Activity of different forms of initiation factor 2 in the vitro synthesis of beta-galactosidase.
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Coliphages↗
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DNA-directed in vitro synthesis of beta-galactosidase: requirement for formylation of methionyl-tRNAf.
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Animals↗
Inhibition of protein synthesis by glucose 6-phosphate and fructose 1,6-diphosphate in lysed rabbit reticulocytes and the reversal of inhibition by NAD+.
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Animals↗
Quantitative comparisons of various biological responses of neutrophils to different active and inactive chemotactic factors.
The effects of chemotactic factors on rabbit neutrophils were evaluated measuring cell migration in modified Boyden chambers and under agarose, in lysosomal enzyme release, leukocyte aggregation, and in vivo neutropenia. Chemotactins employed included the complement-derived C3 and C5 fragments, the bacterial chemotactic factor from culture supernatant fluids of Escherichia coli, and the synthetic chemotactic factors Met-Leu-Phe and formyl-Met-Leu-Phe. A consistent parallelism was found in all the leukocyte responses to a given chemotactic factor. In no instance, with any of the five chemotactic factor preparations, did cells responding in one assay system fail to respond in the four other assay systems, suggesting a common event in all of the cell responses. Boyden chamber chemotaxis was consistently the most sensitive assay; the agarose assay was, in general, less sensitive by a factor of 100 fold. Enzyme release approached, in cell sensitivity to chemotactic factors, that of the Boyden chamber assay. In general, in vitro leukocyte aggregation and in vivo neutropenia were considerably less sensitive assays. Chemotactic factor inactivator (CFI) purified from human serum destroyed in parallel all biological activities of C3 and C5 chemotactic factors but had no effect on the bacterial chemotactic factor and the activities of synthetic chemotactic peptides.
Aminopeptidases↗
Binding of polyanions to carrier ampholytes in isoelectric focusing.
The binding of carrier ampholytes to polyanions is markedly pH-dependent: it is very strong at pH 3, rather weak at pH 5 and abolished at pH 7. Binding is affected by the type of negative charge, its density and spatial orientation on the polyanion. On the basis of the type of negative charge, the binding strength decreases in the following order: polyphosphate greater than polysulphate greater than polycarboxylate. Given the same type of negative charge, the binding is dependent on charge density and its space orientation: thus polyglutamic acid forms stronger complexes than polygalacturonic acid. The minimum length of the polyanion eliciting a measurable binding appears to be of the order of about six negative charges, as demonstrated with hexametaphosphate.
Anions↗
Identification, purification and properties of clone-specific glycoprotein antigens constituting the surface coat of Trypanosoma brucei.
Soluble glycoproteins have been purified from a series of clones of Trypanosoma brucei 427. Each clone yielded a characteristic predominant glycoprotein which induced clone-specific immunity to trypanosome infection in mice. These glycoproteins were shown by specific labelling and enzyme digestion of cells to be the major components of the trypanosome surface coat. Each glycoprotein consisted of a single polypeptide chain having an apparent molecular weight of 65 000 (as measured by SDS-polyacrylamide gel electrophoresis) and containing around 600 amino acid and 20 monosaccharide residues. Preliminary structural studies indicated large changes in amino acid sequence dispersed over a considerable length of the polypeptide chain. Proteolytic activity was demonstrated in semi-purified trypanosome extracts, providing one reason for the heterogeneity sometimes observed in surface glycoprotein antigen preparations.
Animals↗
Direct iodination of specific residues in crystals of yeast formylatable methionine-accepting transfer ribonucleic acid.
Crystals of yeast formylatable methionine-accepting transfer ribonucleic acid (tRNAfMet) were iodinated by using a modification of Commerford's procedure [Commerford, S.L. (1971) Biochemistry 10, 1993]. Chromatographic analysis of nuclease digestion products showed that radioactive iodine binds covalently to the 5 position of three nucleotide residues: U8, C73, and C74. These three iodine substitutions were assigned to three peaks in a difference Fourier synthesis comparing the iodinated derivative with native tRNAfMet. In this way the positions of U8, C73, and C74 were marked in the crystal structure of yeast tRNAfMet, providing guidepoints for the interpretation of a 4.5-A electron density map.
Base Sequence↗
Proton nuclear magnetic resonance study of the effect of pH on tRNA structure.
