scholarly journals Ribosomal protein L3 functions as a ‘rocker switch’ to aid in coordinating of large subunit-associated functions in eukaryotes and Archaea

2008 ◽  
Vol 36 (19) ◽  
pp. 6175-6186 ◽  
Author(s):  
Arturas Meskauskas ◽  
Jonathan D. Dinman
Keyword(s):  
Ribosomal Protein ◽  
Large Subunit ◽  
Associated Functions ◽  
Ribosomal Protein L3

scholarly journals Mutations in the Bacterial Ribosomal Protein L3 and Their Association with Antibiotic Resistance

2015 ◽  
Vol 59 (6) ◽  
pp. 3518-3528 ◽  
Author(s):  
Rasmus N. Klitgaard ◽  
Eleni Ntokou ◽  
Katrine Nørgaard ◽  
Daniel Biltoft ◽  
Lykke H. Hansen ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Ribosomal Protein ◽  
Large Subunit ◽  
23S Rrna ◽  
Wild Type ◽  
Growth Data ◽  
Content Type ◽  
E Coli ◽  
Ribosomal Protein L3 ◽  
The Impact

ABSTRACTDifferent groups of antibiotics bind to the peptidyl transferase center (PTC) in the large subunit of the bacterial ribosome. Resistance to these groups of antibiotics has often been linked with mutations or methylations of the 23S rRNA. In recent years, there has been a rise in the number of studies where mutations have been found in the ribosomal protein L3 in bacterial strains resistant to PTC-targeting antibiotics but there is often no evidence that these mutations actually confer antibiotic resistance. In this study, a plasmid exchange system was used to replace plasmid-carried wild-type genes with mutated L3 genes in a chromosomal L3 deletion strain. In this way, the essential L3 gene is available for the bacteria while allowing replacement of the wild type with mutated L3 genes. This enables investigation of the effect of single mutations inEscherichia coliwithout a wild-type L3 background. Ten plasmid-carried mutated L3 genes were constructed, and their effect on growth and antibiotic susceptibility was investigated. Additionally, computational modeling of the impact of L3 mutations inE. coliwas used to assess changes in 50S structure and antibiotic binding. All mutations are placed in the loops of L3 near the PTC. Growth data show that 9 of the 10 mutations were well accepted inE. coli, although some of them came with a fitness cost. Only one of the mutants exhibited reduced susceptibility to linezolid, while five exhibited reduced susceptibility to tiamulin.

scholarly journals Cloning cDNAs for Genes Preferentially Expressed during Fruit Growth in Cucumber

1999 ◽  
Vol 124 (2) ◽  
pp. 136-139 ◽  
Author(s):  
Takuro Suyama ◽  
Kunio Yamada ◽  
Hitoshi Mori ◽  
Kiyotoshi Takeno ◽  
Shohei Yamaki
Keyword(s):  
Arabidopsis Thaliana ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Ribosomal Protein ◽  
Time Course ◽  
Large Subunit ◽  
Fruit Growth ◽  
Cdna Clones ◽  
Subtraction Hybridization ◽  
Ribosomal Protein L3

A cDNA library was constructed from poly(A)+RNA extracted from pollinated fruit of `PMR-142' cucumber (Cucumis sativus L.). Subtraction hybridization was made between the cDNAs and poly(A)+RNA from unpollinated fruit to isolate cDNA clones that corresponded to the genes preferentially expressed in the pollinated fruit. We isolated three cDNAs, which were 756, 826, and 998 nucleotides long and designated Csf1, Csf2, and Csf3, respectively. When fruit growth was triggered by pollination, auxin treatment and natural parthenocarpy, Csf2 was always expressed. Time course of expression of the Csf2 gene was nearly parallel to that of the fruit growth. Nucleotide sequences of the Csf cDNAs were fully determined. Homology of the deduced amino acid sequence for Csf1 showed 75% identity with a pea extensin. Only 37%, 33%, and 26% homology was found between Csf2 and bell pepper CaSn-2, tobacco FB7-4, and opium poppy gMLP15, respectively. The Csf3 sequence showed 68% identity with the large subunit of 60S ribosomal protein L3 of Arabidopsis thaliana.

