Ribosomal protein S1 and polypeptide chain initiation in bacteria.
(33/527)
Among several subspecies of 30S subunits of Escherichia coli observed by polyacrylamide-agarose gel electrophoresis, only the slow-moving, protein S1-containing subspecies participates in the formation of the 30S initiation complex with coliphage MS2 RNA as mRNA; the other subspecies retain activity with AUG as mRNA; they are also active in the poly(U)-directed binding of Phe-tRNA. Protein S1 from Caulobacter crescentus substitutes for E. coli S1 despite the fact that C. crescentus ribosomes do not bind MS2 RNA. Under appropriate conditions, the entire population of E. coli 30S subunits can be isolated as the S1-containing subspecies. Protein S1 is lost by salt treatment of ribosomes. (+info)
The effect of tRNA derivatives bound with natural or synthetic mRNA on the interaction of Escherichia coli ribosomes with colicin E3.
(34/527)
Ribosomal binding complexes directed by poly(U) or T4 mRNA were formed with aminoacyl-tRNA or its derivatives bound to predominantly the P or A binding site. The defined binding complexes were reacted with colicin E3 and the reaction was assessed by the ability of the complexes to proceed with polypeptide synthesis. The results indicated that only one of the four complexes tested was completely resistant to colicin E3-induced inactivation: that of Phe-tRNA bound in the presence of poly(U) to the A-site. The poly(U) directed complex of AcPhe-tRNA and the T4-mRNA-directed complex at the A-site appeared slightly resistant, while the T4 mRNA initiation complex was inactivated by colicin E3 in a manner similar to non-complexed ribosomes. Colicin E3 added to ribosomes after protein synthesis had been initiated affected the subsequent polymerization in a manner corresponding to the response of the binding complexes. Thus, poly(U)-translating ribosomes were less affected than ribosomes translating the viral mRNA. The vulnerability of natural-mRNA-directed binding complexes to inactivation by colicin E3 is in accord with the mode of inactivation by the colicin in vivo. (+info)
Purification and characterization of homogeneous initiation factor M2A from rabbit reticulocytes.
(35/527)
Rabbit reticulocyte initiation factor M2A has been prepared in homogeneous form. The final preparation was purified 2,300-fold and ran as a single band on polyacrylamide gel electrophoresis in three different buffer systems: alkaline, sodium dodecyl sulfate, and acidic 6.5 M urea. IF-M2A also ran as a single band in polyacrylamide gel isoelectric focusing experiments with an apparent pI of 6.45. The molecular weight of IF-M2A was approximately 125,000 based on determinations by low speed equilibrium centrifugation (118,000), sodium dodecyl sulfate gel electrophoresis (130,000) and s20,w combined with Stokes radius (124,000). The amino acid composition of IF-M2A revealed three unusual features: a) the basic amino acids represented 19.4 mol %; b) glutamicacid (plus glutamine) constituted 18.8 mol%; c) tryptophan and cysteine residues were only 0.4 and 0.7 mol%, respectively. Homogeneous IF-M2A was tested in several initiation assays using either natural or artificial mRNAs. In each assay tested, homogeneous IF-M2A fully substituted for cruder preparations and at concentrations commensurate with its increased purity. IF-M2A was also examined for ribosome-dependent GTP hydrolysis, an assay requiring ribosomes but no other initiation factors. Analysis of the data yielded a Km for GTP of 10 muM and a Vmax for hydrolysis of 1.20pmol/mug IF-M2A/min. In addition, IF-M2A mediated GTP hydrolysis required both 40 S and 60 S subunits for maximal activity. The possibility that IF-M2A is a factor required for the joining of 40 S and 60 S subunits is discussed. (+info)
Polyamines in bacteriophage R17 and its RNA.
(36/527)
Bacteriophage R17 and its RNA were found to contain significant amounts of spermidine but not of putrescine. When isolated at 0.01 M KCl, up to 1,000 molecules of spermidine were associated with the virion. The phage RNA isolated with phenol plus sodium lauryl sulfate contained approximately 70 to 90 molecules of spermidine. The association appeared to be ionic because the bound spermidine could be dissociated by KCl, MgCl2, or both. Effects of polyamines on in vitro translation were studied using both poly(U) and phage R17-RNA as mRNA. Addition of spermidine to the system at suboptimal concentrations of Mg2+ resulted in marked stimulations of the rate of protein synthesis. Putrescine alone had no effect but stimulated the incorporation in the presence of suboptimal concentrations of spermidine plus Mg2+. The isolated amino acid-incorporating system contained suboptimal soluble and bound polyamines. A comparison of incorporation was made in this system using R17-RNA with and without bound spermidine. No effects of these bound cations were detected on the rate or extent of incorporation of valine. The ratio of incorporation of histidine (present in non-coat proteins) to valine (total protein) revealed little difference as a functions of cation in the system or a function of the spermidine present in R17-RNA. (+info)
Photoaffinity polyamines: interactions with AcPhe-tRNA free in solution or bound at the P-site of Escherichia coli ribosomes.
