First class transcription termination factors--functional analogs of aminoacyl tRNA

Reviewed and discussed are the recent data demonstrating profound functional similarity between class-1 translation termination factors (RF1 and RF2 in prokaryotes, aRF1 and eRF1 in Archaea and eukaryotes, respectively) and aminoacyl-tRNA as regards their roles in the course of translation on the ribosome. Functional analogy of these two components of the cell protein-synthesizing machinery was suggested long ago; however, numerous experimental proofs have been obtained only recently. This similarity implies that decoding of the genetic information by the ribosomal machine is performed similarly at all stages of translation, though tRNA plays the main role at initiation and elongation, while the protein is most important for termination. Earlier it was found that nucleic acids (ribozymes) can operate like the protein enzymes, and now we have got evidence for the reverse: a protein (translation termination factor) can act like a nucleic acid (tRNA). Thus one can speak of "exchange" of molecular functions between proteins and nucleic acids. Therefore, the profound chemical difference between proteins and nucleic acids is not an insuperable barrier to their mutual functional replacement in certain situations.     

Prediction of the Secondary Structure Contents of Globular Proteins Based on Three Structural Classes

The prediction of the secondary structural contents (those of -helix and -strand) of a globular protein is of great use in the prediction of protein structure. In this paper, a new prediction algorithm has been proposed based on Chou's database [Chou (1995), Proteins 21, 319–344]. The new algorithm is an improved multiple linear regression method, taking into account the nonlinear and coupling terms of the frequencies of different amino acids and the length of the protein. The prediction is also based on the structural classes of proteins, but instead of four classes, only three classes are considered, the class, class, and the mixed + and / class or simply the class. Thus the ambiguity that usually occurs between + proteins and / proteins is eliminated. A resubstitution examination for the algorithm shows that the average absolute errors are 0.040 and 0.035 for the prediction of -helix content and -strand content, respectively. An examination of cross-validation, the jackknife analysis, shows that the average absolute errors are 0.051 and 0.045 for the prediction of -helix content and -strand content, respectively. Both examinations indicate the self-consistency and the extrapolating effectiveness of the new algorithm. Compared with other methods, ours has the merits of simplicity and convenience for use, as well as high prediction accuracy. By incorporating the prediction of the structural classes, the only input of our method is the amino acid composition and the length of the protein to be predicted.     

The regulation of RNA synthesis in yeast I: Starvation experiments

Summary The synthesis of tRNA in yeast is shown to be under separate control to that of rRNA during amino acid and nitrogen starvation. Inhibitors of the elongation and termination steps of protein synthesis were found to stimulate the synthesis of tRNA in starved yeast cells. This effect appeared to be due to the trickle-charging of tRNA. Two inhibitors of early steps in the initiation of protein synthesis were found to be unable to stimulate RNA synthesis in starved cells. It is proposed that yeast tRNA synthesis is under autoregulatory control and that the level of tRNA charging and the mRNA-ribosome complex are important components of this control system.     

Effects of external phosphorus supply on internal phosphorus concentration and the initiation,growth and exudation of cluster roots in Hakea prostrata R.Br.

The response of internal phosphorus concentration, cluster-root initiation, and growth and carboxylate exudation to different external P supplies was investigated in Hakea prostrata R.Br. using a split-root design. After removal of most of the taproot, equal amounts of laterals were allowed to grow in two separate pots fastened together at the top, so that the separate root halves could be exposed to different conditions. Plants were grown for 10 weeks in this system; one root half was supplied with 1 M P while the other halves were supplied with 0, 1, 25 or 75 M P. Higher concentrations of P supplied to one root half significantly increased the P concentration of those roots and in the shoots. The P concentrations in root halves supplied with 1 M P were invariably low, regardless of the P concentration supplied to the other root half. Cluster root initiation was completely suppressed on root halves supplied with 25 or 75 M P, whereas it continued on the other halves supplied with 1 M P indicating that cluster-root initiation was regulated by local root P concentration. Cluster-root growth (dry mass increment) on root halves supplied with 1 M P was significantly reduced when the other half was either deprived of P or supplied with 25 or 75 M P. Cluster-root growth was favoured by a low shoot P status at a root P supply that was adequate for increased growth of roots and shoots without increased tissue P concentrations. The differences in cluster-root growth on root halves with the same P supply suggest that decreased cluster-root growth was systemically regulated. Carboxylate-exudation rates from cluster roots on root halves supplied with 1 M P were the same, whether the other root half was supplied with 1, 25 or 75 M P, but were approximately 30 times faster when the other half was deprived of P. Estimates of root P-uptake rates suggest a rather limited capacity for down-regulating P uptake when phosphate was readily available.     

