Isolation and Properties of Crystalline Initiation Factor F<sub>1</sub> from <i>Escherichia coli</i> Ribosomes

Lee‐Huang, Sylvia; Sillero, Marı́a A. Günther; Ochoa, Severo

doi:10.1111/j.1432-1033.1971.tb01274.x

Cited by 37 publications

(5 citation statements)

References 32 publications

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“…These results are in good agreement with the total amino acid composition of the protein, but are quite different from the com-position of IF-l from E. COZY Q13 reported in [22]. The most striking differences are the high content of lysine (10 residues) and arginine (8 residues), and the presence of 1 cysteine and 1 tryptophan reported in [22] compared to lysine (5 residues) and arginine' (6 residues) and the complete absence of cysteine and tryptophan in the protein of the present report. Our results, on the other hand, are in good agreement with the amino acid composition of IF-1 reported in [23].…”

Section: Resultssupporting

confidence: 80%

Structure‐function relationships in Escherichia coliinitiation factors

1979

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Section: Resultssupporting

confidence: 80%

Structure‐function relationships in Escherichia coliinitiation factors

1979

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“…Details of this method will be published elsewhere (Muller and H. Noll, in preparation). Purified initiation factor IF-1 was prepared according to Lee-Huang et al (17), IF-2 as described by Remold-O'Donnell and Thach (18), and IF-3 by the method of Dubnoff and Maitra (19). Each preparation gave only one major band upon acrylamide gel electrophoresis; all three factors were required for translation of R17 RNA.…”

Section: Methodsmentioning

confidence: 99%

Initiation Factor IF-3-Dependent Binding of Escherichia coli Ribosomes and N -Formylmethionine Transfer-RNA to Rabbit Globin Messenger

Noll¹,

Noll²,

Lingrel³

1972

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

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An initiation complex has been formed in high yields from E. coli ribosomes, 9S messenger RNA for rabbit hemoglobin, and N-formylmethionine-tRNA. Initiation factor IF-3 is required for the binding and puromycin is required for the release of fMet. Valyl-tRNA fails to bind to the second codon, whereas a mixture of 15 aminoacyl-tRNAs promotes incorporation. Together with quantitative data, the findings suggest that IF-3 directs the ribosomes to an AUG codon on one of the two globin messengers, at a site that is different from the normal starting point for globin synthesis.The mechanism by which ribosomes recognize the AUG start signal at the beginning of a message is not understood. The two most obvious possibilities are: (i) the secondary structure of the messenger exposes only the starting triplet and masks internal AUG triplets, regardless of whether they are in or out of phase; or (ii) the ribosomes respond to an additional signal, for example a specific nucleotide sequence present only near the initial AUG triplet. The second possibility is attractive, because it could explain translational control if we assume that the signal is specific for certain classes of messengers, and if we further postulate specific protein adaptors that allow ribosomes to recognize only their cognate messengers (1-7). Favored candidates for such a role in specificity have been initiation factors required for the correct attachment of ribosomes to natural messengers.Although partial discrimination has been reported with different IF-3 fractions and bacteriophage messengers (2-8),the basis of the specificity has not been established, and the nature of the signal is elusive (9). A more decisive answer could be expected by testing a bacterial translation system with a known messenger from the other end of evolution. The development of a purified system from Escherichia coli in which nearly all the ribosomes are active in initiation with R17 RNA (10) and the availability of messenger RNA for rabbit globin (11) have made such an approach feasible.

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“…EF-Tu (13) was a generous gift from D. Miller of this Institute. Initiation factors and CRP were prepared from a 1 M NH4Cl wash of ribosomes according to methods described previously (14)(15)(16)(17). Gel analysis in the presence of sodium dodecyl sulfate showed that IF-3 and CRP were >90% pure, IF-2 >70% pure, and IF-1 about 40% pure.…”

mentioning

confidence: 99%

DNA-directed synthesis in vitro of beta-galactosidase: requirement for a ribosome release factor.

Kung¹,

Treadwell²,

Spears³

et al. 1977

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

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Tle DNA-directed synthesis of fl-galactosidase in Escherichia coli extracts has been investigated in a partially fractionated system. A dependency was obtained for 3',5'-cyclic AMP receptor protein and also for a factor, from the salt wash of ribosomes, that has been purified to near homogeneity. This factor has been identified with a ribosome release factor previously purified from the supernatant fraction by A. Hirashima and A. Kaji [(1972) Biochemisty 11, 4037-40441. In the coupled transcription-translation system this factor stimulates fl-galactosidase synthesis and total protein synthesis 2-to 4-fold. It is thus clear that the ribosome release factor has a physiological function in translation. It may also affect transcription, because it stimulated total RNA synthesis up to 50% in this in vitro system.In order to better understand the detailed mechanism of gene expression we undertook the purification of the components required for the DNA-dependent synthesis in vitro of f3-galactosidase by a system from Escherichia colh extracts (1-4). In these earlier studies the synthesis was dependent on ribosomal subunits, an NH4Cl wash of the ribosomes, and a supernatant fraction. Subsequently, the roles of the ribosomes and the factors in the salt wash were examined in more detail. Dependencies were obtained for ribosomal proteins L7 and L12 (1), for initiation factors IF-1 and IF-3 (2), and for three factors in the supernatant extract: RNA polymerase, a factor called L factor, and elongation factor (EF) Tu (3, 4). In a preliminary report (5) it was shown that at least two other components of the salt wash of the ribosomes, in addition to the initiation factors, were required for fl-galactosidase synthesis. One was 3',5'-cyclic AMP receptor protein (CRP) and the other, whose function was unknown, was called La.Though it initially seemed likely that La was a novel factor, the present studies have revealed that it is a known but littlestudied factor. In 1969 Modolell and Davis (6) demonstrated that in the presence of fusidic acid ribosomes remain on mRNA after their nascent peptides have been released by puromycin. This finding suggested that the release of nascent peptides does not result in the automatic release of ribosomes from mRNA. It was subsequently found that after the release of polypeptides from purified polysomes by puromycin, the release of the ribosomes from mRNA required a novel supernatant factor (noted in ref. 7). The ribosome release factor (RRF) was meanwhile described in detail, on the basis of a similar activity, by Hirashima and Kaji (8).The similarity between L,, and RRF was not initially noted because La was obtained from the salt wash of the ribosomes, while RRF has been isolated from the ribosome-free superna-The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 ...

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Isolation and Properties of Crystalline Initiation Factor F₁ from Escherichia coli Ribosomes

Cited by 37 publications

References 32 publications

Structure‐function relationships in Escherichia coliinitiation factors

Structure‐function relationships in Escherichia coliinitiation factors

Initiation Factor IF-3-Dependent Binding of Escherichia coli Ribosomes and N -Formylmethionine Transfer-RNA to Rabbit Globin Messenger

DNA-directed synthesis in vitro of beta-galactosidase: requirement for a ribosome release factor.

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Isolation and Properties of Crystalline Initiation Factor F1 from Escherichia coli Ribosomes

Cited by 37 publications

References 32 publications

Structure‐function relationships in Escherichia coliinitiation factors

Structure‐function relationships in Escherichia coliinitiation factors

Initiation Factor IF-3-Dependent Binding of Escherichia coli Ribosomes and N -Formylmethionine Transfer-RNA to Rabbit Globin Messenger

DNA-directed synthesis in vitro of beta-galactosidase: requirement for a ribosome release factor.

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Isolation and Properties of Crystalline Initiation Factor F₁ from Escherichia coli Ribosomes