scholarly journals Effect of α-sarcin and ribosome-inactivating proteins on the interaction of elongation factors with ribosomes

1989 ◽  
Vol 257 (3) ◽  
pp. 723-727 ◽  
Author(s):  
M Brigotti ◽  
F Rambelli ◽  
M Zamboni ◽  
L Montanaro ◽  
S Sperti
Keyword(s):  
Higher Plants ◽  
Ricinus Communis ◽  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Artemia Salina ◽  
Elongation Factors ◽  
Ribosome Inactivating Proteins ◽  
Rabbit Reticulocyte Lysate System ◽  
Reticulocyte Lysate ◽  
Comparable Activity

alpha-Sarcin from Aspergillus giganteus and the ribosome-inactivating proteins (RIPs) from higher plants inactivate the 60 S ribosomal subunit. The former is an RNAase, whereas RIPs are N-glycosidases. The site of cleavage of RNA and that of N-glycosidic depurinization are at one nucleotide distance in 28 S rRNA [Endo & Tsurugi (1987) J. Biol. Chem. 262, 8128-8130]. The effect of alpha-sarcin and that of RIPs on the interaction of elongation factors with Artemia salina (brine shrimp) ribosomes have been investigated. alpha-Sarcin inhibits both the EF1 (elongation factor 1)-dependent binding of aminoacyl-tRNA and the GTP-dependent binding of EF2 (elongation factor 2) to ribosomes, whereas two of the RIPs tested, ricin from Ricinus communis (castor bean) and volkensin from Adenia volkensii (kilyambiti), inhibit only the latter reaction. EF2 protects ribosomes from inactivation by both alpha-sarcin and ricin. The EF1-binding site is affected only by alpha-sarcin. The sensitivity of this site to alpha-sarcin is increased by pretreatment of ribosomes with ricin. A. salina ribosomes were highly resistant to the third RIP tested, namely gelonin from Gelonium multiflorum. All four proteins tested have, however, a comparable activity on the rabbit reticulocyte-lysate system.

Flammulin: A novel ribosome-inactivating protein from fruiting bodies of the winter mushroom Flammulina velutipes

2000 ◽  
Vol 78 (6) ◽  
pp. 699-702 ◽  
Author(s):  
H X Wang ◽  
T B Ng
Keyword(s):  
Deae Cellulose ◽  
Flammulina Velutipes ◽  
Fruiting Bodies ◽  
Terminal Sequence ◽  
Ribosome Inactivating Protein ◽  
Ribosome Inactivating Proteins ◽  
Rabbit Reticulocyte Lysate System ◽  
Free Translation ◽  
Reticulocyte Lysate ◽  
Low Ionic Strength

A protein with a molecular weight of 40 kDa, capable of inhibiting cell-free translation in a rabbit reticulocyte lysate system with an IC50 of 0.25 nM, was isolated from fruiting bodies of the mushroom Flammulina velutipes. The protein, designated flammulin, was devoid of ribonuclease activity. Flammulin was unadsorbed on DEAE-cellulose at neutral pH and low ionic strength and adsorbed on CM-Sepharose and Affi-gel blue gel under similar conditions. Its N-terminal sequence demonstrates sites of similarity to those of plant ribosome-inactivating proteins (RIPs).Key words: mushroom, ribosome-inactivating protein, fruiting body.

scholarly journals Effect of modeccin on the steps of peptide-chain elongation

1978 ◽  
Vol 176 (2) ◽  
pp. 371-379 ◽  
Author(s):  
L Montanaro ◽  
S Sperti ◽  
M Zamboni ◽  
M Denaro ◽  
G Testoni ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Peptide Bond ◽  
Inhibitory Effect ◽  
Artemia Salina ◽  
Chain Elongation ◽  
Peptide Bond Formation ◽  
Experimental Conditions ◽  
Related Plant ◽  
The Individual

Modeccin inhibits polypeptide-chain elongation catalysed by Artemia salina (brine shrimp) ribosomes by inactivating the 60 S ribosomal subunit. Among the individual steps of elongation, peptide-bond formation, catalysed by 60 S peptidyltransferase, is unaffected by the toxin, whereas the binding of EF 2 (elongation factor 2) to ribosomes is strongly inhibited. Modeccin does not affect the poly(U)-dependent non-enzymic binding of either deacylated tRNAPhe or phenylalanyl-tRNA to ribosomes. The inhibitory effect of modeccin on the EF 1 (elongation factor 1)-dependent binding of phenylalanyl-tRNA is discussed, since it is decreased by tRNAPhe, which stimulates the binding reaction. The analysis of the distribution of ribosome-bound radioactivity during protein synthesis shows that modeccin consistently inhibits the radioactivity bound as long-chain peptides, but depending on the experimental conditions, can leave unchanged or even greatly stimulates the radioactivity bound as phenylalanyl-tRNA and/or short-chain peptides. It is concluded that, during the complete elongation cycle, modeccin does not affect the binding of the first aminoacyl-tRNA to ribosomes, but inhibits some step in the subsequent repetitive activity of either EF 1 or EF 2. The results obtained indicate that the mechanism of action of modeccin is very similar to that of ricin and related plant toxins such as abrin and crotin.