Role of the recF gene of Escherichia coli K-12 in lambda recombination

Summary When Escherichia coli K12() lysogens are infected with heteroimmune phage, which are unable to replicate, general recombination between phage and prophage depends on the bacterial recF gene. It has been shown that in E. coli K12 postconjugational recombination, the RecF pathway only works with full efficiency if exonuclease I is absent (Clark 1973). However, results presented in this paper indicate that under conditions in which replication is blocked, the recombination pathway dependant on the recF gene is fully active in producing viral recombinants even, if the phage is Red⁺, in the presence of exonuclease I. In contrast, removal of exonuclease and protein requires elimination of exonuclease I for an efficient RecF pathway. It is concluded that the Red system cooperates with the RecF pathway and that this cooperation involves overcoming the inhibitory effects of exonuclease I. In the absence of exonuclease, protein stimulates recF-dependent recombination but does not suffice to prevent the negative effect of exonuclease I. In the presence of protein, full efficiency of the RecF pathway can be obtained either via cooperation with exonuclease I or, if the viral exonuclease is defective, via inactivation of exonuclease I. Since activity of exonuclease appears necessary to overcome the inhibitory effects of exonuclease I, it is proposed here that exonuclease diverts material from the RecF pathway in a shunt reaction which allows completion of recF-initiated recombinational intermediates via a mechanism insensitive to exonuclease I.When replication is allowed, the Rec system produces viral recombinants mainly via a recF-independent mechanism. However, a major contribution of the RecF pathway to recombination is observed after removal of the Red system and exonuclease I.Obra social de la Caja de Ahorros de Valencia (Director: S. Grisolía)     

Transport number effects in the transverse tubular system and their implications for low frequency impedance measurement of capacitance of skeletal muscle fibers

Summary It has been shown in an earlier paper that the slow transient decrease in conductance, somtimes referred to as creep, obtained with small-to-medium hyperpolarizing current or voltage pulses is due to K⁺ transport number differences across the walls of the transverse tubular system. Using the same basic numerical analysis and the parameters already obtained experimentally in the previous paper for frog skeletal muscle in a sulphate Ringer's solution, this paper predicts the equivalent membrane capacitance and dynamic resistance due to transport number effects for very low amplitude and low frequency sinusoidal currents from the phase lag of the voltage response behind the current. Such sinusoidal currentper se give rise to an equivalent capacitance which increased from less than 1F·cm^–2 at 10 Hz to about 16F·cm^–2 at 0.01 Hz and to an equivalent dynamic membrane resistance which increases from its instantaneous slope resistance value of 11.7kcm² at 10 Hz to about 16kcm² at 0.01 Hz. Similar small sinusoidal components of current superimposed on depolarizing and hyperpolarizing pulses (25–45 mV) give rise to even greater capacitances at low frequencies (e.g., 24–28F·cm^–2 at 0.01 Hz). The response due to large sinusoidal currents was also investigated. These transport number effects help to explain the small discrepancies obtained by some workers between experimental and predicted values of skeletal muscle fiber impedances measured in the 1–10 Hz range and would seem to be critical for the interpretation of any skeletal muscle fiber impedance studies done at frequencies less than 1 Hz.     

