Steven T. Gregory

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.

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.

Mutational Analysis of 16S and 23S rRNA Genes of Thermus thermophilus

Structural studies of the ribosome have benefited greatly from the use of organisms adapted to extreme environments. However, little is known about the mechanisms by which ribosomes or other ribonucleoprotein complexes have adapted to functioning under extreme conditions, and it is unclear to what degree mutant phenotypes of extremophiles will resemble those of their counterparts adapted to more moderate environments. It is conceivable that phenotypes of mutations affecting thermophilic ribosomes, for instance, will be influenced by structural adaptations specific to a thermophilic existence. This consideration is particularly important when using crystal structures of thermophilic ribosomes to interpret genetic results from nonextremophilic species. To address this issue, we have conducted a survey of spontaneously arising antibiotic-resistant mutants of the extremely thermophilic bacterium Thermus thermophilus, a species which has featured prominently in ribosome structural studies. We have accumulated over 20 single-base substitutions in T. thermophilus 16S and 23S rRNA, in the decoding site and in the peptidyltransferase active site of the ribosome. These mutations produce phenotypes that are largely identical to those of corresponding mutants of mesophilic organisms encompassing a broad phylogenetic range, suggesting that T. thermophilus may be an ideal model system for the study of ribosome structure and function.

A structural basis for streptomycin-induced misreading of the genetic code.

During protein synthesis, the ribosome selects aminoacyl-tRNAs with anticodons matching the mRNA codon present in the A-site of the small ribosomal subunit. The aminoglycoside antibiotic streptomycin disrupts decoding by binding close to the site of codon recognition. Here we use X-ray crystallography to define the impact of streptomycin on the decoding site of the Thermus thermophilus 30S ribosomal subunit in complexes with cognate or near-cognate anticodon stem-loop analogs (ASLs) and mRNA. Our crystal structures display a significant local distortion of 16S rRNA induced by streptomycin, including the crucial bases A1492 and A1493 that participate directly in codon recognition. Consistent with kinetic data, we observe that streptomycin stabilizes the near-cognate ASL complex, while destabilizing the cognate ASL complex. These data reveal how streptomycin disrupts the recognition of cognate ASLs and yet improves recognition of a near-cognate ASL.

Thermus thermophilus L11 Methyltransferase, PrmA, Is Dispensable for Growth and Preferentially Modifies Free Ribosomal Protein L11 Prior to Ribosome Assembly

The ribosomal protein L11 in bacteria is posttranslationally trimethylated at multiple amino acid positions by the L11 methyltransferase PrmA, the product of the prmA gene. The role of L11 methylation in ribosome function or assembly has yet to be determined, although the deletion of Escherichia coli prmA has no apparent phenotype. We have constructed a mutant of the extreme thermophile Thermus thermophilus in which the prmA gene has been disrupted with the htk gene encoding a heat-stable kanamycin adenyltransferase. This mutant shows no growth defects, indicating that T. thermophilus PrmA, like its E. coli homolog, is dispensable. Ribosomes prepared from this mutant contain unmethylated L11, as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and are effective substrates for in vitro methylation by cloned and purified T. thermophilus PrmA. MALDI-TOF MS also revealed that T. thermophilus L11 contains a total of 12 methyl groups, in contrast to the 9 methyl groups found in E. coli L11. Finally, we found that, as with the E. coli methyltransferase, the ribosomal protein L11 dissociated from ribosomes is a more efficient substrate for in vitro methylation by PrmA than intact 70S ribosomes, suggesting that methylation in vivo occurs on free L11 prior to its incorporation into ribosomes.

Altered discrimination of start codons and initiator tRNAs by mutant initiation factor 3.

IF3 is essential for ensuring the fidelity of the initiation step of translation in bacterial cells. Mutations at residues R99 and R131 in the C-terminal domain of the factor have previously been shown to increase initiation from the non-canonical GUA codon. Here we show that these mutant forms of IF3 fail to discriminate against initiation from many different non-AUG codons. They also enhance the activity of mutant tRNAs carrying changes in the three consecutive G-C pairs that are conserved in the anticodon stem of initiator tRNAs. In addition, the IF3 mutants stimulate initiations from leaderless mRNAs and from internal initiation codons, in the absence of any SD-anti-SD interaction. These results indicate that IF3 ensures the accuracy of initiation by inspecting both the codon-anticodon pairing and unique features of the initiator tRNA as well as suppressing initiation from other potential start sites within the mRNA.

