Bifunctional transfer-messenger RNA

Transfer-messenger RNA (tmRNA) is a bifunctional RNA that has properties of a tRNA and an mRNA. tmRNA uses these two functions to release ribosomes stalled during translation and target the nascent polypeptides for degradation. This concerted reaction, known as trans-translation, contributes to translational quality control and regulation of gene expression in bacteria. tmRNA is conserved throughout bacteria, and is one of the most abundant RNAs in the cell, suggesting that trans-translation is of fundamental importance for bacterial fitness. Mutants lacking tmRNA activity typically have severe phenotypes, including defects in viability, virulence, and responses to environmental stresses.     

tmRNA of Streptomyces collinus and Streptomyces griseus during the growth and in the presence of antibiotics

Streptomycetes are soil microorganisms with the potential to produce a broad spectrum of secondary metabolities. The production of antibiotics is accompanied by a decrease in protein synthesis, which raises the question of how these bacteria survived the transition from the primary to the secondary metabolism. Translating ribosomes incapable to properly elongate or terminate polypeptide chain activate bacterial trans‐translation system. Abundance and stability of the tmRNA during growth of Streptomyces collinus and Streptomyces griseus producing kirromycin and streptomycin, respectively, was analysed. The level of tmRNA is mostly proportional to the activity of the translational system. We demonstrate that the addition of sub‐inhibitory concentrations of produced antibiotics to the cultures from the beginning of the exponential phase of growth leads to an increase in tmRNA levels and to an incorporation of amino acids into the tag‐peptides at trans‐translation of stalled ribosomes. These findings suggest that produced antibiotics induce tmRNA that facilitate reactivation of stalled complex of ribosomes and maintain viability. The effect of antibiotics that inhibit the cell‐wall turnover, DNA, RNA or protein synthesis on the level of tmRNA was examined. Antibiotics interfering with ribosomal target sites are more effective at stimulation of the tmRNA level in streptomycetes examined than those affecting the synthesis of DNA, RNA or the cell wall.     

Beyond ribosome rescue: tmRNA and co-translational processes

tmRNA is a unique bi-functional RNA that acts as both a tRNA and an mRNA to enter stalled ribosomes and direct the addition of a peptide tag to the C terminus of nascent polypeptides. Despite a reasonably clear understanding of tmRNA activity, the reason for its absolute conservation throughout the eubacteria is unknown. Although tmRNA plays many physiological roles in different bacterial systems, recent studies suggest a general role for trans-translation in monitoring protein folding and perhaps other co-translational processes. This review will focus on these new hypotheses and the data that support them.     

顺式和反式-AT型聚酮合酶研究进展及应用

聚酮类化合物因广泛应用于医药等方面而被大家所熟知,Ⅰ型聚酮合酶（Polyketide Synthase,PKS）在催化聚酮类化合物的生物合成中起着重要的作用。根据不同的酰基转移酶（Acyltransferase,AT）结构域,I型PKS可分为顺式-AT （cis-Acyltransferase,cis-AT）型PKS和反式-AT （trans-Acyltransferase,trans-AT）型PKS,目前cis-AT型PKS研究得比较透彻,trans-AT型PKS相关研究成为当今热点。本文总结了cis-AT型PKS和trans-AT型PKS的联系与区别、工程进展、相关应用以及目前存在的问题,以期为了解cis-AT型PKS和trans-AT型PKS在聚酮化合物合成中的作用提供参考。     

SmpB functions in various steps of trans-translation

tmRNA has a dual function as a tRNA and an mRNA to facilitate trans-translation, in which a ribosome can switch between translation of a truncated mRNA and the tmRNA’s tag sequence. SmpB is a tmRNA binding protein that has been identified to be essential for trans-translation in vivo. To further study the function of SmpB, an S30 fraction from an Escherichia coli strain, in which the set of genes for SmpB and tmRNA has been deleted from the genome, and His-tagged SmpB active in trans-translation were prepared. The SmpB-depleted S30 fraction had an ability to facilitate poly(U)-dependent tag-peptide synthesis in vitro when purified His-tagged SmpB was exogenously added together with tmRNA, although SmpB was not required for in vitro poly(U)-dependent poly(Phe) synthesis. It was also found that depletion of SmpB leads to a decrease in the level of tmRNA in the cell. In addition, SmpB considerably enhanced the aminoacylation of tmRNA by alanyl-tRNA synthetase in vitro. The aminoacylation enhancement by SmpB, the binding of SmpB to tmRNA and the effect of depletion of SmpB on the expression level of tmRNA in the cell were all affected by some mutations in the tRNA-like domain which cause a defect in ribosome binding leading to a trans-translation deficiency. These results demonstrate that, via binding to the tRNA-like domain of tmRNA, SmpB plays various roles: rescuing the tmRNA molecule from degradation in the cell, enhancing the aminoacylation of tmRNA and mediating the binding of tmRNA to ribosome.     