On the calculation of birth rates and death rates in fluctuating populations with continuous recruitment

Summary A two-instar and a three-instar model for the calculation of birth and death rates is proposed which both use only a minimum of assumptions. It is shown that the proposed approach is a generalization of the methods of Edmondson (1960, 1968), Caswell (1972), and Paloheimo (1974). The validity of the two methods is compared with two-instar and three-instar models according to Edmondson, Caswell and Paloheimo. Since it is impossible to use field data for the comparison, a set of 27 computer simulations was run. From these simulations the true and estimated values were obtained. The comparisons showed that it is better to use the new two-instar model in an environment with no instarspecific mortality. But it is better to use three-instar models when strong instarspecific selection prevails. Among the three-instar models the best results are obtained by the method of Paloheimo when predicting birth rates of young and of adults. It proved that the bias produced by the several methods is mainly influenced by the amount of deviation from the situation of steady state.The first chapter of this paper is part of a doctoral thesis (Seitz, 1977) submitted to the University of Munich     

The Origin and Evolution of Operons: The Piecewise Building of the Proteobacterial Histidine Operon

The structure and organization of 470 histidine biosynthetic genes from 47 different proteobacteria were combined with phylogenetic inference to investigate the mechanisms responsible for assembly of the his pathway and the origin of his operons. Data obtained in this work showed that a wide variety of different organization strategies of his gene arrays exist and that some his genes or entire his operons are likely to have been horizontally transferred between bacteria of the same or different proteobacterial branches. We propose a piecewise model for the origin and evolution of proteobacterial his operons, according to which the initially scattered his genes of the ancestor of proteobacteria coded for monofunctional enzymes (except possibly for hisD) and underwent a stepwise compacting process that reached its culmination in some -proteobacteria. The initial step of operon buildup was the formation of the his core, a cluster consisting of four genes (hisBHAF) whose products interconnect histidine biosynthesis to both de novo synthesis of purine metabolism and that occurred in the common ancestor of the // branches, possibly after its separation from the one. The following step was the formation of three mini-operons (hisGDC, hisBHAF, hisIE) transcribed from independent promoters, that very likely occurred in the ancestor of the /-branch, after its separation from the one. Then the three mini-operons joined together to give a compact operon. In most -proteobacteria the two fusions involving the gene pairs hisN–B and hisI–E occurred. Finally the -proteobacterial his operon was horizontally transferred to other proteobacteria, such as Campylobacter jejuni. The biological significance of clustering of his genes is also discussed.[Reviewing Editor: Dr. Martin Kreitman]     

Effect of chronic desipramine treatment on neurotransmitter-thyrotropin releasing hormone—thyrotropin interactions in the rat

Long-term administration of the antidepressant drug, desipramine (20 mg/kg/day, orally for 28 days), decreased the stimulatory effect of the ₂-adrenoceptor agonist, clonidine (250 g/kg, i.p.) on thyrotropin (TSH) secretion in the rat, but did not alter basal TSH secretion. -Adrenoceptor-mediated inhibition of TSH secretion by isoproterenol (1 mg/ kg, i.p.) was unaffected by chronic desipramine treatment, as were the stimulatory effect of TSH-releasing hormone (TRH, 5 g/kg, i.v.) on TSH release and its inhibition by the -adrenoceptor antagonist, phentolamine (2 mg/kg, i.p.). These findings suggest that chronic desipramine treatment induces subsensitivity of ₂-adrenoceptors which modulate TSH secretion in the rat while not affecting -adrenoceptor-mediated inhibition of TSH release. These findings suggest that pituitary TRH receptors are unchanged but that changes occurred at the hypothalamic level in ₂-adrenoceptor-mediated stimulation of TRH release. Although cerebral -adrenoceptors have been shown convincingly to be down-regulated after chronic desipramine treatment, their function in the hypothalamic TRH system after 28 days of treatment with desipramine appears to be unimpaired.     

