Correlation between poly(U) misreading and poly(dT) translation efficiency in E coli cell-free systems

A positive correlation between poly(U) misreading and efficiency of poly(dT) translation has been revealed in cell-free systems from wild-type E coli and streptomycin--resistant mutants with altered ribosomal protein S12. Different factors promoting misreading of poly(U) such as aminoglycoside antibiotics and Mg2+ ions also stimulate poly(dT) translation. The effect of the antibiotics on poly(U) translation efficiency and misreading as well as on poly(dT) decoding is characterised by the same order: neomycin greater than kanamycin greater than streptomycin. S12 mutants ribosomes are less erroneous in poly(U) translation and less efficient in poly(dT) decoding. The data obtained are in good agreement with the hypothesis of stereospecific stabilization of codon-anticodon complexes by the ribosome decoding centre.     

Effect of streptomycin on the stoichiometry of GTP hydrolysis in a poly(U)-dependent cell-free translation system

The technique of Sepharose-bound template translation has been used to estimate the stoichiometry of GTP hydrolysis during peptide elongation in the presence of streptomycin. The presence of streptomycin has been shown to have no great effect on the elongation rate and the stoichiometry of GTP hydrolysis during codon-specific peptide elongation in the poly(U)-directed translation system: the molar ratio of hydrolysed GTP to incorporated phenylalanine was about 2. At the same time streptomycin exerted a significant effect during misreading when a ribosome-bound peptide in the poly(U)-programmed system was elongated by leucine or isoleucine residues: the miselongation was stimulated and hence the ratio of hydrolysed GTP per peptide bond was strongly reduced, as compared with the excessive GTP hydrolysis which is characteristic of the misreading system in the absence of streptomycin [(1984) FEBS Lett. 178, 283-287]. The conclusion has been made that streptomycin blocks the stage of correction ('proof-reading') following GTP hydrolysis during EF-Tu-dependent aminoacyl-tRNA binding.     

Elongation factor Tu: a molecular switch in protein biosynthesis

Elongation factor Tu (EF-Tu), the most abundant protein in Escherichia coli, is a guanine nucleotide-binding protein that in the 'on' state acts as a carrier of amino acyl-tRNA to the ribosome. Our knowledge of this essential component of translation has brought substantial progress in the past decade thanks to the co-ordinated application of biochemical, physico-chemical and genetic methods. Crystallographic analysis at 2.6 A resolution and site-directed mutagenesis have revealed structural and functional similarities between the guanine nucleotide-binding domains of EF-Tu and human H-ras p21 protein. The regulation of the expression of the two EF-Tu-encoding genes in E. coli, particularly that of tufB, has been shown to involve diverse mechanisms. Several aspects of the functions of EF-Tu in the elongation cycle have been reinvestigated, leading to new insights. These studies have emphasized the manifold aspects of the mechanisms regulating the activity of EF-Tu in the bacterial cell.     

Factor-free and one-factor-promoted poly(U,C)-dependent synthesis of polypeptides in cell-free systems from Escherichia coli

α-Tropomyosin from rat cardiac muscle was shown by two-dimensional gel electrophoresis to become phosphorylated when tissue slices were incubated in Eagle's medium supplemented with ³²P_i. In the adult rat and mouse heart the level of phosphorylation was ～30%, but the level was much higher in the foetal heart (60–70%). A similar developmental trend was observed in skeletal muscle from the rat and mouse, where phosphorylated forms of both α- and β-tropomyosins were observed. When rat cardiac cells were grown in tissue culture in the presence of ³²P_i, radioactivity was incorporated into the region of the gel containing tropomyosin.     

Differential features of ribosomes and of poly(U)-programmed cell-free systems derived from sulphur-dependent archaebacterial species

