ABSTRACT
Staphylococcus aureus is a major opportunistic and versatile pathogen. Because the bacteria rapidly evolve multi-resistances towards antibiotics, there is an urgent need to find novel targets and alternative strategies to cure bacterial infections. Here, we provide a brief overview on the knowledge acquired on S. aureus ribosomes, which is one of the major antibiotic targets. We will show that subtle differences exist between the translation at the initiation step of Gram-negative and Gram-positive bacteria although their ribosomes display a remarkable degree of resemblance. In addition, we will illustrate using specific examples the diversity of mechanisms controlling translation initiation in S. aureus that contribute to shape the expression of the virulence factors in a temporal and dynamic manner.
ABSTRACT
The assembly of the protein synthesis machinery occurs during translation initiation. In bacteria, this process involves the binding of messenger RNA(mRNA) start site and fMet-tRNA(fMet) to the ribosome, which results in the formation of the first codon-anticodon interaction and sets the reading frame for the decoding of the mRNA. This interaction takes place in the peptidyl site of the 30S ribosomal subunit and is controlled by the initiation factors IF1, IF2 and IF3 to form the 30S initiation complex. The binding of the 50S subunit and the ejection of the IFs mark the irreversible transition to the elongation phase. Visualization of these ligands on the ribosome has been achieved by cryo-electron microscopy and X-ray crystallography studies, which has helped to understand the mechanism of translation initiation at the molecular level. Conformational changes associated with different functional states provide a dynamic view of the initiation process and of its regulation.
Subject(s)
Bacteria/genetics , Peptide Chain Initiation, Translational , Protein Biosynthesis , Bacteria/metabolism , Models, Molecular , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Nucleic Acid Conformation , Peptide Initiation Factors/chemistry , Peptide Initiation Factors/genetics , Peptide Initiation Factors/metabolism , Protein Conformation , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Transfer, Met/chemistry , Ribosome Subunits, Small, Bacterial/chemistry , Ribosome Subunits, Small, Bacterial/genetics , Ribosome Subunits, Small, Bacterial/metabolismABSTRACT
Using X-ray crystallography, we have directly observed the path of mRNA in the 70S ribosome in Fourier difference maps at 7 A resolution. About 30 nucleotides of the mRNA are wrapped in a groove that encircles the neck of the 30S subunit. The Shine-Dalgarno helix is bound in a large cleft between the head and the back of the platform. At the interface, only about eight nucleotides (-1 to +7), centered on the junction between the A and P codons, are exposed, and bond almost exclusively to 16S rRNA. The mRNA enters the ribosome around position +13 to +15, the location of downstream pseudoknots that stimulate -1 translational frame shifting.
Subject(s)
Nucleic Acid Conformation , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Ribosomes/chemistry , Ribosomes/metabolism , Bacteriophage T4/genetics , Base Pairing , Base Sequence , Binding Sites , Codon/genetics , Crystallography, X-Ray , DNA-Binding Proteins/genetics , Escherichia coli/genetics , Fourier Analysis , Frameshifting, Ribosomal , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Protein Subunits , RNA, Messenger/genetics , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 16S/metabolism , Ribosomes/genetics , Thermus thermophilus/chemistry , Viral Proteins/geneticsABSTRACT
We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound messenger RNA and transfer RNAs (tRNAs) at 5.5 angstrom resolution. All of the 16S, 23S, and 5S ribosomal RNA (rRNA) chains, the A-, P-, and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 angstrom movements associated with tRNA translocation with intersubunit movement.
Subject(s)
RNA, Messenger/chemistry , RNA, Ribosomal/chemistry , RNA, Transfer, Amino Acid-Specific/chemistry , RNA, Transfer/chemistry , Ribosomal Proteins/chemistry , Ribosomes/chemistry , Ribosomes/ultrastructure , Anticodon , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Base Sequence , Binding Sites , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Biosynthesis , Protein Conformation , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Messenger/metabolism , RNA, Ribosomal/metabolism , RNA, Transfer/metabolism , RNA, Transfer, Amino Acid-Specific/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism , Thermus thermophilus/chemistry , Thermus thermophilus/ultrastructureSubject(s)
Ligands , Ribosomes/ultrastructure , Binding Sites , Nucleic Acid Conformation , Protein Conformation , RNA, Messenger/chemistry , RNA, Messenger/metabolism , RNA, Ribosomal/chemistry , RNA, Ribosomal/ultrastructure , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Ribosomal Proteins/chemistry , Sensitivity and Specificity , Thermus thermophilusABSTRACT
Ribosomal protein S8, which is essential for the assembly of the central domain of 16S rRNA, is one of the most thoroughly studied RNA-binding proteins. To map its surrounding RNA in the ribosome, we carried out directed hydroxyl radical probing of 16S rRNA using Fe(II) tethered to nine different positions on the surface of protein S8 in 70S ribosomes. Hydroxyl radical-induced cleavage was observed near the classical S8-binding site in the 620 stem, and flanking the other S8-footprinted regions of the central domain at the three-helix junction near position 650 and the 825 and 860 stems. In addition, cleavage near the 5' terminus of 16S rRNA, in the 300 region of its 5' domain, and in the 1070 region of its 3'-major domain provide information about the proximity to S8 of RNA elements not directly involved in its binding. These data, along with previous footprinting and crosslinking results, allowed positioning of protein S8 and its surrounding RNA elements in a 7.8-A map of the Thermus thermophilus 70S ribosome. The resulting model is in close agreement with the extensive body of data from previous studies using protein-protein and protein-RNA crosslinking, chemical and enzymatic footprinting, and genetics.