scholarly journals Identification of Functionally Important Amino Acids of Ribosomal Protein L3 by Saturation Mutagenesis

2005 ◽  
Vol 25 (24) ◽  
pp. 10863-10874 ◽  
Author(s):  
Arturas Meskauskas ◽  
Alexey N. Petrov ◽  
Jonathan D. Dinman
Keyword(s):  
Ribosomal Protein ◽  
Protein Interactions ◽  
Ribosomal Proteins ◽  
Large Scale ◽  
Mutational Analysis ◽  
Core Protein ◽  
Large Subunit ◽  
Saturation Mutagenesis ◽  
Functional Regions ◽  
Ribosomal Protein L3

ABSTRACT There is accumulating evidence that many ribosomal proteins are involved in shaping rRNA into their functionally correct conformations through RNA-protein interactions. Moreover, although rRNA seems to play the central role in all aspects of ribosome function, ribosomal proteins may be involved in facilitating communication between different functional regions in ribosome, as well as between the ribosome and cellular factors. In an effort to more fully understand how ribosomal proteins may influence ribosome function, we undertook large-scale mutational analysis of ribosomal protein L3, a core protein of the large subunit that has been implicated in numerous ribosome-associated functions in the past. A total of 98 different rpl3 alleles were genetically characterized with regard to their effects on killer virus maintenance, programmed −1 ribosomal frameshifting, resistance/hypersensitivity to the translational inhibitor anisomycin and, in specific cases, the ability to enhance translation of a reporter mRNA lacking the 5′ 7mGppp cap structure and 3′ poly(A) tail. Biochemical studies reveal a correlation between an increased affinity for aminoacyl-tRNA and the extent of anisomycin resistance and a decreased peptidyltransferase activity and increased frameshifting efficiency. Immunoblot analyses reveal that the superkiller phenotype is not due to a defect in the ability of ribosomes to recruit the Ski-complex, suggesting that the defect lies in a reduced ability of mutant ribosomes to distinguish between cap+/poly(A)+ and cap−/poly(A)− mRNAs. The results of these analyses are discussed with regard to how protein-rRNA interactions may affect ribosome function.

scholarly journals Yeast NOP2 encodes an essential nucleolar protein with homology to a human proliferation marker.

1994 ◽  
Vol 127 (6) ◽  
pp. 1799-1813 ◽  
Author(s):  
E de Beus ◽  
J S Brockenbrough ◽  
B Hong ◽  
J P Aris
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Ribosomal Protein ◽  
Nucleolar Protein ◽  
Mrna Levels ◽  
Multicopy Plasmid ◽  
Yeast Saccharomyces Cerevisiae ◽  
Protein Levels ◽  
Significant Amino Acid Sequence ◽  
Ribosomal Protein L3

We have isolated a gene (NOP2) encoding a nucleolar protein during a search for previously unidentified nuclear proteins in the yeast Saccharomyces cerevisiae. The protein encoded by NOP2 (Nop2p) has a predicted molecular mass of 70 kD, migrates at 90 kD by SDS-PAGE, and is essential for cell viability. Nop2p shows significant amino acid sequence homology to a human proliferation-associated nucleolar protein, p120. Approximately half of Nop2p exhibits 67% amino acid sequence identity to p120. Analysis of subcellular fractions indicates that Nop2p is located primarily in the nucleus, and nuclear fractionation studies suggest that Nop2p is associated with the nucleolus. Indirect immunofluorescence localization of Nop2p shows a nucleolar-staining pattern, which is heterogeneous in appearance, and a faint staining of the cytoplasm. The expression of NOP2 during the transition from stationary phase growth arrest to rapid growth was measured, and compared to the expression of TCM1, which encodes the ribosomal protein L3. Nop2p protein levels are markedly upregulated during the onset of growth, compared to the levels of ribosomal protein L3, which remain relatively constant. NOP2 mRNA levels also increase during the onset of growth, accompanied by a similar increase in the levels of TCM1 mRNA. The consequences of overexpressing NOP2 from the GAL10 promoter on a multicopy plasmid were investigated. Although NOP2 overexpression produced no discernible growth phenotype and had no effect on ribosome subunit synthesis, overexpression was found to influence the morphology of the nucleolus, as judged by electron microscopy. Overexpression caused the nucleolus to become detached from the nuclear envelope and to become more rounded and/or fragmented in appearance. These findings suggest roles for NOP2 in nucleolar function during the onset of growth, and in the maintenance of nucleolar structure.