scholarly journals Effect of the antibiotic purpuromycin on cell-free protein-synthesizing systems

1989 ◽  
Vol 259 (1) ◽  
pp. 307-310 ◽  
Author(s):  
F Rambelli ◽  
M Brigotti ◽  
M Zamboni ◽  
M Denaro ◽  
L Montanaro ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Elongation Factor ◽  
Eukaryotic Cell ◽  
Artemia Salina ◽  
Culture Broth ◽  
Gram Positive Bacteria ◽  
Reticulocyte Lysate ◽  
Directed System ◽  
Bacteria And Fungi ◽  
Ribosomal Binding Site

Purpuromycin, an antibiotic isolated from the culture broth of Actinoplanes ianthinogenes, which is very active against Gram-positive bacteria and fungi, inhibits protein synthesis in both prokaryotic and eukaryotic cell-free systems. The ID50 was 9 microM with the endogenous mRNA-directed rabbit reticulocyte lysate, 17 microM with a poly(U)-directed system from Escherichia coli and 69 microM with a poly(U)-directed system from Artemia salina cysts. Of the three steps of elongation, purpuromycin does not affect the peptidyl-transferase reaction, inhibits the elongation factor 1 (EF-1) dependent binding of phenylalanyl-tRNA and stimulates the GTP-dependent binding of EF-2. When protein synthesis is stopped by the addition of purpuromycin, the nascent peptide chains are found in the puromycin-reactive P site. The results suggest that the mechanism of action of purpuromycin is similar to that of fusidic acid. Both antibiotics would seem to produce a stable guanine nucleotide-ribosome-EF-2 complex which allows one round of translocation but prevents, because of a common or overlapping ribosomal binding site for the two elongation factors, the subsequent EF-1-dependent binding of aminoacyl-tRNA.

scholarly journals Relationship between elongation factor 1- and elongation factor 2-dependent guanosine triphosphatase activities of ribosomes. Inhibition of both activities by ricin

1975 ◽  
Vol 148 (3) ◽  
pp. 447-451 ◽  
Author(s):  
S Sperti ◽  
L Montanaro ◽  
A Mattioli ◽  
G Testoni
Keyword(s):  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Elongation Factors ◽  
Toxic Protein ◽  
Elongation Factor 2 ◽  
Guanosine Triphosphatase ◽  
Elongation Factor 1 ◽  
Ricin A

The elongation factor 1- and elongation factor 2-dependent GTPase (guanosine triphosphatase) activities of ribosomes are inhibited by ricin, a toxic protein known to inactivate the 60S ribosomal subunit. It is suggested that also in eukaryotic ribosomes a “GTPase site’, located on the larger subunit, is common to the two elongation factors.

A Homodimeric Sporamin-Type Trypsin Inhibitor with Antiproliferative, HIV Reverse Transcriptase-Inhibitory and Antifungal Activities from Wampee (Clausena lansium) Seeds

2003 ◽  
Vol 384 (2) ◽  
pp. 289-293 ◽  
Author(s):  
T.B. Ng ◽  
S.K. Lam ◽  
W.P. Fong
Keyword(s):  
Reverse Transcriptase ◽  
Trypsin Inhibitor ◽  
Simple Procedure ◽  
Inhibitory Effect ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Human Leukemia ◽  
Hiv Reverse Transcriptase ◽  
Rabbit Reticulocyte Lysate System ◽  
Reticulocyte Lysate

Abstract A homodimeric trypsin inhibitor with a molecular mass of 54 kDa was isolated from the seeds of Clausena lansium (Lour) Skeels with a very simple procedure comprising extraction with an aqueous buffer and ion exchange chromatography on CM-cellulose. It inhibited trypsin with an IC50 of 2.2 nM but was without any inhibitory effect on chymotrypsin and proteinase K. The uptake of MTT by human leukemia HL60 and hepatoma Hep G2 cells was inhibited with an IC50 of 100 uM. Translation in the cellfree rabbit reticulocyte lysate system was inhibited with an IC50 of 3.6 uM. The activity of HIV-1 reverse transcriptase was reduced in the presence of the trypsin inhibitor. The trypsin inhibitor exerted antifungal activity toward Physalospora piricola but not Mycosphaerella arachidicola, Botrytis cinerea, Fusarium oxysporum or Coprinus comatus.

scholarly journals Plastid-Localized EMB2726 Is Involved in Chloroplast Biogenesis and Early Embryo Development in Arabidopsis

2021 ◽  
Vol 12 ◽  
Author(s):  
Chuanling Li ◽  
Jian-Xiu Shang ◽  
Chenlei Qiu ◽  
Baowen Zhang ◽  
Jinxue Wang ◽  
...  
Keyword(s):  
Chloroplast Development ◽  
Developmental Process ◽  
Higher Plants ◽  
Critical Role ◽  
Elongation Factor ◽  
Early Embryo ◽  
The Body ◽  
Insertion Mutant ◽  
Cell Stage ◽  
Dna Insertion