Structural features of the tmRNA–ribosome interaction

Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA–ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.     

Substrate selectivity of lipases in the esterification of cis/trans-isomers and positional isomers of conjugated linoleic acid (CLA)

The substrate selectivity of several microbial lipases has been examined in the esterification of the conjugated linoleic acid (CLA) isomers cis-9,trans-11-, cis-9,cis-11-, trans-9,trans-11- and trans-10,cis-12-octadecadienoic acid with n-butanol in n-hexane. Lipases from Candida cylindracea and Mucor miehei had a preference for the cis-9,trans-11-octadecadienoic acid, while Chirazyme L-5, a Candida antarctica lipase A, accepted the trans-9,trans-11-fatty acid with a high selectivity. Moreover, lipase from Candida cylindracea and Chirazyme L-5 catalysed the esterification of the cis-9,trans-11-octadecadienoic acid with n-butanol faster than the corresponding reaction of the trans-10,cis-12-fatty acid.     

Trans-sialidase genes expressed in mammalian forms of Trypanosoma cruzi evolved from ancestor genes expressed in insect forms of the parasite

The trans-sialidase of Trypanosoma cruzi mammalian forms transfers sialic acids from host's cell-surface glycoconjugates to acceptor molecules on parasite cell surface. To investigate the mechanism by which the mammalian stages of Trypanosoma cruzi have acquired their trans-sialidase, we compared the nucleotide and predicted amino acid sequences of trans-sialidase genes expressed in different developmental stages and strains of Trypanosoma cruzi with the sialidase gene of Trypanosoma rangeli and the sialidase genes of the prokaryotic genera Clostridium, Salmonella, and Actinomyces. The trans-sialidase gene products of Trypanosoma cruzi have a significant degree of structural and biochemical similarity to the sialidases found in bacteria and viruses, which would hint that horizontal gene transfer occurred in Trypanosome cruzi trans-sialidase evolutionary history. The comparison of inferred gene trees with species trees suggests that the genes encoding the T. cruzi trans-sialidase of mammalian forms might be derived from genes expressed in the insect forms of the genus Trypanosome. The branching order of trees inferred from T. cruzi trans-sialidase sequences, the sialidase from Trypanosoma rangeli, and bacterial sialidases parallels the expected branching order of the species and suggests that the divergence times of these sequences are remarkably long. Therefore, a vertical inheritance from a hypothetical eukaryotic trans-sialidase gene expressed in insect forms of trypanosomes is more likely to have occurred than the horizontal gene transfer from bacteria, and thus explains the presence of this enzyme in the mammalian infective forms of Trypanosoma cruzi.Correspondence to: M.R.S. Briones     

Competition of cyclooctenes and cyclooctadienes for ethylene binding and activity in plants

trans-Cyclooctene, cis,trans-1,5-cyclooctadiene, and cis,trans-1,3-cyclooctadiene have been compared with the cis and cis,cis isomers and with 2,5-norbornadiene for competition with ethylene for binding in mung bean sprouts and tobacco and for action (induction of chlorophyll degradation) in banana. The compounds containing a trans double bond were much more effective in competition for binding and action than the cis and cis,cis compounds. trans-Cyclooctene and cis,trans-1,3-cyclooctadiene were in the general range of 50–90 times more effective than 2,5-norbornadiene.R.J. Reynolds Research Apprentice     

