The path of the growing peptide chain through the 23S rRNA in the 50S ribosomal subunit; a comparative cross-linking study with three different peptide families

1. The 30S ribosomal subunit of the extreme halophile Halobacterium cutirubrum is unstable and loses 75% of its ribosomal protein when the 70S ribosome is dissociated into the two subunits. A stable 30S subunit is obtained if the dissociation of the 70S particle is carried out in the presence of the soluble fraction. 2. A fractionation procedure was developed for the selective removal of groups of proteins from the 30S and 50S subunits. When the ribosomes, which are stable in 4m-K+ and 0.1m-Mg2+, were extracted with low-ionic-strength buffer 75–80% of the 30S proteins and 60–65% of the 50S proteins as well as the 5S rRNA were released. The proteins in this fraction are the most acidic of the H. cutirubrum ribosomal proteins. Further extraction with Li+–EDTA releases additional protein, leaving a core particle containing either 16S rRNA or 23S rRNA and about 5% of the total ribosomal protein. The amino acid composition, mobility on polyacrylamide gels at pH4.5 and 8.7, and the molecular-weight distribution of the various protein fractions were determined. 3. The s values of the rRNA are 5S, 16S and 23S. The C+G contents of the 16S and 23S rRNA were 56.1 and 58.8% respectively and these are higher than C+G contents of the corresponding Escherichia coli rRNA (53.8 and 54.1%).

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Ketolide Antimicrobial Activity Persists after Disruption of Interactions with Domain II of 23S rRNA

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.10.3677-3683.2004 ◽

2004 ◽

Vol 48 (10) ◽

pp. 3677-3683 ◽

Cited By ~ 20

Author(s):

Guy W. Novotny ◽

Lene Jakobsen ◽

Niels M. Andersen ◽

Jacob Poehlsgaard ◽

Stephen Douthwaite

Keyword(s):

Antimicrobial Activity ◽

Ribosomal Subunit ◽

23S Rrna ◽

Alkyl Aryl ◽

Interaction Sites ◽

Enhanced Resistance ◽

Low Levels ◽

Domain V ◽

Block Protein Synthesis ◽

Do So

ABSTRACT Ketolides are the latest derivatives developed from the macrolide erythromycin to improve antimicrobial activity. All macrolides and ketolides bind to the 50S ribosomal subunit, where they come into contact with adenosine 2058 (A2058) within domain V of the 23S rRNA and block protein synthesis. An additional interaction at nucleotide A752 in the rRNA domain II is made via the synthetic carbamate-alkyl-aryl substituent in the ketolides HMR3647 (telithromycin) and HMR3004, and this interaction contributes to their improved activities. Only a few macrolides, including tylosin, come into contact with domain II of the rRNA and do so via interactions with nucleotides G748 and A752. We have disrupted these macrolide-ketolide interaction sites in the rRNA to assess their relative importance for binding. Base substitutions at A752 were shown to confer low levels of resistance to telithromycin but not to HMR3004, while deletion of A752 confers low levels of resistance to both ketolides. Mutations at position 748 confer no resistance. Substitution of guanine at A2058 gives rise to the MLSB (macrolide, lincosamide, and streptogramin B) phenotype, which confers resistance to all the drugs. However, resistance to ketolides was abolished when the mutation at position 2058 was combined with a mutation in domain II of the same rRNA. In contrast, the same dual mutations in rRNAs conferred enhanced resistance to tylosin. Our results show that the domain II interactions of telithromycin and HMR3004 differ from each other and from those of tylosin. The data provide no indication that mutations within domain II, either alone or in combination with an A2058 mutation, can confer significant levels of telithromycin resistance.

