Ribosome stalling and peptidyl-tRNA drop-off during translational delay at AGA codons

Minigenes encoding the peptide Met–Arg–Arg have been used to study the mechanism of toxicity of AGA codons proximal to the start codon or prior to the termination codon in bacteria. The codon sequences of the ‘mini-ORFs’ employed were initiator, combinations of AGA and CGA, and terminator. Both, AGA and CGA are low-usage Arg codons in ORFs of Escherichia coli but, whilst AGA is translated by the scarce tRNA^Arg4, CGA is recognized by the abundant tRNA^Arg2. Overexpression of minigenes harbouring AGA in the third position, next to a termination codon, was deleterious to the cell and led to the accumulation of peptidyl-tRNA^Arg4 and of the peptidyl-tRNA cognate to the preceding CGA or AGA Arg triplet. The minigenes carrying CGA in the third position were not toxic. Minigene-mediated toxicity and peptidyl-tRNA accumulation were suppressed by overproduction of tRNA^Arg4 but not by overproduction of peptidyl-tRNA hydrolase, an enzyme that is only active on substrates that have been released from the ribosome. Consistent with these findings, peptidyl-tRNA^Arg4 was identified to be mainly associated with ribosomes in a stand-by complex. These and previous results support the hypothesis that the primary mechanism of inhibition of protein synthesis by AGA triplets in pth⁺ cells involves sequestration of tRNAs as peptidyl-tRNA on the stalled ribosome.     

ftsE(Ts) Affects Translocation of K+-Pump Proteins into the Cytoplasmic Membrane of Escherichia coli

The ftsE(Ts) mutation of Escherichia coli causes defects in cell division and cell growth. We expressed alkaline phosphatase (PhoA) fusion proteins of KdpA, Kup, and TrkH, all of which proved functional in vivo as K⁺ ion pumps, in the mutant cells. During growth at 41°C, these proteins were progressively lost from the membrane fraction. The reduction in the abundance of these proteins inversely correlated with cell growth, but the preformed proteins in the membrane were stable at 41°C, indicating that the molecules synthesized at the permissive temperature were diluted in a growth-dependent manner at a high temperature. Pulse-chase experiments showed that KdpA-PhoA was synthesized, but the synthesized protein did not translocate into the membrane of the ftsE(Ts) cells at 41°C and degraded very rapidly. The loss of KdpA-PhoA from the membrane fractions of ftsE(Ts) cells was suppressed by a multicopy plasmid carrying the ftsE⁺ gene. While cell growth stopped when the abundance of these proteins decreased 15-fold, the addition of a high concentration of K⁺ ions specifically alleviated the growth defect of ftsE(Ts) cells but not cell division, and the cells elongated more than 100-fold. We conclude that one of the causes of growth cessation in the ftsE(Ts) mutants is a defect in the translocation of K⁺-pump proteins into the cytoplasmic membrane.     

Screening of mitochondrial mutations and insertion–deletion polymorphism in gestational diabetes mellitus in the Asian Indian population

In this study we scrutinized the association between the A8344G/A3243G mutations and a 9-bp deletion polymorphism with gestational diabetes mellitus (GDM) in an Asian Indian population. The A3243G mutation in the mitochondrial tRNA^Leu(UUR) causes mitochondrial encephalopathy myopathy, lactic acidosis, and stroke-like episodes (MELAS), while the A8344G mutation in tRNA^Lys causes myoclonus epilepsy with ragged red fibers (MERRF). We screened 140 pregnant women diagnosed with GDM and 140 non-GDM participants for these mutations by PCR-RFLP analysis. Both A3243G and A8344G were associated with GDM (A3243: OR-3.667, 95% CI = 1.001–13.43, p = 0.03; A8344G: OR-11.00, 95% CI = 0.6026–200.8, p = 0.04). Mitochondrial DNA mutations contribute to the development of GDM. Our results conclude that mitochondrial mutations are associated with the GDM women in our population. Thus it is important to screen other mitochondrial mutations in the GDM women.     

An rne-1 pnp-7 Double Mutation Suppresses the Temperature-Sensitive Defect of lacZ Gene Expression in a divE Mutant

A divE mutant, which has a temperature-sensitive mutation in the tRNA₁^Ser gene, exhibits differential loss of the synthesis of certain proteins, such as β-galactosidase and succinate dehydrogenase, at nonpermissive temperatures. In Escherichia coli, the UCA codon is recognized only by tRNA₁^Ser. Several genes containing UCA codons are normally expressed after a temperature shift to 42°C in the divE mutant. Therefore, it is unlikely that the defect in protein synthesis at 42°C is simply caused by a defect in the decoding function of the mutant tRNA₁^Ser. In this study, we sought to determine the cause of the defect in lacZ gene expression in the divE mutant. It has also been shown that the defect in lacZ gene expression is accompanied by a decrease in the amount of lacZ mRNA. To examine whether inactivation of mRNA degradation pathways restores the defect in lacZ gene expression, we constructed divE mutants containing rne-1, rnb-500, and pnp-7 mutations in various combinations. We found that the defect was almost completely restored by introducing an rne-1 pnp-7 double mutation into the divE mutant. Northern hybridization analysis showed that the rne-1 mutation stabilized lacZ mRNA, whereas the pnp-7 mutation stabilized mutant tRNA₁^Ser, at 44°C. We present a mechanism that may explain these results.     

Biochemical Characterization and Validation of a Catalytic Site of a Highly Thermostable Ts2631 Endolysin from the Thermus scotoductus Phage vB_Tsc2631

Phage vB_Tsc2631 infects the extremophilic bacterium Thermus scotoductus MAT2631 and uses the Ts2631 endolysin for the release of its progeny. The Ts2631 endolysin is the first endolysin from thermophilic bacteriophage with an experimentally validated catalytic site. In silico analysis and computational modelling of the Ts2631 endolysin structure revealed a conserved Zn²⁺ binding site (His³⁰, Tyr⁵⁸, His¹³¹ and Cys¹³⁹) similar to Zn²⁺ binding site of eukaryotic peptidoglycan recognition proteins (PGRPs). We have shown that the Ts2631 endolysin lytic activity is dependent on divalent metal ions (Zn²⁺ and Ca²⁺). The Ts2631 endolysin substitution variants H30N, Y58F, H131N and C139S dramatically lost their antimicrobial activity, providing evidence for the role of the aforementioned residues in the lytic activity of the enzyme. The enzyme has proven to be not only thermoresistant, retaining 64.8% of its initial activity after 2 h at 95°C, but also highly thermodynamically stable (T_m = 99.82°C, ΔH_cal = 4.58 × 10⁴ cal mol^-1). Substitutions of histidine residues (H30N and H131N) and a cysteine residue (C139S) resulted in variants aggregating at temperatures ≥75°C, indicating a significant role of these residues in enzyme thermostability. The substrate spectrum of the Ts2631 endolysin included extremophiles of the genus Thermus but also Gram-negative mesophiles, such as Escherichia coli, Salmonella panama, