A Trichodermin-Resistant Mutant of Saccharomyces cerevisiae with an Abnormal Distribution of Native Ribosomal Subunits (original) (raw)
Related papers
European Journal of Biochemistry, 1975
Quantitative binding studies of [G-3H]anisomycin and [~cetyZ-~~C]trichodermin to sensitive and resistant 80-S ribosomes from yeasts are described in this work. A single mutation, most probably affecting the ribosome peptidyl transferase centre, appears to have pleiotropic effects on the ribosome leading to resistance to trichodermin and anisomycin and to an increased sensitivity to sparsomycin. Resistance to trichodermin is due to a reduced affinity of ribosomes from the mutant for the antibiotic. Ribosomes from the sensitive strain (Y166) bind [a~etyl-~~C]trichodermin with a dissociation constant of 0.99 pM while those from the resistant one (TR,) bind [~cetyZ-'~C]trichodermin with a dissociation constant of 15.4 pM. Similar results are obtained when the binding of [acetyZ-14C]trichodermin to Y166 and TR, 60-S subunits is studied.
Alterations in the Primary Structures of Ribosomal Proteins in Acquired Drug Resistance
2012
Acquired drug resistance is a multifactorial process that is one of the major causes for cancer treatment failure. The anticancer drug, mitoxantrone, was recently determined to inhibit ribosome biogenesis. Changes in ribosomal protein composition and efficiency with which the ribosomes incorporate 35 S-methionine has been noted in a mitoxantrone resistant MCF7 cell line when compared with a drugsusceptible parental cell line. This dissertation evaluated three proteomic workflows in order to successfully characterize the changes in the primary structures of cytoplasmic ribosomal proteins isolated from a mitoxantrone resistant breast cancer cell line that could serve some functional significance to the resistance when compared with a parental drug-susceptible cell line. A combination of the data from the three workflows allowed for the identification of 76 of the 79 human ribosomal proteins with an average sequence coverage of 76%. The N-terminal ends of 52 of the ribosomal proteins were identified using bottom-up and middle-down mass spectrometric approaches. An additional 7 N-terminal fragments were identified by top-down high resolution mass spectrometric analysis. Forty of the 52 N-terminal peptides were observed to have lost their N-terminal methionine and 19 were acetylated. Identification of the Nterminal peptides was most successful using the middle-down approach. Internal acetylations (on lysine) and phosphorylations were only noted with trypsin in-gel digestion and HPLC fraction analysis. Gel arrays of the two ribosomal populations illustrated differences in the protein compositions. Comparative densitometry imaging software indicated the presence of two novel protein spots in the drug resistant cell line as well six additional spots with increased and decreased abundances. High coverage bottom-up mass spectrometric analysis allowed for these protein spots to be assigned as isoform pairs of RPS3, RPS10, RPL11 and RPL23A. Molecular masses and top-down analyses were used to define the alterations in the ribosomal proteins in conjunction with high coverage bottom up and middle-down analyses. The change in the primary structures of these four ribosomal proteins is believed to alter access to the mRNA tunnel in the ribosome. This suggests that these ribosomes may participate in differential selective translation to allow for the cell to produce the necessary proteins during cellular stress.
Mutations Outside the Anisomycin-Binding Site Can Make Ribosomes Drug-Resistant
Journal of Molecular Biology, 2008
Eleven mutations have been identified in 23S rRNA that make Haloarcula marismortui resistant to anisomycin, an antibiotic that competes with the amino acid side chains of aminoacyl tRNAs for binding to the A-site cleft of the large ribosomal unit. The correlation observed between the sensitivity of H. marismortui to anisomycin and the affinity of its large ribosomal subunits for the drug indicates that its response to anisomycin is determined primarily by binding of the drug to its large ribosomal subunit. The structures of large ribosomal subunits containing resistance mutations show that these mutations can be divided into two classes: (1) those that interfere with specific drug-ribosome interactions, and (2) those that stabilize the apo-conformation of the A-site cleft of the ribosome relative to its drug-bound conformation. The conformational effects of some mutations of the second kind propagate through the ribosome for considerable distances, and are reversed when A-site substrates bind to the ribosome.