The low-field 220-MHz proton nuclear magnetic resonance (NMR) spectra of four tRNA molecules, Escherichia coli tRNAPhe, tRNA1Val, and tRNAfMet1, and yeast tRNAPhe, at neutral and mildly acidic pH are compared. We find a net increase in the number of resonances contributing to the -9.9-ppm peak (downfield from sodium 4,4-dimethyl-4-silapentanesulfonate) in three of these tRNAs at pH 6, while tRNAfMet1 does not clearly exhibit this behavior. The increase in intensity at this resonance position is half-completed at pH 6.2 in the case of yeast tRNAPhe. An alteration at the 5'-phosphate terminus is not involved, since removal of the terminal phosphate does not affect the gain in intensity at -9.9 ppm. Based on a survey of the tertiary interactions in the four molecules, assuming that they possess tertiary structures like that of yeast tRNAPhe at neutral pH, we tentatively attribute this altered resonance in E. coli and yeast tRNAPhe to the protonation of the N3 of the adenine residue at position 9 which results in the stabilization of the tertiary triple A23-U12-A9. This intepretation is supported by model studies on the lowfield proton NMR spectrum of AN oligomers at acid pH, which reveal an exchanging proton resonance at -9.4 ppm if the chain length N greater than or equal to 6.
Circular Dichroism↗
Activation of the rabbit polymorphonuclear leukocyte membrane "Na+, K+"-ATPase by chemotactic factor.
Addition of the synthetic chemotactic factor, formyl-methionyl-leucyl-phenylala-nine (F-Met-Leu-Phe) to medium containing magnesium, sodium, and potassium results in a doubling of the "Na+, K+"-ATPase activity of the plasma membrane fraction from polymophonuclear leukocytes (PMN). This activation is sensitive to ouabain inhibition and is dose dependent, maximal activity occuring at 10(-9)MF-Met-Leu-Phe. Equivalent activation was observed with the nonformylated derivative Met-Leu-Phe at 10(-9)M. The dipeptide, carbobenzoxy-methionylphenylalanine, which acts as an antagonist for F-Met-Leu-Phe, prevents the stimulation of the "Na+, K+"-ATPase by F-Met-Leu-Phe.
Adenosine Triphosphatases↗
Stability of the unique anticodon loop conformation of E.coli tRNAfMet.
Initiator tRNAs have an anticodon loop conformation distinct from that of elongation tRNAs as detected by susceptibility to S1 nuclease. We now find the anticodon loop conformation of E. coli tRNAfMet to be stable under different salt conditions as detected by using S1 nuclease as a structural probe. In contrast, a conformational change is observed in the T- and D- loop of this tRNA in the absence of added Mg2+. This change can be suppressed by spermine. Even under those conditions effecting a change in T- and D- loop conformation, the anticodon loop does not change. This suggests that the conformational shift is controlled by Mg2+ and restricted to the D- and T- loop region only without affecting the anticodon domain. The use of S1 nuclease as a conformational probe requires the use of kinetic studies to determine the initial cleavage sites. Thus, the use of a strong inhibitor which immediately stops the action of this nuclease is necessary. ATP is shown to be such an inhibitor.
Anticodon↗
Nucleotide sequence of formylmethionine tRNA from an extreme thermophile, Thermus thermophilus HB8.
The nucleotide sequence of formylmethionine tRNA from an extreme thermophile, Thermus thermophilus HB8, was determined by a combination of classical methods using unlabeled samples to determine the sequences of the oligonucleotides of RNase T1 and RNase A digests and a rapid sequencing gel technique using 5'-32P labeled samples to determine overlapping sequences. Formylmethionine tRNA from T. thermophilus is composed of two species, tRNAf1Met and tRNAf2Met. Their nucleotide sequences are almost identical, and are also almost identical with that of E. coli tRNAfMet, except for slight modifications and replacements. Both species have modifications at three points which do not exist in E. coli tRNAfMet: 2'-O-methylation at G19, N-1-methylation at A59 and 2-thiolation at T55. Moreover U51 in E. coli tRNAfMet is replaced by C51 in both species, so that a G-C pair is formed between this C51 and G65. tRNAf2Met has a reversed G-C pair at positions 52 and 64 compared with those in tRNAf1Met and E. coli tRNAfMet. Other regions are mostly the same as those in all prokaryotic initiator tRNAs so far reported. The thermostability of these thermophile initiator tRNAs is discussed in relation to their unique modifications.
Base Sequence↗
Blocks in elongation and initiation of protein synthesis induced by interferon treatment in mouse L cells.
Synthesis of polypeptide chains coded by exogenous messenger RNAs is inhibited in cell-free extracts from interferon-treated mouse L cells, due to a "deficiency" in some specific tRNA species. A detailed analysis shows that polypeptide chain elongation is blocked and incomplete chains are formed. After a few minutes, however, initiation of new polypeptide chains is also blocked. Messenger RNA still binds to ribosomes but initiator Met-tRNA(FMET) binding is inhibited. The block in initiation appears to be secondary to the block in elongation.
Animals↗
Role of divalent ions in folding of tRNA.