Embryogenesis is a critical developmental process that establishes the body organization of higher plants. During this process, the biogenesis of chloroplasts from proplastids is essential. A failure in chloroplast development during embryogenesis can cause morphologically abnormal embryos or embryonic lethality. In this study, we isolated a T-DNA insertion mutant of the Arabidopsis gene EMBRYO DEFECTIVE 2726 (EMB2726). Heterozygous emb2726 seedlings produced about 25% albino seeds with embryos that displayed defects at the 32-cell stage and that arrested development at the late globular stage. EMB2726 protein was localized in chloroplasts and was expressed at all stages of development, such as embryogenesis. Moreover, the two translation elongation factor Ts domains within the protein were critical for its function. Transmission electron microscopy revealed that the cells in emb2726 embryos contained undifferentiated proplastids and that the expression of plastid genome-encoded photosynthesis-related genes was dramatically reduced. Expression studies of DR5:GFP, pDRN:DRN-GFP, and pPIN1:PIN1-GFP reporter lines indicated normal auxin biosynthesis but altered polar auxin transport. The expression of pSHR:SHR-GFP and pSCR:SCR-GFP confirmed that procambium and ground tissue precursors were lacking in emb2726 embryos. The results suggest that EMB2726 plays a critical role during Arabidopsis embryogenesis by affecting chloroplast development, possibly by affecting the translation process in plastids.

scholarly journals Arabidopsis small nucleolar RNA monitors the efficient pre-rRNA processing during ribosome biogenesis

2016 ◽  
Vol 113 (42) ◽  
pp. 11967-11972 ◽  
Author(s):  
Pan Zhu ◽  
Yuqiu Wang ◽  
Nanxun Qin ◽  
Feng Wang ◽  
Jia Wang ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Higher Plants ◽  
Ribosomal Subunit ◽  
Rrna Processing ◽  
Developmental Defects ◽  
Protein Mutants ◽  
5.8S Rrna ◽  
Important Regulator ◽  
Nucleolar Rna

Ribosome production in eukaryotes requires the complex and precise coordination of several hundred assembly factors, including many small nucleolar RNAs (snoRNAs). However, at present, the distinct role of key snoRNAs in ribosome biogenesis remains poorly understood in higher plants. Here we report that a previously uncharacterized C (RUGAUGA)/D (CUGA) type snoRNA, HIDDEN TREASURE 2 (HID2), acts as an important regulator of ribosome biogenesis through a snoRNA–rRNA interaction. Nucleolus-localized HID2 is actively expressed in Arabidopsis proliferative tissues, whereas defects in HID2 cause a series of developmental defects reminiscent of ribosomal protein mutants. HID2 associates with the precursor 45S rRNA and promotes the efficiency and accuracy of pre-rRNA processing. Intriguingly, disrupting HID2 in Arabidopsis appears to impair the integrity of 27SB, a key pre-rRNA intermediate that generates 25S and 5.8S rRNA and is known to be vital for the synthesis of the 60S large ribosomal subunit and also produces an imbalanced ribosome profile. Finally, we demonstrate that the antisense-box of HID2 is both functionally essential and highly conserved in eukaryotes. Overall, our study reveals the vital and possibly conserved role of a snoRNA in monitoring the efficiency of pre-rRNA processing during ribosome biogenesis.

The ribosome as a molecular machine: the mechanism of tRNA–mRNA movement in translocation

2011 ◽  
Vol 39 (2) ◽  
pp. 658-662 ◽  
Author(s):  
Marina V. Rodnina ◽  
Wolfgang Wintermeyer
Keyword(s):  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Thermal Fluctuations ◽  
Spontaneous Movement ◽  
Conformational States ◽  
Gtp Hydrolysis ◽  
Time Resolved ◽  
Directional Bias ◽  
Loosely Coupled ◽  
Dynamic Events

Translocation of tRNA and mRNA through the ribosome is one of the most dynamic events during protein synthesis. In the cell, translocation is catalysed by EF-G (elongation factor G) and driven by GTP hydrolysis. Major unresolved questions are: how the movement is induced and what the moving parts of the ribosome are. Recent progress in time-resolved cryoelectron microscopy revealed trajectories of tRNA movement through the ribosome. Driven by thermal fluctuations, the ribosome spontaneously samples a large number of conformational states. The spontaneous movement of tRNAs through the ribosome is loosely coupled to the motions within the ribosome. EF-G stabilizes conformational states prone to translocation and promotes a conformational rearrangement of the ribosome (unlocking) that accelerates the rate-limiting step of translocation: the movement of the tRNA anticodons on the small ribosomal subunit. EF-G acts as a Brownian ratchet providing directional bias for movement at the cost of GTP hydrolysis.

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