Initiation of Protein Synthesis in Escherichia coli, I. Purification and Properties of the Initiation Factors

Wahba, Amy; Chae, Younbyoung; Iwasaki, Kentaro; Mazumder, Rajarshi; Miller, Martha J.; Sabol, Steven L.; Sillero, Marı́a A. Günther

doi:10.1101/sqb.1969.034.01.034

Cited by 24 publications

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“…Since the complex is not homogeneous and contains other proteins, this is to be considered a minimal value. It is, nevertheless, much larger than the lo/,, value reported by Wahba et al [14].…”

Section: Demonstration Of F3 Presence In Association With F2contrasting

confidence: 48%

“…GTP is required for the binding of fMet-tRNA to ribosomes [13]. Since this binding can be observed under certain conditions with F2 alone [5] attempts were made in several laboratories to demonstrate a GTP binding to protein F2 [14,15]. These attempts showed, however, that, if any, only minute amounts of GTP could be bound (in the order of lo/o of the expected value [14]).…”

Section: Demonstration Of F3 Presence In Association With F2mentioning

confidence: 99%

“…The stoichiometry of this binding was calculated from the data shown in Fig. 3 : assuming for the complex a molecular weight of about 100000 (estimated by filtration through Sephadex G-200, and in keeping with the known molecular weights of F2 and F3 [14]), 0.25 mole GTP are retained per mole protein. Since the complex is not homogeneous and contains other proteins, this is to be considered a minimal value.…”

Section: Demonstration Of F3 Presence In Association With F2mentioning

confidence: 99%

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A Novel Form of Initiation Factors from Escherichia coil which Binds Formylmethionyl tRNA and GTP: “F2 · F3 Complex”

Groner

Revel

1971

European Journal of Biochemistry

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A fraction containing a complex between initiation factors F2 and F3 was isolated from Escherichia coli ribosomes. I n contrast to the free factors, this F2 -F3 complex makes a stable ternary association with formylmethionyl-tRNA and GTP. F2 -F3 complex can be formed on the ribosomes during the process of initiation. It acts preferentially on the 30 S ribosomal subunit to which it confers a much larger affinity for fMet-tRNA than observed with the free factors. A model for the function of F2 F3 complex as an intermediate in the initiation cycle is proposed.During initiation of protein synthesis, formylmethionyl-tRNA is attached to the ribosome to donate the first amino acid of the peptide chain [l]. During elongation, all other aminoacyl-tRNAs are successively bound to the ribosome after forming a complex with elongation factors and GTP [2]. Ono et al. [3] demonstrated that elongation factors recognize and bind aminoacyl tRNAs with the exception of fMet-tRNA. Since binding of this initiator tRNA is promoted by the initiation factors and GTP [4,5], it was tempting to verify if indeed these proteins specifically recognize fMet-tRNA and to search for the formation of a complex similar to that found in the case of elongation factors. Until now, however, all attempts to demonstrate such a complex with initiation factors Fl(A), F2(B) or F3(C) remained have unsuccessful.While purifying initiation factors from Escherichia coli we observed the existence of a fraction containing both initiation proteins F2 and F3 activities.This fraction designated "F2 -F3 complex" was partially purified and its properties are reported here. I n contrast to the free factors, F2 * F3 can form a stable complex with GTP and fMet-tRNA in the absence of ribosomes. No other aminoacyl-tRNAs are bound. The data reported here suggest that F2 -F3 complex participates on the 3 0 s ribosome in the process of peptide chain initiation. MATERIALS AND METHODSThe preparation of high salt (2 M NH4C1) washed ribosomes from E. coli MRE 600 was described previously [5]. Ribosomal subunits from washed ribosomes were prepared according to . Formyl [36S]methionyl-tRNA from E . coli was prepared as before [5]. T factor preparation was a gift from Dr. A. Panet. Fractionation and Purification of Initiation FactorsInitiation factors were purified from the 1 M NH,C1 ribosomal wash fluid [7] obtained from 1200 g E. coli MRE 600. The protein precipitated by SOo/, saturation of ammonium sulfate were dissolved in 20 mM potassium phosphate buffer pH 7.2, 0.2 mM MgCl,, 7 mM 2-mercaptoethanol and 501, glycerol (buffer P). Initiation factor activities Fl(A), F2(C) and F3(B) were separated by chromatography on DEAE-cellulose [5]. F1 was further p d e d on Sephadex G-50 and chromatography on phosphocellulose according to Hershey et al. [S]. Factor F2 fractions from DEAE-cellulose were further purified on DEAE-Sephadex, hydroxylapatite and Sephadex G-200 [9]. F3 was further fractionated and purified on DEAE-Sephadex and phosphocellulose as described elsewhere [7]. Isolation o...

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Section: Demonstration Of F3 Presence In Association With F2contrasting

confidence: 48%

Section: Demonstration Of F3 Presence In Association With F2mentioning

confidence: 99%

Section: Demonstration Of F3 Presence In Association With F2mentioning

confidence: 99%

See 1 more Smart Citation

A Novel Form of Initiation Factors from Escherichia coil which Binds Formylmethionyl tRNA and GTP: “F2 · F3 Complex”

Groner

Revel

1971

European Journal of Biochemistry

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“…As seen in Figs. 2C and F, highly purified initiation factor F3(B) (18,19) indeed is a powerful inhibitor of single ribosome accumulation: its presence in the reaction mixture leads to the appearance of large amounts of 32P-labeled 50 and 30S ribosomal subunits instead of single ribosomes, while few, if any, 3H-labeled subunits are converted to single ribosomes.…”