The native structure of tRNA is not achieved in low salt (4.5 mM Na+, 25 degrees C), but can be restored by addition of divalent ions. We have explored the structure of the central region in Escherichia coli tRNAfMet by absorption and emission spectroscopy of 4-thiouracil, and the structure of the anticodon loop in yeast tRNAPhe by fluorescence of the 'Y' base, versus the number of manganese ions bound to tRNA, which was derived from electron spin resonance. The fluorescence of the reduced 8-13 photoproduct (in which 4-thiouracil at position 8 is crosslinked to cytosine at position 13) was also analysed. In low salt (e.g. 4.5 mM Na+), the region of 4-thiouracil is affected strongly as the first eight Mn2+ bind to tRNA, whereas the fluorescence of the 'Y' base is affected only after four Mn2+ are bound. Considering the structural similarities of the two tRNAs, this suggests that the reorganisation brought about by divalent ions starts in the central region, the anticodon loop being affected later. The binding of divalent ions to each region starts together with its restructuration. Monovalent ions can substitute for divalent ions in this process, a 15 mM sodium concentration being equivalent to the binding of the first five Mn2+. If divalent ions are then added, even the first ones distribute themselves between both the central and the anticodon region. Alternatively, the renaturation may be achieved by monovalent ions only, implying that no sites exist whose occupancy by divalent ions is crucial for the native structure. These observations suggest that the role and means of divalent ion binding to tRNA are largely explainable in terms of a simple maganese-phosphate binding supplemented by electrostatic interaction with distant phosphates.
Anticodon↗
Translation of MS2 RNA in vitro in the absence of initiation factor IF-3.
An Escherichia coli cell-free translational system, deprived of initiation factor IF-3, has been used to study the role of the factor in protein synthesis. In this system, 30-S ribosomal subunits are preincubated together with MS2 phage RNA in a small volume in the presence of 10 mM Mg(Ac)2; the missing components required for protein synthesis are then added and assembly of elongating ribosomes is allowed to occur. This stepwise assembly process permits formation of functional complexes which can carry out protein synthesis in the complete absence of IF-3. The translational products, obtained in the absence of IF-3, have been analysed and shown to be similar to those synthesized in the presence of the factor. The main product observed is the phage coat protein.
Coliphages↗
Preferential charging of tRNA-Met-f in Escherichia coli K12.
The charging of tRNA-Met-f and tRNA-Met-m in vivo and in vitro and initiation of polysomes during methionine limitation were studied in two strains of Escherichia coli K12. In the wild-type strain the distribution of polysomes as well as the kinetic parameters of methionyl-tRNA synthetase indicate preferential acylation of tRNA-Met-f. This preferential charging of tRNAM-et-f does not take place in a mutant strain which is also defective in initiation of polysomes during methionine limitation.
Adenosine Triphosphate↗
Protons and Mg2+ cations as probes in investigating the role of GTP in initiation complex formation.
fMet-tRNAfMet binding to both 30-S subunits and to 70-S particles is dependent on both pH AND Mg2+ concentration: for fMet-tRNAfMet binding to 70-S particles, variations of pH and Mg2+ concentration are tightly interdependent. This behavior can be interpreted by the polyelectrolyte theory as a direct consequence of the fact that the binding occurs in a polyanionic micro-environment. The pH-dependent binding to 70-S particles clearly shows the involvement of two prototropic groups which appear to be those carrying out GTP hydrolysis, therefore directly linked to initiation complex formation; in the presence of a non-hydrolyzable analogue to GTP, guanosine 5'-[beta, gamma-imido]triphosphate, the binding of fMet-tRNAfMet shows much less interdependence between variation of pH and Mg2+ concentration.
Binding Sites↗
Cell-free synthesis of the mitochondrial ADP/ATP carrier protein of Neurospora crassa.
ADP/ATP carrier protein was synthesized in heterologous cell-free systems programmed with Neurospora poly(A)-containing RNA and homologous cell-free systems from Neurospora. The apparent molecular weight of the product obtained in vitro was the same as that of the authentic mitochondrial protein. The primary translation product obtained in reticulocyte lysates starts with formylmethionine when formylated initiator methionyl-tRNA (fMet-tRNAfMet) was present. The product synthesized in vitro was released from the ribosomes into the postribosomal supernatant. The evidence presented indicates that the ADP/ATP carrier is synthesized as a polypeptide with the same molecular weight as the mature monomeric protein and does not carry an additional sequence.
Animals↗
In vitro translation of Harvey murine sarcoma virus RNA.
The viral RNA of the Harvey strain of murine sarcoma virus (Ha-SV), which does not encode for any known viral structural polypeptides, has been translated in a nuclease-digested, cell-free system. The major protein product of the in vitro translation reaction has a molecular weight of 21,000 and is initiated faithfully with [35S]formylmethionine from formyl-[35S]methionyl-tRNAFMET. This polypeptide is clearly distinct from the RNA of the Moloney strain of type C helper virus used to pseudotype the Ha-SV. The intensity of the 21,000-dalton polypeptide on gels correlates well to the concentration of Ha-SV RNA in different viral RNA preparations. These experiments indicate that a polypeptide marker for Ha-SV is now available for the first time. The possibility that this protein is the product of the rat portion of the Ha-SV genome is discussed.
Cell Line↗