Onset of nucleic acid synthesis during germination of Pisum sativum L.

Summary Measurments of total nucleic acid content of the embryonic axis indicated that massive net synthesis of both DNA and RNA was initiated at approximately 30 h after the onset of germination. The onset of net nucleic acid synthesis was marked by an increase in the rate of incorporation of [³H]thymidine into DNA, and of [³H]orotic acid and [³H]uridine into both DNA and RNA. rRNA was usually more heavily labelled than tRNA, but was not preferentially accumulated, suggesting a grater rate of turnover of rRNA than tRNA. Some incorporation of precursors occurred prior to the onset of net nucleic acid synthesis, particularly into RNA. This was taken to represent nucleic acid turnover. There was no evidence that the scavenging pathways for nucleotide biosynthesis were more important than the normal pathways in contributing precursors for net nucleic acid synthesis.     

Inhibition of chlorophyll synthesis inHordeum vulgare by 3-amino 2,3-dihydrobenzoic acid (gabaculin)

Gabaculin (3-amino 2,3-dihydrobenzoic acid) is shown to be a very potent inhibitor of chlorophyll formation inHordeum vulgate. Exposure of leaf segments to 30/M gabaculin results in an 80% inhibition of chlorophyll synthesis, and this is paralleled by a decrease in carotenoid. Dual-inhibitor studies with dioxoheptanoic acid, which is an inhibitor of inolaevulinic acid dehydratase, show that gabaculin inhibits an earlier step than dioxoheptanoic acid and affects -aminolaevulinic acid synthesis rather than its subsequent metabolism.     

K-current fluctuations in inward-rectifying channels of frog skeletal muscle

Summary K currents and K-current fluctuations were recorded in inwardly rectifying K channels of frog skeletal muscle under voltage-clamp conditions. External application of 0.2 to 10mm Cs reduces the inward mean K current but produces a distinct increase of the spectral density of K-current fluctuations. The additional fluctuations arise from the random blocking by Cs ions. From the variance of current fluctuations, the steady-state current and the probability of the open unblocked channel an effective single-channel conductance ^* was calculated. ^* strongly depends on the external Cs concentration (7.8 pS at 0.2mm Cs, 2.1 pS at 10mm Cs). This dependence is interpreted in terms of a two-step blocking process: (1) a fast exchange of Cs ions between the external solution and a first binding site inside the channel which leads to the Cs-modulated effective single-channel conductance, and (2) a slow Cs binding to a second site deeper in the channel which produces the observed current fluctuations. With this hypothesis we obtained a real single-channel conductance of 10 pS and a real density ofn4 inwardly rectifying channels per m² of muscle surface area.     

Rapid fold and structure determination of the archaeal translation elongation factor 1β from Methanobacterium thermoautotrophicum

The tertiary fold of the elongation factor, aEF-1, from Methanobacterium thermoautotrophicum was determined in a high-throughput fashion using a minimal set of NMR experiments. NMR secondary structure prediction, deuterium exchange experiments and the analysis of chemical shift perturbations were combined to identify the protein fold as an alpha-beta sandwich typical of many RNA binding proteins including EF-G. Following resolution of the tertiary fold, a high resolution structure of aEF-1 was determined using heteronuclear and homonuclear NMR experiments and a semi-automated NOESY assignment strategy. Analysis of the aEF-1 structure revealed close similarity to its human analogue, eEF-1. In agreement with studies on EF-Ts and human EF-1, a functional mechanism for nucleotide exchange is proposed wherein Phe46 on an exposed loop acts as a lever to eject GDP from the associated elongation factor G-protein, aEF-1. aEF-1 was also found to bind calcium in the groove between helix 2 and strand 4. This novel feature was not observed previously and may serve a structural function related to protein stability or may play a functional role in archaeal protein translation.     