(37/527)
Two photoreactive derivatives of spermine, azidobenzamidino (ABA)-spermine and azidonitrobenzoyl (ANB)-spermine, were used for mapping of polyamine binding sites in AcPhe-tRNA free in solution or bound at the P-site of Escherichia coli poly(U)-programmed ribosomes. Partial nuclease digestion indicated that the deep pocket formed by nucleosides of the D-stem and the variable loop, as well as the anticodon stem, are preferable polyamine binding sites for AcPhe-tRNA in the free state. ABA-spermine was a stronger cross-linker than ANB-spermine. Both photoprobes were linked to AcPhe-tRNA with higher affinity when the latter was non-enzymatically bound to poly(U)-programmed ribosomes. In particular, the cross-linking at the TpsiC stem and acceptor stem was substantially promoted. The photolabeled AcPhe-tRNA.poly(U).ribosome complex exhibited moderate reactivity towards puromycin. The attachment of photoprobes to AcPhe-tRNA was mainly responsible for this defect. A more complicated situation was revealed when the AcPhe-tRNA.poly(U).ribosome complex was formed in the presence of translation factors; the reactivity towards puromycin was stimulated by irradiating such a complex in the presence of photoprobes at 50 microM, with higher concentrations being inhibitory. The stimulatory effect was closely related with the binding of photoprobes to ribosomes. The results are discussed on the basis of possible AcPhe-tRNA conformational changes induced by the incorporation of photoprobes. (+info)
A study of codon-dependent binding of aminoacyl-tRNA with the ribosomal 30-S subparticle of Escherichia coli. Determination of the active-particle fraction and binding constants in different media.
(38/527)
Titration of isolated Escherichia coli ribosomal 30-S particles with [14C]phenylalanyl-tRNA in the presence of poly(uridylic acid) was used for a quantitative assay of codon-dependent binding of aminoacyl-tRNA with the small ribosomal subparticle. The technique has allowed the estimation both of the fraction of "active" 30-S subparticles capable of forming the 30-S - poly(U) - phenylalanyl-tRNA complexes and the equilibrium constants of phenylalanyl-tRNA binding in different media. Heterogeneity of the ternary complexes formed has been revealed: at least two classes of complexes differing in stability have been observed. The stability of the 30-S - poly(U) - phenylalanyl-tRNA complexes has been shown to decrease with the lowering of the Mg2+ concentration, the increase of K+ concentration and the addition of urea. The stability of the complexes increases with the increase of Mg2+ concentration, with the addition of ethanol and decrease of temperature. It is demonstrated that the fraction of actively binding 30-S particles also varies in different medium conditions; it decreases with the increase of ionic strength (K+) and with the addition of urea, and increases with the increase of Mg2+ concentration and addition of ethanol. (+info)
Pools of ribosomal proteins in Escherichia coli. Studies on the exchange of proteins between pools and ribosomes.
(39/527)
A labelling technique in vivo has been introduced which allows the tritiation of cell components with high specific activity during growth in rich medium. By this technique the pool size of each protein can be measured directly in the supernatant from centrifugation at 150000 times g. A measurable pool was found for the proteins S1, S2, S10, L1, L4, L7, L8/9, L10, L12, L21, and L25. Experiments on migration of ribosomal proteins from the supernatant to ribosomes (i.e. association) and vice versa (dissociation) demonstrate a remarkable constancy in the composition of the ribosome. There is no significant difference between ribosomes engaged or not engaged in poly-(Phe) synthesis. (+info)
The interaction of fusidic acid with peptidyl-transfer-ribonucleic-acid - ribosome complexes.
(40/527)
The inhibitory action of fusidic acid on peptide-chain elongation was studied with systems in vitro directed by either polyuridylic acid or endogenous messenger (Escherichia coli polysomes washed with 1 M NH4Cl) or R17 RNA, and supplemented with either crude or purified elongation factors. In all cases strong inhibition of synthesis required high concentrations of the antibiotic (approx. 1 mM), while a similar inhibition of the EF-G-plus-ribosome-dependent GTP hydrolysis required between 10 and 100 times less antibiotic. Since most of the GTP hydrolysis observed was presumably due to free ribosomes (without aminoacyl-tRNA or peptidyl-tRNA), fusidic acid seemed to interact far more easily with these ribosomes than with ribosomes engaged in peptide-chain elongation. The role of the GDP-EF-G-ribosome-fusidic acid complex in the inhibition of polypeptide synthesis was assessed by measuring formation of this complex on polysomes engaged in peptide-chain elongation. Using purified elongation factors the complex formed on only 25-35% of ribosomes, as measured either by retention of [3H]GDP or by hydrolysis of [3H, gamma-32P]GTP. In contrast, with crude factors (S 100 extract) it formed on more than 70% of ribosomes. The results are compatible with the postulated role of the complex in polypeptide synthesis inhibition (blockade of the ribosomal acceptor site and subsequent inhibition of aminoacyl-tRNA binding) and indicate that formation of the complex takes place by overriding the control that prevents interaction of EF-G when the donor site is occupied by peptidyl-tRNA. In the polyuridylic-acid-directed system for synthesis of oligophenylalanine the antibiotic inhibits every round of peptide elongation, including dipeptide formation, to roughly the same extent. (+info)