Subject(s)
RNA, Ribosomal, 16S/chemistry , Ribosomal Proteins/chemistry , Ribosomes/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Crystallography, X-Ray , Hydroxyl Radical/chemistry , Models, Molecular , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Protein Conformation , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Ribosomal, 16S/metabolism , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Ribosomes/metabolism , Static Electricity , Thermus thermophilus/genetics , Thermus thermophilus/metabolismABSTRACT
Structures of 70S ribosome complexes containing messenger RNA and transfer RNA (tRNA), or tRNA analogs, have been solved by x-ray crystallography at up to 7.8 angstrom resolution. Many details of the interactions between tRNA and the ribosome, and of the packing arrangements of ribosomal RNA (rRNA) helices in and between the ribosomal subunits, can be seen. Numerous contacts are made between the 30S subunit and the P-tRNA anticodon stem-loop; in contrast, the anticodon region of A-tRNA is much more exposed. A complex network of molecular interactions suggestive of a functional relay is centered around the long penultimate stem of 16S rRNA at the subunit interface, including interactions involving the "switch" helix and decoding site of 16S rRNA, and RNA bridges from the 50S subunit.
Subject(s)
RNA, Ribosomal/chemistry , RNA, Transfer/chemistry , Ribosomes/chemistry , Ribosomes/physiology , Thermus thermophilus/chemistry , Anticodon/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Base Pairing , Binding Sites , Crystallization , Crystallography, X-Ray , Fourier Analysis , Models, Molecular , Nucleic Acid Conformation , Peptide Elongation Factors/metabolism , Protein Biosynthesis , Protein Conformation , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Messenger/chemistry , RNA, Messenger/metabolism , RNA, Ribosomal/metabolism , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 23S/chemistry , RNA, Transfer/metabolism , Ribosomal Proteins/chemistry , Ribosomal Proteins/metabolism , Ribosomes/ultrastructure , Thermus thermophilus/ultrastructureABSTRACT
The 7.8 angstrom crystal structure of the 70S ribosome reveals a discrete double-helical bridge (B4) that projects from the 50S subunit, making contact with the 30S subunit. Preliminary modeling studies localized its contact site, near the bottom of the platform, to the binding site for ribosomal protein S15. Directed hydroxyl radical probing from iron(II) tethered to S15 specifically cleaved nucleotides in the 715 loop of domain II of 23S ribosomal RNA, one of the known sites in 23S ribosomal RNA that are footprinted by the 30S subunit. Reconstitution studies show that protection of the 715 loop, but none of the other 30S-dependent protections, is correlated with the presence of S15 in the 30S subunit. The 715 loop is specifically protected by binding free S15 to 50S subunits. Moreover, the previously determined structure of a homologous stem-loop from U2 small nuclear RNA fits closely to the electron density of the bridge.
Subject(s)
RNA, Bacterial/chemistry , RNA, Ribosomal, 23S/chemistry , Ribosomal Proteins/metabolism , Ribosomes/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Escherichia coli/chemistry , Hydroxyl Radical , Nucleic Acid Conformation , Protein Conformation , RNA, Bacterial/metabolism , RNA, Ribosomal, 23S/metabolism , RNA, Small Nuclear/chemistry , RNA, Small Nuclear/metabolism , Ribosomal Proteins/chemistry , Ribosomes/metabolism , Ribosomes/ultrastructure , Thermus thermophilus/chemistryABSTRACT
During reverse transcription of the genomic RNA of human immunodeficiency virus type 1 (HIV-1) into double-stranded DNA, reverse transcriptase (RT) must accommodate RNA-RNA, DNA-RNA, RNA-DNA and DNA-DNA hybrids as primer-template. In this study, we examined extension of RNA-tRNA3Lys, and DNA-tRNA3Lys complexes by HIV-1 RT. When the 3' end of tRNA3Lys is annealed to oligoribonucleotides, tRNA3Lys, but not the complementary RNAs, is extended by HIV-1 RT, indicating that tRNA3Lys is efficiently used as primer and RNA as template. An opposite primer usage is observed when tRNA3Lys is annealed to complementary oligodeoxyribonucleotides. In this case, the oligodeoxyribonucleotides are efficiently used as primer and tRNA3Lys as template. This result indicates that the nature of nucleic acid bound to tRNA3Lys determines which strand of the RNA-tRNA3Lys and DNA-tRNA3Lys hybrids is extended by HIV-1 RT. When an oligoribonucleotide is annealed to an unmodified transcript of tRNA3Lys, both nucleic acids are extended by HIV-1 RT, indicating that specific selection of tRNA3Lys as primer requires the post-transcriptional modifications of tRNA3Lys.