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A comparison of the unfolding and dissociation of the large ribosome subunits from Rhodopseudomonas·spheroides N.C.I.B. 8253 and Escherichia coli M.R.E. 600

Biochemical Journal ◽

10.1042/bj1330739 ◽

1973 ◽

Vol 133 (4) ◽

pp. 739-747 ◽

Cited By ~ 2

Author(s):

A. Robinson ◽

J. Sykes

Keyword(s):

Escherichia Coli ◽

Protein Interactions ◽

Ribosomal Proteins ◽

Ribosomal Subunit ◽

23S Rrna ◽

Core Particle ◽

Protein Loss ◽

E Coli ◽

Rrna Species ◽

Rhodopseudomonas Spheroides

1. The behaviour of the large ribosomal subunit from Rhodopseudomonas spheroides (45S) has been compared with the 50S ribosome from Escherichia coli M.R.E. 600 (and E. coli M.R.E. 162) during unfolding by removal of Mg2+ and detachment of ribosomal proteins by high univalent cation concentrations. The extent to which these processes are reversible with these ribosomes has also been examined. 2. The R. spheroides 45S ribosome unfolds relatively slowly but then gives rise directly to two ribonucleoprotein particles (16.6S and 13.7S); the former contains the intact primary structure of the 16.25S rRNA species and the latter the 15.00S rRNA species of the original ribosome. No detectable protein loss occurs during unfolding. The E. coli ribosome unfolds via a series of discrete intermediates to a single, unfolded ribonucleoprotein unit (19.1S) containing the 23S rRNA and all the protein of the original ribosome. 3. The two unfolded R. spheroides ribonucleoproteins did not recombine when the original conditions were restored but each simply assumed a more compact configuration. Similar treatments reversed the unfolding of the E. coli 50S ribosomes; replacement of Mg2+ caused the refolding of the initial products of unfolding and in the presence of Ni2+ the completely unfolded species (19.1S) again sedimented at the same rate as the original ribosomes (44S). 4. Ribosomal proteins (25%) were dissociated from R. spheroides 45S ribosomes by dialysis against a solution with a Na+/Mg2+ ratio of 250:1. During this process two core particles were formed (21.2S and 14.2S) and the primary structures of the two original rRNA species were conserved. This dissociation was not reversed. With E. coli 50S approximately 15% of the original ribosomal protein was dissociated, a single 37.6S core particle was formed, the 23S rRNA remained intact and the ribosomal proteins would reassociate with the core particle to give a 50S ribosome. 5. The ribonuclease activities in R. spheroides 45S and E. coli M.R.E. 600 and E. coli M.R.E. 162 50S ribosomes are compared. 6. The observations concerning unfolding and dissociation are consistent with previous reports showing the unusual rRNA complement of the mature R. spheroides 45S ribosome and show the dependence of these events upon the rRNA and the importance of protein–protein interactions in the structure of the R. spheroides ribosome.

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The structural basis for inhibition of ribosomal translocation by viomycin

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2002888117 ◽

2020 ◽

Vol 117 (19) ◽

pp. 10271-10277

Author(s):

Ling Zhang ◽

Ying-Hui Wang ◽

Xing Zhang ◽

Laura Lancaster ◽

Jie Zhou ◽

...

Keyword(s):

Conformational Changes ◽

Binding Sites ◽

Ribosomal Subunit ◽

Structural Basis ◽

23S Rrna ◽

Subunit Dissociation ◽

Classical State ◽

State Transfer ◽

70S Ribosome ◽

Intersubunit Rotation

Viomycin, an antibiotic that has been used to fight tuberculosis infections, is believed to block the translocation step of protein synthesis by inhibiting ribosomal subunit dissociation and trapping the ribosome in an intermediate state of intersubunit rotation. The mechanism by which viomycin stabilizes this state remains unexplained. To address this, we have determined cryo-EM and X-ray crystal structures of Escherichia coli 70S ribosome complexes trapped in a rotated state by viomycin. The 3.8-Å resolution cryo-EM structure reveals a ribosome trapped in the hybrid state with 8.6° intersubunit rotation and 5.3° rotation of the 30S subunit head domain, bearing a single P/E state transfer RNA (tRNA). We identify five different binding sites for viomycin, four of which have not been previously described. To resolve the details of their binding interactions, we solved the 3.1-Å crystal structure of a viomycin-bound ribosome complex, revealing that all five viomycins bind to ribosomal RNA. One of these (Vio1) corresponds to the single viomycin that was previously identified in a complex with a nonrotated classical-state ribosome. Three of the newly observed binding sites (Vio3, Vio4, and Vio5) are clustered at intersubunit bridges, consistent with the ability of viomycin to inhibit subunit dissociation. We propose that one or more of these same three viomycins induce intersubunit rotation by selectively binding the rotated state of the ribosome at dynamic elements of 16S and 23S rRNA, thus, blocking conformational changes associated with molecular movements that are required for translocation.