Cloning of Trypanosoma cruzi trans-Sialidase and Expression in Pichia pastoris

Trypanosoma cruzi, the agent causing Chagas' disease, expresses an enzyme that transfers sialic acids among glycoproteins and glycolipids both from the host cell surface and its own surface. This enzyme, called trans-sialidase, is different from higher eukaryotic sialyltransferases in that it does not accept cytidine 5′-monophospho-N-acetylneuraminic acid as a donor substrate. Also, the common glycosyltransferase structure is not present. To study this enzyme, an active member was cloned and expressed in higher eukaryotic cells. Expression of recombinant enzyme was achieved in the methylotrophic yeast Pichia pastoris. The N-terminal fusion of a secretion signal and the C-terminal addition of an epitope tag resulted not only in high expression levels, but also enabled easy detection and purification. Using P. pastoris, we obtained about 5 mg of enzymatically active trans-sialidase per liter of induced culture medium.     

trans-Stilbene degradation by Arthrobacter sp. SL3 cells

trans-Stilbene degradation was examined by the reaction using resting cells of microorganisms isolated through the enrichment culture using trans-stilbene. The strain SL3, showing the highest trans-stilbene-degrading activity, was identified as Arthrobacter sp. One of the reaction products was identified to be cis,cis-muconic acid. Arthrobacter sp. SL3 cells also transformed benzaldehyde, benzoic acid and catechol into cis,cis-muconic acid, suggesting that one benzene ring of trans-stilbene was converted into cis,cis-muconic acid via benzaldehyde formed by its C_α=C_β bond cleavage.     

First evidence of trans-resveratrol production in cell suspension cultures of cotton (Gossypium hirsutum L.)

For the first time, trans-resveratrol, a stilbene, has been identified in cotton cell suspensions. Cell suspensions of Coker 312, a cultivar which produces embryogenic structures, acccumulate trans-resveratrol contrary to those of cultivar R405-2000, which do not. This stilbene may be a good phenolic marker for induction of somatic embryogenesis in cotton.     

Trans labilization of am(m)ine ligands from platinum(II) complexes by cancer cell extracts

Cisplatin, cis-[Pt(NH₃)₂Cl₂], is an effective anticancer agent in wide clinical use whose efficacy is affected by cellular interactions with sulfur-containing nucleophiles. These interactions can potentially enhance the efficacy of the drug by mediating its delivery to nuclear DNA or inactivate the drug by binding to it irreversibly or by labilizing the NH₃ ligands. Despite the potential importance of trans-labilization reactions in the mechanism of action of the drug, few detailed studies on trans labilization of the ammines have been conducted. We used 2D NMR to show that some trans labilization occurs in proliferating cells and that aqueous extracts of cancer cells labilized 20% of the amine ligands of cis-[PtCl₂(¹³CH₃NH₂)₂] after a 12-h incubation. Both low molecular mass nucleophiles (less than 3 kDa) and high molecular mass nucleophiles (more than 3 kDa) labilize the amines with similar efficiency. Studies with model compounds show that thiols and thioethers bind to platinum(II) at similar rates, but thioethers are significantly more efficient at labilizing the am(m)ine at lower pH. N-Acetylcysteine is a more efficient trans-labilizer than glutathione, suggesting that the displacement of the amine proceeds through an associative mechanism. The lag time, the time that elapses from the formation of the Pt–S bond till the release of the amine trans to the sulfur, depends on the pH (for thiols), increasing at lower pH. Quantification of the platinum adducts obtained from incubation of cisplatin with cell extracts indicates that two thirds of the platinum is bound to cellular components with molecular mass greater than 3 kDa. D. Gibson is a member of the David R. Bloom Center for Pharmacy.     

Rejection of tmRNA·SmpB after GTP hydrolysis by EF-Tu on ribosomes stalled on intact mRNA

Messenger RNAs lacking a stop codon trap ribosomes at their 3′ ends, depleting the pool of ribosomes available for protein synthesis. In bacteria, a remarkable quality control system rescues and recycles stalled ribosomes in a process known as trans-translation. Acting as a tRNA, transfer-messenger RNA (tmRNA) is aminoacylated, delivered by EF-Tu to the ribosomal A site, and accepts the nascent polypeptide. Translation then resumes on a reading frame within tmRNA, encoding a short peptide tag that targets the nascent peptide for degradation by proteases. One unsolved issue in trans-translation is how tmRNA and its protein partner SmpB preferentially recognize stalled ribosomes and not actively translating ones. Here, we examine the effect of the length of the 3′ extension of mRNA on each step of trans-translation by pre-steady-state kinetic methods and fluorescence polarization binding assays. Unexpectedly, EF-Tu activation and GTP hydrolysis occur rapidly regardless of the length of the mRNA, although the peptidyl transfer to tmRNA decreases as the mRNA 3′ extension increases and the tmRNA·SmpB binds less tightly to the ribosome with an mRNA having a long 3′ extension. From these results, we conclude that the tmRNA·SmpB complex dissociates during accommodation due to competition between the downstream mRNA and the C-terminal tail for the mRNA channel. Rejection of the tmRNA·SmpB complex during accommodation is reminiscent of the rejection of near-cognate tRNA from the ribosome in canonical translation.     

