Suppression in vitro: Identification of a Serine-sRNA as a "Nonsense" Suppressor

Capecchi, Mario R.; Gussin, Gary N.

doi:10.1126/science.149.3682.417

Cited by 216 publications

(41 citation statements)

References 23 publications

(1 reference statement)

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“…Soon after the identification of nonsense codons, nonsense suppression was demonstrated in vitro and in vivo through mutations in bacterial tRNA genes (Capecchi and Gussin, 1965). Since, suppression became the main genetic approach to investigate translation mechanisms and has been widely used in different organisms.…”

Section: Concepts and Facts Came First From Prokaryotesmentioning

confidence: 99%

Eukaryotic release factors (eRFs) history

Инге-Вечтомов

Zhouravleva

Maury

2003

Biology of the Cell

110

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In the present review, we describe the history of the identification of the eukaryotic translation termination factors eRF1 and eRF3. As in the case of several proteins involved in general and essential processes in all cells (e.g., DNA replication, gene expression regulation...) the strategies and methodologies used to identify these release factors were first established in prokaryotes. The genetic investigations in Saccharomyces cerevisiae have made a major contribution in the field. A large amount of data have been produced, from which it was concluded that the SUP45 and SUP35 genes were controlling translation termination but were also involved in other functions important for the cell organization and the cell cycle accomplishment. This does not seem to be restricted to yeast but is also probably the case in eukaryotes in general. The biochemical studies of the proteins encoded by the higher eukaryote homologs of SUP45 and SUP35 were efficient and permitted the identification of eRF1 as being the key protein in the termination process, eRF3 having a stimulating role. Around 25 years were needed after the identification of sup45 and sup35 mutants for the characterization of their gene products as eRF1 and eRF3, respectively. It also has to be pointed out that if the results came first from bacteria, the identification of RF3 and eRF3 was made practically at the same time. Moreover, eRF1 was the first crystal structure obtained for a class-1 release factor, the bacterial RF2 structure came later. The goal is now to understand at the molecular level the roles of both eRF1 and eRF3 in addition to their translation termination functions.

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Section: Concepts and Facts Came First From Prokaryotesmentioning

confidence: 99%

Eukaryotic release factors (eRFs) history

Инге-Вечтомов

Zhouravleva

Maury

2003

Biology of the Cell

110

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“…Complex I could have further functions : it could act as a precursor for phage and phage-like particles [8] as it was reported for the filamentous DNAbacteriophage fd [16]. I n this case the binding of a few fd-protein subunits, with or without a minor component, to fd DNA seems to be an inescapable condition for phage self assembly.…”

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confidence: 83%

“…Different types of RNA have catalytic function for the capsid formation and are packed into the capsid. A low amount of infectivity can be restored if fr RNA or the RNA of related phages is used, however, the bulk of particles remains non-infectious, fails to adsorb to the bacterial host and exposes part of its RNA to ribonuclease digestion, properties similar to those of defective particles obtained from phages mutant in the maturation factor [5-71.I n the case of the related phages MS2, R17 and f2 it was shown that with amounts of phage RNA increasing the I : 180 ratio, another complex is formed consisting of 5-6 protein subunits per RNA strand [2, Complex I could have further functions : it could act as a precursor for phage and phage-like particles [8] as it was reported for the filamentous DNAbacteriophage fd [16]. I n this case the binding of a few fd-protein subunits, with or without a minor component, to fd DNA seems to be an inescapable condition for phage self assembly.…”

mentioning

confidence: 99%

“…I n the case of the related phages MS2, R17 and f2 it was shown that with amounts of phage RNA increasing the I : 180 ratio, another complex is formed consisting of 5-6 protein subunits per RNA strand [2, Complex I could have further functions : it could act as a precursor for phage and phage-like particles [8] as it was reported for the filamentous DNAbacteriophage fd [16]. I n this case the binding of a few fd-protein subunits, with or without a minor component, to fd DNA seems to be an inescapable condition for phage self assembly.…”

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confidence: 99%

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Studies on a Possible Precursor in the Self Assembly of the Bacteriophage fr.

Höhn

1969

European Journal of Biochemistry

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Complex I , a complex consisting of I RNA strand and 6 protein subunits of the icosahedral RNA-phage fr was formed. The protein subunits bind on more than one site to the RNA. Complex I is very specific in respect to the RNA partner and its formation renders the RNA neither more nor less advantageous in forming phage-like particles with additional protein.The icosahedral capsid of the RNA bacteriophage fr and of related phages can be reconstituted in vitro [l-41. Different types of RNA have catalytic function for the capsid formation and are packed into the capsid. A low amount of infectivity can be restored if fr RNA or the RNA of related phages is used, however, the bulk of particles remains non-infectious, fails to adsorb to the bacterial host and exposes part of its RNA to ribonuclease digestion, properties similar to those of defective particles obtained from phages mutant in the maturation factor [5-71.I n the case of the related phages MS2, R17 and f2 it was shown that with amounts of phage RNA increasing the I : 180 ratio, another complex is formed consisting of 5-6 protein subunits per RNA strand [2, Complex I could have further functions : it could act as a precursor for phage and phage-like particles [8] as it was reported for the filamentous DNAbacteriophage fd [16]. I n this case the binding of a few fd-protein subunits, with or without a minor component, to fd DNA seems to be an inescapable condition for phage self assembly.I n contrast, complex1 could be a type of RNA not to be used in bacteriophage formation, in order to preserve its modulated messenger function.Results described in this paper show that complex I can be incorporated into phage-like particles, although it is not a necessary precursor. Compared with RNA, complex1 shows neither an advantage nor a disadvantage in being incorporated into the phage capsid.During an attempt to further characterize complex I , results were obtained strongly indicating that coat protein is bound t o RNA a t more than one site. Isolation of RNA and RNA Fragments Phage RNA was isolated by a %fold phenol extraction for 10 min a t 4'. As an alternative method phage was kept for 5 min a t 37' in 1 O l 0 sodium dodecyl sulfate with subsequent centrifugation in a sucrose gradient and collecting of the material sedimenting a t 28 S [ZO]. To obtain RNA fragments, 1 mg RNA dissolved in 500 pl H,O was treated for 5 min at 37" with 400 pg ribonuclease. Sucrose gradient centrifugation was performed for 2 hours a t 4" and 50,000 rev./ min in the SW50 rotor in a Spinco L2 centrifuge. RNA fragments sedimenting in distinct peaks a t 18S, 11 S, and 6 S were collected. MATERIALS AND METHODS IsolationAll preparations of RNA and RNA fragments were purified by ethanol precipitation.MS2-RNA was a kind gift of T. Sugiyama, tobacco mosaic virus RNA and turnip yellow mosaic virus RNA from K. W. Mundry and L. Bockstahler. ~51.Isolation of fr Protein 500pg fr, optionally labelled with 35S was dissolved in loop1 H,O and added to 2 0 0 4 ice cold glacial acetic acid. After 20 min the...

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“…They act di rectly as structural genes for the tRNAs which are altered in the anticodon [1,2,7,15] and translate as specific amino acids the three nonsense triplets, UAG (amber) [26,30], UAA (ochre) [12] and UGA (opal) [9,21] that code as signals for termination of the polypeptide chains.…”

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confidence: 99%