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Precise determination of RNA–protein contact sites in the 50 S ribosomal subunit of Escherichia coli

Biochemical Journal ◽

10.1042/bj3340039 ◽

1998 ◽

Vol 334 (1) ◽

pp. 39-42 ◽

Cited By ~ 18

Author(s):

Bernd THIEDE ◽

Henning URLAUB ◽

Helga NEUBAUER ◽

Gerlinde GRELLE ◽

Brigitte WITTMANN-LIEBOLD

Keyword(s):

Escherichia Coli ◽

Ribosomal Proteins ◽

Ribosomal Subunit ◽

Laser Desorption ◽

Precise Determination ◽

Cross Linking ◽

Laser Desorption Ionization ◽

Precise Data ◽

Contact Sites

RNA–protein cross-linked complexes were isolated and purified to obtain precise data about RNA–protein contact sites in the 50 S ribosomal subunit of Escherichia coli. N-terminal microsequencing and matrix-assisted laser desorption ionization MS were used to identify the cross-linking sites at the amino acid and nucleotide levels. In this manner the following contact sites of five ribosomal proteins with the 23 S rRNA were established: Lys-67 of L2 to U-1963, Tyr-35 of L4 to U-615, Lys-97 of L21 to U-546, Lys-49 of L23 to U-139 or C-140 and Lys-71 and Lys-74 of L27 to U-2334.

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Resistance to Linezolid Caused by Modifications at Its Binding Site on the Ribosome

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05702-11 ◽

2011 ◽

Vol 56 (2) ◽

pp. 603-612 ◽

Cited By ~ 192

Author(s):

Katherine S. Long ◽

Birte Vester

Keyword(s):

Binding Site ◽

Ribosomal Proteins ◽

Resistance Mechanism ◽

Ribosomal Subunit ◽

Resistance Mechanisms ◽

Drug Binding ◽

23S Rrna ◽

Detailed Knowledge ◽

Linezolid Resistance ◽

Almost All

ABSTRACTLinezolid is an oxazolidinone antibiotic in clinical use for the treatment of serious infections of resistant Gram-positive bacteria. It inhibits protein synthesis by binding to the peptidyl transferase center on the ribosome. Almost all known resistance mechanisms involve small alterations to the linezolid binding site, so this review will therefore focus on the various changes that can adversely affect drug binding and confer resistance. High-resolution structures of linezolid bound to the 50S ribosomal subunit show that it binds in a deep cleft that is surrounded by 23S rRNA nucleotides. Mutation of 23S rRNA has for some time been established as a linezolid resistance mechanism. Although ribosomal proteins L3 and L4 are located further away from the bound drug, mutations in specific regions of these proteins are increasingly being associated with linezolid resistance. However, very little evidence has been presented to confirm this. Furthermore, recent findings on the Cfr methyltransferase underscore the modification of 23S rRNA as a highly effective and transferable form of linezolid resistance. On a positive note, detailed knowledge of the linezolid binding site has facilitated the design of a new generation of oxazolidinones that show improved properties against the known resistance mechanisms.

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Retapamulin Inhibition of Translation and 50S Ribosomal Subunit Formation in Staphylococcus aureus Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00475-07 ◽

2007 ◽

Vol 51 (9) ◽

pp. 3385-3387 ◽

Cited By ~ 18

Author(s):

W. Scott Champney ◽

Ward K. Rodgers

Keyword(s):

Staphylococcus Aureus ◽

16S Rrna ◽

Cell Viability ◽

Protein Biosynthesis ◽

Ribosomal Subunit ◽

Inhibitory Effect ◽

23S Rrna ◽

Specific Inhibition ◽

Methicillin Resistant

ABSTRACT Retapamulin inhibited protein biosynthesis and cell viability in methicillin-sensitive and methicillin-resistant Staphylococcus aureus organisms. A specific inhibitory effect on 50S ribosomal subunit formation was also found. Pulse-chase labeling experiments confirmed the specific inhibition of 50S subunit biogenesis. Turnover of 23S rRNA was found, with no effect on 16S rRNA amounts.

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