Section: Formation Of Single Ribosomes From Subunitsmentioning

confidence: 99%

“…Accordingly, the dissociation of single ribosomes would be required for protein synthesis to proceed. One of the three known initiation factors for protein synthesis found associated with 30S ribosomal subunits (15), F3(B)* (16)(17)(18)(19), also appears to dissociate single ribosomes under defined conditions in vitro (18)(19)(20)(21), and was postulated to mediate the dissociation step (20,21).…”

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confidence: 99%

Control of Single Ribosome Formation by an Initiation Factor for Protein Synthesis

Kaempfer

1971

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACT30 and 50S ribosomal subunits, released from polysomes upon polypeptide chain termination, possess a high affinity for each other and readily form single ribosomes. Highly purified initiation factor F3(B), acting stoichiometrically, prevents the formation of single ribosomes without promoting their dissociation. These findings are interpreted in terms of a ribosome cycle in which a limiting amount of factor F3(B) controls the number of ribosomes active in protein synthesis, in response to metabolic changes in the cell, by regulating the flow of ribosomal subunits into polysomes or into a sidetrack pool of synthetically inactive single ribosomes. The reported apparent ribosome dissociation activity of F3(B) is explained.In a cell, ribosomes occur in different functional states: ribosomes actively engaged in protein synthesis (present in polysomes), large and small ribosomal subunits, and single ribosomes. Single ribosomes, in contrast to monosomes, do not carry messenger RNA or growing polypeptide chains and are not participating in protein synthesis. Both in proand eukaryotic cells, translating ribosomes frequently undergo exchange of their two subunits, apparently by dissociation after each passage over messenger RNA and reformation from a pool of ribosomal subunits that continuously recycle through polysomes (1-5). By contrast, single ribosomes, at least in mammalian cells, are not in rapid equilibrium with polysomes or ribosomal subunits (6-9).When the over-all rate of protein synthesis decreases, single ribosomes accumulate at the expense of polysomes, while the size of the ribosomal subunit pool remains essentially unchanged (6,8,10,11). The accumulation of single ribosomes was first interpreted as evidence that they are released from polysomes on polypeptide chain termination, and thus are obligatory intermediates of the ribosome cycle intervening between polysomes and the ribosomal subunit pool (11)(12)(13)(14). Accordingly, the dissociation of single ribosomes would be required for protein synthesis to proceed. One of the three known initiation factors for protein synthesis found associated with 30S ribosomal subunits (15), F3(B)* (16-19), also appears to dissociate single ribosomes under defined conditions in vitro (18-21), and was postulated to mediate the dissociation step (20,21).Recently, however, evidence was presented that ribosomes dissociate directly into subunits upon termination of protein synthesis, and that single ribosomes are formed by association of subunits present in the pool (22). Protein synthesis in Escherichia coli extracts diluted sufficiently to prevent the reformation of ribosomes from subunits results in the conversion of polysomes to ribosomal subunits, not single ribosomes, even though single ribosomes do not noticeably dissociate under these conditions. When protein synthesis is allowed to occur in a more concentrated mixture of isotopically heavy and excess light cell extracts, single ribosomes accumulate; those originating from fully heavy polysomes are entirely of ...

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Die Translation der genetischen Information am Ribosom

Schreiber

1971

Angewandte Chemie

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Die in der Basensequenz der Nucleinsäuren enthaltene Information für die Proteinstruktur wird am Ribosom in die Aminosäurensequenz der Proteine übersetzt. Diese Übersetzung, Translation genannt, läßt sich in den Kettenstart, die Kettenverlängerung und den Kettenabschluß gliedern. An jedem Abschnitt sind mehrere spezifische Proteinfaktoren und Nucleinsäuren beteiligt. – Beim Kettenstart werden aus der Startaminosäure‐tRNA, der mRNA mit dem Startsignal und der kleinen und großen Untereinheit eines Ribosoms Startkomplexe gebildet. Dabei wirken GTP und die Startfaktoren mit. – Bei der Kettenverlängerung wird in einem Reaktionszyklus jeweils eine Aminosäure aus der Bindung an die tRNA in eine Bindung in der Polypeptidkette überführt. Zunächst wird die zu inkorporierende Aminosäure als Aminoacyl‐tRNA an das Ribosom gebunden, wozu GTP und Proteinfaktoren erforderlich sind. Die anschließende Entstehung der Peptidbindung wird durch die Peptidyltransferase der großen Ribosomenuntereinheit katalysiert. Danach wird die nunmehr um eine Aminosäure verlängerte Peptidyl‐tRNA auf dem Ribosom von der Aminosäure‐Acceptorstelle A an die Peptidyl‐Donorstelle P verlagert. Hierzu sind ein weiterer Proteinfaktor und die Spaltung von GTP in GDP und Phosphat notwendig. – Der Kettenabschluß wird eingeleitet, sobald eines der drei Terminatortripletts UAA, UAG oder UGA auf der sich relativ zum Ribosom vom 5′‐ zum 3′‐Ende bewegenden mRNA das Ribosom erreicht. Die Ablösung der fertigen Polypeptidketten vom Ribosom hängt von den Ablösefaktoren ab. Vor dem Start einer neuen Polypeptidkette dissoziieren die Ribosomen in ihre Untereinheiten.

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Initiation of Protein Synthesis in Escherichia coli, I. Purification and Properties of the Initiation Factors

Cited by 24 publications

References 0 publications

A Novel Form of Initiation Factors from Escherichia coil which Binds Formylmethionyl tRNA and GTP: “F2 · F3 Complex”

A Novel Form of Initiation Factors from Escherichia coil which Binds Formylmethionyl tRNA and GTP: “F2 · F3 Complex”

Control of Single Ribosome Formation by an Initiation Factor for Protein Synthesis

Die Translation der genetischen Information am Ribosom

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