scholarly journals Expression of a Truncated Form of Ribosomal Protein L3 Confers Resistance to Pokeweed Antiviral Protein and the Fusarium Mycotoxin Deoxynivalenol

2005 ◽  
Vol 18 (8) ◽  
pp. 762-770 ◽  
Author(s):  
Rong Di ◽  
Nilgun E. Tumer
Keyword(s):  
Ribosomal Protein ◽  
Fusarium Species ◽  
Common Mechanism ◽  
Pokeweed Antiviral Protein ◽  
Wild Type ◽  
Antiviral Protein ◽  
Trichothecene Mycotoxin ◽  
Truncated Form ◽  
Fusarium Mycotoxin ◽  
Ribosomal Protein L3

The contamination of important agricultural products such as wheat, barley, or maize with the trichothecene mycotoxin deoxynivalenol (DON) due to infection with Fusarium species is a worldwide problem. Trichothecenes inhibit protein synthesis by targeting ribosomal protein L3. Pokeweed antiviral protein (PAP), a ribosome-inactivating protein binds to L3 to depurinate the α–sarcin/loop of the large rRNA. Plants transformed with the wild-type PAP show lesions and express very low levels of PAP because PAP autoregulates its expression by destabilizing its own mRNA. We show here that transgenic tobacco plants expressing both the wild-type PAP and a truncated form of yeast L3 (L3δ) are phenotypically normal. PAP mRNA and protein levels are very high in these plants, indicating that L3δ suppresses the autoregulation of PAP mRNA expression. Ribosomes are not depurinated in the transgenic plants expressing PAP and L3δ, even though PAP is associated with ribosomes. The expression of the endogenous tobacco ribosomal protein L3 is up-regulated in these plants and they are resistant to the Fusarium mycotoxin DON. These results demonstrate that expression of an N-terminal fragment of yeast L3 leads to trans-dominant resistance to PAP and the trichothecene mycotoxin DON, providing evidence that both toxins target L3 by a common mechanism.

scholarly journals Resistance to the Peptidyl Transferase Inhibitor Tiamulin Caused by Mutation of Ribosomal Protein L3

2003 ◽  
Vol 47 (9) ◽  
pp. 2892-2896 ◽  
Author(s):  
Jacob Bøsling ◽  
Susan M. Poulsen ◽  
Birte Vester ◽  
Katherine S. Long
Keyword(s):  
Ribosomal Protein ◽  
Drug Binding ◽  
Mechanisms Of Resistance ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Resistance Determinants ◽  
Drug Binding Site ◽  
Gene Coding ◽  
Bacterial Ribosome ◽  
Ribosomal Protein L3

ABSTRACT The antibiotic tiamulin targets the 50S subunit of the bacterial ribosome and interacts at the peptidyl transferase center. Tiamulin-resistant Escherichia coli mutants were isolated in order to elucidate mechanisms of resistance to the drug. No mutations in the rRNA were selected as resistance determinants using a strain expressing only a plasmid-encoded rRNA operon. Selection in a strain with all seven chromosomal rRNA operons yielded a mutant with an A445G mutation in the gene coding for ribosomal protein L3, resulting in an Asn149Asp alteration. Complementation experiments and sequencing of transductants demonstrate that the mutation is responsible for the resistance phenotype. Chemical footprinting experiments show a reduced binding of tiamulin to mutant ribosomes. It is inferred that the L3 mutation, which points into the peptidyl transferase cleft, causes tiamulin resistance by alteration of the drug-binding site. This is the first report of a mechanism of resistance to tiamulin unveiled in molecular detail.

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