiotics thiostrepton and micrococcin, which bind to part of the EF-G binding site and interfere with the function of the factor, also affect the function of IF2. While thiostrepton is a strong inhibitor of EF-G-dependent GTP hydrolysis, GTP hydrolysis by IF2 is stimulated by the drug. Micrococcin stimulates GTP hydrolysis by both factors. We show directly that these drugs act by destabilizing the interaction of EF-G with the ribosome, and provide evidence that they have similar effects on IF2.

Ribosomal RNA and protein mutants resistant to spectinomycin.

We have compared the influence of spectinomycin (Spc) on individual partial reactions during the elongation phase of translation in vitro by wild‐type and mutant ribosomes. The data show that the antibiotic specifically inhibits the elongation factor G (EF‐G) cycle supported by wild‐type ribosomes. In addition, we have reproduced the in vivo Spc resistant phenotype of relevant ribosome mutants in our in vitro translation system. In particular, three mutants with alterations at position 1192 in 16S rRNA as well as an rpsE mutant with an alteration of protein S5 were analysed. All of these ribosomal mutants confer a degree of Spc resistance for the EF‐G cycle in vitro that is correlated with the degree of growth rate resistance to the antibiotic in culture.