Error-Prone and Error-Restrictive Mutations Affecting Ribosomal Protein S12

Ribosomal protein S12 plays a pivotal role in decoding functions on the ribosome. X-ray crystallographic analyses of ribosomal complexes have revealed that S12 is involved in the inspection of codon-anticodon pairings in the ribosomal A site, as well as in the succeeding domain rearrangements of the 30S subunit that are essential for accommodation of aminoacyl-tRNA. A role for S12 in tRNA selection is also well supported by classical genetic analyses; mutations affecting S12 are readily isolated in bacteria and organelles, since specific alterations in S12 confer resistance to the error-inducing antibiotic streptomycin, and the ribosomes from many such streptomycin-resistant S12 mutants display decreased levels of miscoding. However, substitutions that confer resistance to streptomycin likely represent a very distinct class of all possible S12 mutants. Until recently, the technical difficulties in generating random, unselectable mutations in essential genes in complex operons have generally precluded the analysis of other classes of S12 alterations. Using a recombineering approach, we have targeted the Escherichia coli rpsL gene, encoding S12, for random mutagenesis and screened the resulting mutants for effects on decoding fidelity. We have recovered over 40 different substitutions located throughout the S12 protein that alter the accuracy of translation without substantially affecting the sensitivity to streptomycin. Moreover, this collection includes mutants that promote miscoding, as well as those that restrict decoding errors. These results affirm the importance of S12 in decoding processes and indicate that alterations in this essential protein can have diverse effects on the accuracy of decoding.

Inactivation of the RluD Pseudouridine Synthase Has Minimal Effects on Growth and Ribosome Function in Wild-Type Escherichia coli and Salmonella enterica

The Escherichia coli rluD gene encodes a pseudouridine synthase responsible for the pseudouridine (Ψ) modifications at positions 1911, 1915, and 1917 in helix 69 of 23S rRNA. It has been reported that deletion of rluD in K-12 strains of E. coli is associated with extremely slow growth, increased readthrough of stop codons, and defects in 50S ribosomal subunit assembly and 30S-50S subunit association. Suppressor mutations in the prfB and prfC genes encoding release factor 2 (RF2) and RF3 that restore the wild type-growth rate and also correct the ribosomal defects have now been isolated. These suppressors link helix 69 Ψ residues with the termination phase of protein synthesis. However, further genetic analysis reported here also reveals that the slow growth and other defects associated with inactivation of rluD in E. coli K-12 strains are due to a defective RF2 protein, with a threonine at position 246, which is present in all K-12 strains. This is in contrast to the more typical alanine found at this position in most bacterial RF2s, including those of other E. coli strains. Inactivation of rluD in E. coli strains containing the prfB allele from E. coli B or in Salmonella enterica, both carrying an RF2 with Ala246, has negligible effects on growth, termination, or ribosome function. The results indicate that, in contrast to those in wild bacteria, termination functions in E. coli K-12 strains carrying a partially defective RF2 protein are especially susceptible to perturbation of ribosome-RF interactions, such as that caused by loss of h69 Ψ modifications.

Recognition of ribosomal protein L11 by the protein trimethyltransferase PrmA.

Bacterial ribosomal protein L11 is post‐translationally trimethylated at multiple residues by a single methyltransferase, PrmA. Here, we describe four structures of PrmA from the extreme thermophile Thermus thermophilus. Two apo‐PrmA structures at 1.59 and 2.3 Å resolution and a third with bound cofactor S‐adenosyl‐L‐methionine at 1.75 Å each exhibit distinct relative positions of the substrate recognition and catalytic domains, revealing how PrmA can position the L11 substrate for multiple, consecutive side‐chain methylation reactions. The fourth structure, the PrmA–L11 enzyme–substrate complex at 2.4 Å resolution, illustrates the highly specific interaction of the N‐terminal domain with its substrate and places Lys39 in the PrmA active site. The presence of a unique flexible loop in the cofactor‐binding site suggests how exchange of AdoMet with the reaction product S‐adenosyl‐L‐homocysteine can occur without necessitating the dissociation of PrmA from L11. Finally, the mode of interaction of PrmA with L11 explains its observed preference for L11 as substrate before its assembly into the 50S ribosomal subunit.

Severity of the Streptomycin Resistance and Streptomycin Dependence Phenotypes of Ribosomal Protein S12 of Thermus thermophilus Depends on the Identity of Highly Conserved Amino Acid Residues

The structural basis for the streptomycin dependence phenotype of ribosomal protein S12 mutants is poorly understood. Here we describe the application of site-directed mutagenesis and gene replacement of Thermus thermophilus rpsL to assess the importance of side chain identity and tertiary interactions as phenotypic determinants of drug-dependent mutants.