Mouse FKBP23 mediates conformer-specific functions of BiP by catalyzing Procis/trans isomerization

FK506-binding proteins (FKBPs) are cellular receptors for the immunosuppressant FK506 and rapamycin. They belong to the ubiquitous peptidyl-prolyl cis/trans isomerases (PPIases) family, which can catalyze the cis/trans isomerization of peptidyl-prolyl bond in peptides and proteins. In previous work, we revealed that mouse FKBP23 binds immunoglobulin binding protein (BiP), the major heat shock protein (Hsp) 70 chaperone in the ER, and the binding is interrelated with [Ca²⁺]. Furthermore, the binding can suppress the ATPase activity of BiP through the PPIase activity of FKBP23. In this work, FKBP23 is demonstrated to mediate functions of BiP by catalyzing the Pro¹¹⁷cis/trans conformational interconversion in the ATPase domain of BiP. This result may provide new understanding to the novel role of PPIase as a molecular switch.     

Rapid determination of trans-fatty acids in human adipose tissue: Comparison of attenuated total reflection infrared spectroscopy and gas chromatography

A rapid attenuated total reflection (ATR) infrared (IR) spectroscopy procedure was used for quantitating the levels of total trans-fatty acid methyl ester (FAME) derivatives in neat (without solvent) test samples isolated from human adipose tissue. This procedure requires no weighing of the laboratory sample. The single-beam spectrum of the trans-containing FAMEs was ‘ratioed' against that of a reference material having only cis double bonds in order to obtain a symmetric absorption band at 966 cm⁻¹ on a horizontal background. A single-reflection ATR diamond cell that requires only about 1 μl of neat FAMEs was used. The average level of trans-fatty acids in human adipose tissue found by ATR (3.07±0.27%) was generally higher than that obtained by gas chromatography (2.59±0.20%). Reasons for such a difference are discussed.     

The multi sex combs gene of Drosophila melanogaster is required for proliferation of the germline

We present a genetic analysis showing that the Drosophila melanogaster gene multi sex combs (mxc; Santamaria and Randsholt 1995) is needed for proliferation of the germline. Fertility is the feature most easily affected by weak hypomorphic mutations of this very pleiotropic locus. Pole cell formation and early steps of gonadogenesis conform to the wild-type in embryos devoid of zygotic mxc ⁺ product. mxc mutant gonad phenotypes and homozygous mxc germline clones suggest a role for mxc ⁺ in control of germ cell proliferation during the larval stages. mxc ⁺ requirement is germ cell autonomous and specific in females, whilst in males mxc ⁺ product is also needed in somatic cells of the gonads. Although mxc can be classified among the Polycomb group (Pc-G) of genes, negative trans-regulators of the ANT-C and BX-C gene complexes, germline requirement for mxc appears independent of a need for other Pc-C gene products, and mxc gonad phenotypes are different from those induced by mutations in BX-C genes. We discuss the possible functions of the mxc ⁺ product which helps to maintain homeotic genes repressed and prevents premature larval haemocyte differentiation and neoplasic overgrowth, but promotes growth and differentiation of male and female gonads.F.D. and O.S. should be considered as equal first authors     

Auxospore formation inLicmophora (Bacillariophyta)

InLicmophora gracilis var.anglica two auxospores are produced per pair of mother-cells, through the allogamic fusion of migratory and stationary gametes. Both active gametes are produced from the same mother-cell and hence both zygotes are formed in the other mother-cell. Pairing can occur between two stalked cells, or between a stalked cell and a detached cell; in the latter case the migratory gametes derive from the detached cell. The auxospores expand parallel to one another and to the apical axis of the donor mother-cell. Behavioural anisogamy of this kind, which may be termed thecis-type, seems to be characteristic of most araphid pennates and contrasts with thetrans-type exhibited byCymbella, Gomphonema and some other raphid taxa, where each mother-cell produces one migratory and one stationary gamete.