Molec. Gen Genet., 1978
Mutants ctl Schi:o.sutclrurottt.t'tr'.s potrrbc rverc isolated as resistant eithcr to trichode rmtn or to anisomycin. Growth tests showed that thc rnajority ol'mutants isolatecl wcrc cross resistant to both drugs rund also to cyclohe xirnide. A limitcd genetic analysis showcd that nlutants at least fbur loci, tri3, tri4. arri I ancl utti2, ltad this phenotype as wils also the case fbr rnutants at thrcc cycloheximiclc resistant loci, t.t'h2. t.t'ltJ and r'.r'ir4 rcportcd previously (lbrahim and Clodclington. 1976). Allclism lests showcd tl.rat thc lli 3, utri2 lrnd cr'[ 4 strains wcre allelic. A rrrutant at anoLhcr trichodcrntin resistant lclcus, /r.i5. was cross rcslstanl to anlsonrycin but sensitive to cyclo-Iicximidc. Ribosornes l}om rvilci type and selcctcd strains we rc analyscd in a poly L.l directed cell frec protein synthcsising systeln. Thrce strains, r'.r'/r I C 7, rrru I F I ancl lli-N l5 (probably a lli 5 allelc) possessed ribo-sornes which wcrc more rcsistant than the wilcl typc to thc clrugs usccl in their isolation. In each case the sitc of'thc resistancc was in thc 60S subunit. Ribo-sorttcs f'rom thc t',r'lr2, c.t'lt3 and ct'i4 strarns welc as sensi{ivc to cycloheximiclc as those from wild type. br Sprirtgcr-\'er llg l()1f be said to specify a ribosornal contponent. Hcncc the next step is usually to tcst various contbinations ol-ribosornes and supernatant cnzyntes fl-orn the resistant and wild type strains in a poly U directcd ccll free systcm in clrder to allocate thc sitc of'resistance to thc ribosontc.
L22 Ribosomal Protein and Effect of Its Mutation on Ribosome Resistance to Erythromycin
Journal of Molecular Biology, 2002
The ribosomal protein L22 is a core protein of the large ribosomal subunit interacting with all domains of the 23 S rRNA. The triplet Met82-Lys83-Arg84 deletion in L22 from Escherichia coli renders cells resistant to erythromycin which is known as an inhibitor of the nascent peptide chain elongation. The crystal structure of the Thermus thermophilus L22 mutant with equivalent triplet Leu82-Lys83-Arg84 deletion has been determined at 1.8 Å resolution. The superpositions of the mutant and the wild-type L22 structures within the 50 S subunits from Haloarcula marismortui and Deinococcus radiodurans show that the mutant b-hairpin is bent inward the ribosome tunnel modifying the shape of its narrowest part and affecting the interaction between L22 and 23 S rRNA. 23 S rRNA nucleotides of domain V participating in erythromycin binding are located on the opposite sides of the tunnel and are brought to those positions by the interaction of the 23 S rRNA with the L22 b-hairpin. The mutation in the L22 b-hairpin affects the orientation and distances between those nucleotides. This destabilizes the erythromycin-binding "pocket" formed by 23 S rRNA nucleotides exposed at the tunnel surface. It seems that erythromycin, while still being able to interact with one side of the tunnel but not reaching the other, is therefore unable to block the polypeptide growth in the drug-resistant ribosome.
2002
Summary A derivative of Mycobacterium smegmatis , which car- ries only one functional rRNA ( rrn ) operon, was used to isolate mutants resistant to the ribosome-targeted antibiotic linezolid. Isolation and characterization of linezolid-resistant clones revealed two classes of mutants. Ribosomes from class I mutants are resis- tant to oxazolidinones in an in vitro peptidyl trans- ferase assay, indicating that resistance maps to the ribosome component. In contrast, ribosomes from class II mutants show wild-type susceptibility to a linezolid derivative in vitro , pointing to a non-riboso- mal mechanism of resistance. Introduction of a wild- type ribosomal RNA operon into linezolid-resistant strains restored linezolid sensitivity in class I mutants, indicating that resistance (i) maps to the rRNA and (ii) is recessive. Sequencing of the entire rrn operon identified a single nucleotide alteration in 23S rRNA of class I mutant strains, 2447G Æ Æ Æ Æ T ( Escherichia coli numbering). Introd...
Antibiotics Targeting Ribosomes: Crystallographic Studies
Current Drug Target -Infectious Disorders, 2002
Resistance to antibiotics is a major problem in modern therapeutics. Ribosomes, the cellular organelle catalyzing the translation of the genetic code into proteins, are targets for several clinically relevant antibiotics. The ribosomes from eubacteria are excellent pathogen models. High resolution structures of the large and small ribosomal subunits were used as references that allowed unambiguous localization of almost a dozen antibiotic drugs, most of which are clinically relevant. Analyses of these structures showed a great diversity in the antibiotics' modes of action, such as interference with substrate binding, hindrance of the mobility required for the biosynthetic process and the blockage of tunnel which provides the path of exit for nascent proteins. All antibiotics studied by us were found to bind primarily to ribosomal RNA and, except for one allosteric effect, their binding did not cause major conformational changes. Antibiotics of the small ribosomal subunit may hinder tRNA binding, decoding, translocation, and the initiation of the entire biosynthetic process. The large subunit agents may target the GTPase center, interfere with peptide bond formation, or block the entrance of the nascent protein exit tunnel. The overall structure of the peptidyl transferase center and the modes of action of the antibiotic agents indicate that the ribosome serves as a template for proper positioning of tRNAs, rather than participating actively in the catalytic events associated with the creation of peptide bonds.