The properties of poly(U)-directed cell-free systems developed from the sulphur-dependent, thermophilic archaebacteria Desulfurococcus mobilis, Thermoproteus tenax, Sulfolobus solfataricus, Thermococcus celer and Thermoplasma acidophilum have been compared. All systems are truly thermophilic in requiring incubation at temperatures close to the physiological optimum for cell growth. Under optimized conditions the error frequency in tRNA selection is less than 0.4% at 80 degrees C, and synthetic efficiencies (Phe residues polymerized per ribosome in 40 min) span from 4 for Tp. tenax, to 10 for Tc. celer, to 20-25 for D. mobilis and T. acidophilum and to 40 for S. solfataricus. According to requirements for polypeptide synthesis and to degree of stability of the ribosomal subunits' association, sulphur-dependent thermophiles cluster into two groups. Group I organisms (D. mobilis, Tp. tenax, S. solfataricus) harbour 70-S monomers composed of weakly associated subunits, whose poly(Phe)-synthesizing capacity is totally dependent on added spermine while being drastically inhibited by monovalent cations. Group II organisms (Tc. celer and T. acidophilum) contain 70-S particles composed of tightly bonded subunits, whose synthetic capacity is independent of spermine while being totally dependent on monovalent cations. Spermine promotes poly(Phe) synthesis on ribosomes of group I organisms by converting the peptidyltransferase center into an active conformation, while monovalent cations are inhibitory by preventing the interaction between the free ribosomal subunits. The closeness between Tc. celer and T. acidophilum ribosomes provides new insight on the phylogenetic placement of Thermococcaceae.     

Involvement of 30S ribosomal protein S1 in poly(U)-directed polyphenylalanine synthesis.

The effect of 30S ribosomal protein S1 on poly(U)-directed polyphenylalanine synthesis was studied using a highly purified cell-free system which was devoid of endogenous S1. The system consisted of homogeneous preparations of EF-Tu, EF-Ts, and EF-G, and 70S ribosomes from which protein S1 had been removed by poly(U)-cellulose column chromatography. It was found that protein S1 was indispensable for translation of poly(U) by an S1-depleted system at low concentrations of poly(U). On the other hand, at higher concentrations of poly(U), a considerable amount of polyphenylalanine was synthesized in the absence of added S1. The stimulatory effect of S1 was observed at all Mg2+ concentrations examined but was most pronounced at 10 mM Mg2+. Some physicochemical properties of the protein were also studied. It was demonstrated that the protein has an elongated shape with an axial ratio of approximately 8.5.     

The accuracy of poly(U) translation by different eukaryotic tRNAs

In this work we present comparative data on rates of phenylalanine and leucine incorporation into the poly(U) dependent product of cell-free translation by different eukaryotic tRNAs at high Mg2+ concentration. The frequency of translation errors has been found to depend upon the value of the tRNAPhe:tRNALeu ratio and the peculiarities of isoacceptor tRNAsLeu of different origin.     

Elongation factor G with effector loop from elongation factor Tu is inactive in translocation

Elongation factors Tu and G (EF-Tu and EF-G) alternately interact with the ribosome during the elongation phase of protein biosynthesis. The function of both factors depends on GTP binding, and the factors are ascribed to a superfamily of G-proteins. All G-proteins contain the effector loop, a structural element that is important for the protein's interaction with its target molecule. In this study the effector loop of EF-G was replaced by the loop taken from EF-Tu. The EF-G with EF-Tu loop has markedly decreased GTPase activity and did not catalyze translocation. We conclude that these loops are not functionally interchangeable since the factors interact with different states of the ribosome.     

5'-poly(A) sequence as an effective leader for translation in eukaryotic cell-free systems

Poly(A) sequence of 25 adenylic residues placed immediately before the start codons of the green fluorescent protein (GFP) and firefly luciferase (Luc) mRNAs is shown to provide a high rate of translation of the heterologous messages in eukaryotic cell-free translation systems. Also the poly(A) leader is found to provide the abolition of the inhibition of translation at excess mRNA concentrations. The possibility of the practical use of the constructs with the poly(A) leader for preparative protein production is demonstrated in the wheat germ continuous-exchange cell-free (CECF) translation system.     