Collagen and proteoglycan in a sea urchin ligament with mutable mechanical properties

Summary The problematic ligament of sea urchins is a connective tissue which crosses the ball-and-socket joint between spine and body wall. The problem of this ligament is that it is composed of parallel collagen fibrils, yet normally undergoes rapid and dramatic alterations in mechanical properties and in length. Previous work has suggested that the collagen fibrils of the ligament are able to slide past one another during length changes but are inhibited from sliding when the ligament is in catch. In this model of the ligament both the collagen fibrils and the interfibrillar matrix are mechanically important. We have found that the collagen fibrils of the spine ligament of the pencil urchin Eucidaris tribuloides are discontinuous and end by tapering within the body of the ligament. Intact fibrils that have been isolated from the ligament vary by more than an order of magnitude in length and in radius but have a constant length/radius (aspect) ratio of about 5300. This is the first determination of the aspect ratio of collagen fibrils from any source. The constant aspect ratio of the fibrils is consistent with their functioning as the discontinuous fiber phase in a fiber-reinforced composite material, while the high value of the aspect ratio indicates that the nonfibrillar matrix, which must act to transfer stress between fibrils, can produce a stiff and strong ligament even if it is several orders of magnitude weaker and more compliant than the fibrils. Moreover, the tensile properties of the ligament may be determined by the properties of the matrix. A prominent component of the interfibrillar matrix is a proteoglycan which associates with specific bands at the surface of the collagen fibrils through noncovalent binding of its core protein. The glycosaminoglycan moiety of this proteoglycan is partly comprised of chondroitin sulfate/dermatan sulfate polymers. These results are consistent with the sliding fibril hypothesis and suggest that the proteoglycan may be an important component of the stress-transfer matrix.     

Translational control of protein synthesis by tRNA unrelated to changes in tRNA concentration

Summary The rate of translation of mRNA into protein is controlled, at least in part, by the concentration of tRNA. However, as one tRNA molecule can recognize more than one codon, different codons translated by the same tRNA may be translated at different rates. The difference in response of a tRNA molecule to different codons could also regulate the rate of protein synthesis. Evidence for this theory is presented, and Crick's wobble hypothesis is extended to include the relative strengths of different codon-anticodon interactions.     

Size-fractionated primary production in the open Southern Ocean in austral spring

Size-fractionated primary production was measured by carbon-14 uptake incubations on three transects between 47°S and 59°30S along 6°W in October/November 1992. Open Antarctic Circumpolar Current and ice-covered Weddell Gyre water showed comparable low productivity (0.3 gCm^–2 day^–1) and size distribution. Picoplankton (<2 m) was the dominant size fraction, contributing approximately half to the total water column production. The significance of larger (>20 m) phytoplankton was only minor. Productivity in the Polar Front Zone north of 50°S, with higher water column stability, was up to 10 times higher with microplankton (>20 m) being predominant. No ice-edge bloom occurred over the 2 months study period; this is explained by non-favourable hydrographic conditions for blooming and the lack of melt-water lenses upon ice retreat. Picoplankton tended to make higher contributions at lower water column stability, and microplankton to make higher contributions at higher stability. Mixing, together with light climate, are discussed as the driving forces for Antarctic primary production and for its size structure.     

The pentafunctional FAS1 genes of Saccharomyces cerevisiae and Yarrowia lipolytica are co-linear and considerably longer than previously estimated

Summary The fatty acid synthetase (FAS) gene FAS1 of the alkane-utilizing yeast Yarrowia lipolytica was cloned and sequenced. The gene is represented by an intron-free reading frame of 6228 by encoding a protein of 2076 amino acids and 229980 Da molecular weight. This protein exhibits a 58% sequence similarity to the corresponding Saccharomyces cerevisiae FAS -subunit. The sequential order of the five FAS1-encoded enzyme domains, acetyl transferase, enoyl reductase, dehydratase and malonyl/palmityl-transferase, is co-linear in both organisms. This finding agrees with available evidence that the functional organization of FAS genes is similar in related organisms but differs considerably between unrelated species. In addition, previously reported conflicting data concerning the 3 end of S. cerevisiae FAS1 were re-examined by genomic and cDNA sequencing of the relevant portion of the gene. Thereby, the translational stop codon was shown to lie considerably downstream of both published termination sites. The S. cerevisiae FAS1 gene thus has a corrected length of 6153 by and encodes a protein of 2051 amino acids and 228667 Da molecular weight.     