Subject(s)
HIV-1/enzymology , RNA Processing, Post-Transcriptional , RNA, Transfer, Lys/metabolism , RNA-Directed DNA Polymerase/metabolism , Base Sequence , DNA, Viral/genetics , HIV Reverse Transcriptase , HIV-1/genetics , Humans , Molecular Sequence Data , RNA, Transfer, Lys/genetics , RNA, Viral/genetics , RNA-Directed DNA Polymerase/geneticsABSTRACT
trans-Diamminedichloroplatinum(II) was used to induce reversible cross-links between Escherichia coli initiation factor 2 (IF-2) and fMet-tRNA(f)(Met). Two distinct cross-links between IF-2 and the initiator tRNA were produced. Analysis of the cross-linking regions on both RNA and protein moieties reveals that the T arm of the tRNA is in the proximity of a region of the C-terminal domain of IF-2 (residues Asn611-Arg645). This cross-link is well-correlated with the fact that the C-domain of IF-2 contains the fMet-tRNA binding site and that the cross-linked RNA fragment precisely maps in a region which is protected by IF-2 from chemical modification and enzymatic digestion. Rather unexpectedly, a second cross-link was characterized which involves the anticodon arm of fMet-tRNA(f)(Met) and the N-terminal part of IF-2 (residues Trp215-Arg237).
Subject(s)
Cisplatin/pharmacology , Escherichia coli/metabolism , Eukaryotic Initiation Factor-2/metabolism , RNA, Transfer, Met/metabolism , Amino Acid Sequence , Base Sequence , Cross-Linking Reagents , Electrophoresis, Polyacrylamide Gel , Escherichia coli/drug effects , Eukaryotic Initiation Factor-2/chemistry , Eukaryotic Initiation Factor-2/isolation & purification , Kinetics , Molecular Sequence Data , Nucleic Acid Conformation , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Protein Conformation , RNA, Transfer, Met/chemistry , RNA, Transfer, Met/isolation & purification , Substrate SpecificityABSTRACT
The synthesis and purification of short model messenger RNAs modified with undecagold cluster are described. A monoamino undecagold cluster was introduced on the oxidized 3' cis-glycol group of the mRNA followed by reduction of the formed Schiff's base. The stability of the modified mRNA under the conditions used for in vitro messenger RNA translation is studied. The possibility of the formation of a specific translational initiation complex with bacterial ribosomes and modified mRNAs is shown. The results of these experiments indicate that the attachment of an undecagold cluster to a mRNA is a useful tool for electron microscopic and crystallographic studies.
Subject(s)
Organometallic Compounds/chemical synthesis , Organometallic Compounds/metabolism , RNA, Messenger/chemical synthesis , RNA, Messenger/metabolism , Ribosomes/metabolism , Base Sequence , Escherichia coli/metabolism , Gold/metabolism , Models, Molecular , Molecular Sequence Data , Organogold Compounds , Organometallic Compounds/chemistry , Protein Biosynthesis/drug effects , Protein Biosynthesis/genetics , RNA, Messenger/chemistry , RNA, Transfer, Met/metabolismABSTRACT
70S ribosomes from Thermus thermophilus are able to form ternary complexes with N-AcPhe-tRNAPhe from either Thermus thermophilus or Escherichia coli, in the presence of a short oligo(U) of six or nine uridines. A complex of N-AcPhe-tRNAPhe/(U)9/70S ribosome from Th. thermophilus was crystallized under the same conditions used for the growth of crystals from isolated ribosomes (S.D. Trakhanov, et al., (1987) FEBS Lett. 220, 319-322).
Subject(s)
RNA, Transfer, Phe/ultrastructure , Ribosomes/ultrastructure , Thermus thermophilus/ultrastructure , Crystallography , Macromolecular Substances , Protein Biosynthesis , RNA, Messenger/ultrastructureSubject(s)
Escherichia coli/metabolism , Peptide Biosynthesis , Peptides , Protein Biosynthesis , RNA, Transfer, Amino Acyl/metabolism , Ribosomes/metabolism , Amino Acids/metabolism , Carbon Radioisotopes , Indicators and Reagents , Kinetics , Polylysine/biosynthesis , Radioisotope Dilution Technique , Templates, GeneticABSTRACT
Misacylated phenylalanyl-tRNALys, just as lysyl-tRNALys, but not phenylalanyl-tRNAPhe, have been shown to serve as substrates for ribosomal synthesis of polypeptides (polyphenylalanine and polylysine, respectively) in the absence of a template polynucleotide (poly(A)). The conclusion was made that it is the structure of tRNA that determines the ability of the aminoacyl-tRNALys to participate in peptide elongation on ribosomes without codon-anticodon interactions.