Picornavirus IRESes and the poly(A) tail jointly promote cap-independent translation in a mammalian cell-free system

In eukaryotic cells, efficient translation of most cellular mRNAs requires the synergistic interplay between the m7GpppN cap structure and the poly(A) tail during initiation. We have developed and characterized a cell-free system from human HeLa cells that recapitulates this important feature, displaying more than one order of magnitude of translational synergism between the cap structure and the poly(A) tail. The stimulation of cap-dependent translation by the poly(A) tail is length-dependent, but not mediated by changes in mRNA stability. Using this system, we investigated the effect of the poly(A) tail on the translation of picornaviral RNAs, which are naturally polyadenylated but initiate translation via internal ribosome entry sites (IRESs). We show that translation driven by the IRESs of poliovirus (PV), encephalomyocarditis virus (EMCV), and hepatitis A virus is also significantly augmented by a poly(A) tail, ranging from an approximately 3-fold stimulation for the EMCV-IRES to a more than 10-fold effect for the PV IRES. These results raise interesting questions concerning the underlying molecular mechanism(s). The cell-free system described here should prove useful in studying these questions as well as providing a general biochemical tool to examine the translation initiation pathway in a more physiological setting.     

Elongation factor Tu is methylated in response to nutrient deprivation in Escherichia coli.

It has been shown previously that starvation of a mid-logarithmic-phase culture of Escherichia coli B/r for an essential nutrient results in the methylation of a membrane-associated protein (P-43) (C. C. Young and R. W. Bernlohr, J. Bacteriol. 172:5147-5153, 1990). In this communication, the purification of P-43 and sequence analysis of cyanogen bromide-generated peptide fragments identified P-43 as elongation factor Tu (EF-Tu). This was confirmed by the ability of anti-EF-Tu antibody to precipitate P-43. We propose that the nutrient-dependent methylation of EF-Tu may be involved in the regulation of growth, possibly as a principal component of an unidentified signal transduction pathway in bacteria.     

Stoichiometry of GTP hydrolysis in a poly(U)-dependent cell-free translation system. Determination of GTP/peptide bond ratios during codon-specific elongation and misreading

Pancreatic secretory factor (PSF), a 17.5-kDa protein purified from the venom of Gila monster (Heloderma suspectum), stimulated amylase secretion from dispersed rat pancreatic acini more efficiently than CCK-8, bombesin, carbachol and secretin, and without increasing 45Ca2+ efflux and cyclic AMP levels. The secretory action was dependent on the presence of extracellular calcium and was additive to the secretion induced by agents acting via cyclic AMP or via Ca2+ efflux.     

Structure of Poly (U).poly (A).poly (U)

The molecular structure of poly (U).poly (A).poly (U) has been determined and refined using the continuous x-ray intensity data on layer lines in the diffraction pattern obtained from an oriented fiber of the RNA. The final R-value for the preferred structure is 0.24, far lower than that for the plausible alternatives. The polymer forms an 11-fold right-handed triple-helix of pitch 33.5A and each base triplet is stabilized by Crick-Watson-Hoogsteen hydrogen bonds. The ribose rings in the three strands have C3'-endo, C2'-endo and C2'-endo conformations, respectively. The helix derives additional stability through systematic interchain hydrogen bonds involving ribose hydroxyls and uracil bases. The relatively grooveless cylindrical shape of the triple-helix is consistent with the lack of lateral organization.     

Cap-Poly(A) synergy in mammalian cell-free extracts. Investigation of the requirements for poly(A)-mediated stimulation of translation initiation

The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to stimulate synergistically translation initiation in vivo, a phenomenon observed to date in vitro only in translation systems containing endogenous competitor mRNAs. Here we describe nuclease-treated rabbit reticulocyte lysates and HeLa cell cytoplasmic extracts that reproduce cap-poly(A) synergy in the absence of such competitor RNAs. Extracts were rendered poly(A)-dependent by ultracentrifugation to partially deplete them of ribosomes and associated initiation factors. Under optimal conditions, values for synergy in reticulocyte lysates approached 10-fold. By using this system, we investigated the molecular mechanism of poly(A) stimulation of translation. Maximal cap-poly(A) cooperativity required the integrity of the eukaryotic initiation factor 4G-poly(A)-binding protein (eIF4G-PABP) interaction, suggesting that synergy results from mRNA circularization. In addition, polyadenylation stimulated uncapped cellular mRNA translation and that driven by the encephalomyocarditis virus internal ribosome entry segment (IRES). These effects of poly(A) were also sensitive to disruption of the eIF4G-PABP interaction, suggesting that 5'-3' end cross-talk is functionally conserved between classical mRNAs and an IRES-containing mRNA. Finally, we demonstrate that a rotaviral non-structural protein that evicts PABP from eIF4G is capable of provoking the shut-off of host cell translation seen during rotavirus infection.