Translation of the genetic message

Self Cite

Section: F Assaymentioning

confidence: 94%

Section: F Assaymentioning

confidence: 99%

See 1 more Smart Citation

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

Lee‐Huang

Sillero

Ochoa

1971

Self Cite

“…Crude initiation factor was prepared from ribosomal wash [13,14]. The ribosome wash was mixed with protamine sulfate (Calbiochem) to final concentration of 0.077%, and placed in ice for 30 min.…”

mentioning

confidence: 99%

Requirement for Ribosome‐Releasing Factor for the Release of Ribosomes at the Termination Codon

Ogawa

Kaji

1975

With the use of 3H-labeled R17 amB2 phage RNA having an UAG codon at the seventh triplet of the coat cistron, release of the RNA from ribosomes at the termination codon was studied. The ribosome-releasing factor previously described was shown to stimulate the process of mRNA release at the termination codon. The stimulatory effect of the factor on this process was dependent on the termination factor (RF-1). GTP was required for this process and guanosine 5'-(P7y-methylene)triphosphate could not replace GTP. No apparent change of size of R17 RNA was observed during the release of the R17 RNA from the ribosomes. The ribosome-releasing factor is distinct from the known termination codon-specific factor such as RF-1.In the preceding publications [l -31 it was demonstrated that elongation factor G(EF-G), a previously unknown factor (tentatively named ribosome-releasing factor), and GTP were necessary for release of ribosomes from naturally occurring polysome which had been treated with puromycin. The ribosomereleasing factor has been purified to electrophoretic homogeneity and has a molecular weight of approximately 18000. It was proposed that this factor may play some role in the release of ribosomes from messenger RNA at the termination point of each cistron.In this communication, evidence is presented that ribosome-releasing factor can function for the release of ribosomes at the termination codon as well as other points in the messenger RNA. With the use of amB, R17RNA which has an amber codon at the seventh triplet of coat cistron [4-61, it was demonstrated that the factor catalyzed the release of mRNA from ribosomes at the amber codon. The released RNA was not fragmented during the release from ribosomes. Definirion. A,, unit, the quantitity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a 1-cm pathlength cell. EXPERIMENTAL PROCEDURE Preparation of Various FactorsThe ribosome-releasing factor was purified to electrophoretic homogeneity as described previously [l]. The crude soluble fraction of Escherichia coli containing the termination factor RF-1, was prepared free of ribosome-releasing factor as follows. The ribosome-free supernatant fluid (1 1, 9.26 g) prepared as described previously (step one of the purification of the factor by Hirashima and Kaji [I]). The protein fraction precipitated between 45 and 75 % saturation of ammonium sulfate, collected by centrifugation at 8500 rev./min for 15 min in a Sorvall GSA rotor, dissolved in 80ml of 10mM Tris-HC1 (pH 7.8), l.OmM magnesium acetate, 20 mM ammonium chloride, 0.5 mM dithiothreitol, 5 % glycerol, and dialyzed against the same buffer overnight. Insoluble material was removed by centrifugation and the solution (80 ml) containing 5.33 g of protein was placed on a DEAEcellulose column (2.6 x 86 cm), which had been equilibrated with the same buffer. The loaded column was washed with the same buffer. The ribosome-releasing factor was eluted during this washing process. The proteins retained on the column were...

“…It is further substantiated by the separation of the E.coli factor IF-3 into subfractions differing in their activity towards different cistrons in mRNAs, as well as isolation of 'interference' proteins modulating the specificity of this factor [8,9]. However, binding of synthetic mRNAs, such as poly(A,U,G), poly(U) [10,11] as well as formaldehyde-treated MS2 RNA to E.coli 70-S ribosomes or 3 0 3 subunits proceeds in the absence of initiation factors [12]. On the other hand, addition of IF-1, IF-2 and IF-3 to a system containing poly(A, U, G) and 3 0 4 subunits considerably enhances binding of f!Met-tRNA [13].…”

mentioning

confidence: 99%

Recognition of Initiation Codons in Modified f2 RNA by Escherichia coli Ribosomes

Szkopińska¹,

Zagórski²,

L³

et al. 1975

In the presence of E. coli ribosomes one molecule of modified f2 RNA binds about 50 times more fMettRNA than the native f2 RNA molecule. Our experiments show that E. colz ribosomes deprived of initiation factors are able to select initiation codons in modified f2 RNA. MATERIALS AND METHODS ChemicalsAll reagents were of analytical grade. Amino acids, GTP, alumina type 305 were purchased from Sigma Chemicals Co. (U.S.A.), sodium dodecylsulphate, 2-mercaptoethanol and Tris were from Kcch-Light Laboratories Ltd (England), CsCl was from Lachema (Czechoslovakia), RNase-free deoxyribonuclease and salt-free lysozyme were from Worthington Biochemical