Synthetic substrate analogues for UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme

UDP-GlcNAc: Man1-6R (1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complexN-glycans. We have tested a series of synthetic analogues of the substrate Man1-6(GlcNAc1-2Man1-3)Man-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attachesN-acetylglucosamine in 1-2 linkage to the 2-OH of the Man1-6 residue. The 2-deoxy analogue is a competitive inhibitor (K _i=0.13mm). The 2-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3-, 4- and 6-OH groups are not essential for binding or catalysis since the 3-, 4- and 6-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3-position to pentyl and substituted pentyl groups causes competitive inhibition (K _i=1.0–2.5mm). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3-O-(4,4-azo)pentyl group and a 3-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man1-6 residue are essential for binding although the 2- and 3-OH face the catalytic site of the enzyme. The 4-OH group of the Man-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind. This contrasts with GlcNAc-T I which cannot bind to the 4-deoxy-Man- substrate analogue. The data are compatible with our previous observations that a bisectingN-acetylglucosamine at the 4-OH position prevents both GlcNAc-T I and GlcNAc-T II catalysis. However, in the case of GlcNAc-T II, the bisectingN-acetylglucosamine prevents binding due to steric hindrance rather than to removal of an essential OH group. The 3-OH of the Man1-3 is an essential group for GlcNAc-T II since the 3-deoxy derivative does not bind to the enzyme. The trisaccharide GlcNAc1-2Man1-3Man-O-octyl is a good inhibitor (K _i=0.9mm). The above data together with previous studies indicate that binding of the GlcNAc1-2Man1-3Man- arm of the branched substrate to the enzyme is essential for catalysis. Abbreviations: GlcNAc-T I, UDP-GlcNAc:Man1-3R (1-2)-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GlcNAc-T II, UDP-GlcNAc:Man1-6R (1-2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143); MES, 2-(N-morpholino)ethane sulfonic acid monohydrate.     

Quantitative analysis of type IV collagen alpha chains in the basement membrane of human urogenital epithelium

Type IV collagen is a major component of the basement membrane (BM), which consists of six genetically distinct (IV) chains. In this study the expression of these six (IV) chains was demonstrated immunohistochemically. In addition, the 2(IV) and 5(IV) chains were analysed quantitatively by confocal laser scanning microscopy in human urogenital epithelial BM. The 1/2(IV) and 5/6(IV) chains were immunoreactive in the epithelial BM, whereas, 3/4(IV) chains were not. The quantitative analysis revealed that the amount of 2(IV) and 5(IV) chains differed in each urogenital epithelial BM. The content of 5(IV) chains in the epithelial BM of the bladder was differentially high, and that of the foreskin was differentially low. It is concluded that the elasticity of epithelial BM of the bladder may be structurally related to the high content of 5/6(IV) chains.     

The relationship between guanosine tetraphosphate,polysomes and RNA synthesis in amino acid starved escherichia coli

ArelA⁺ strain ofE. coli with four amino acid requirements was starved separately for each amino acid, after which the levels of polysomes, guanosine-5-diphosphate-3-diphosphate and the residual net synthesis of RNA were determined. The polysome level and guanosine-5-diphosphate-3-diphosphate production were coordinately affected by starvation for the different amino acids, whereas no correlation was found between these two parameters and residual RNA synthesis. The main conclusion stemming from these results is that guanosine-5-diphosphate-3-diphosphate cannot act as the sole effector molecule in stringent control of RNA synthesis.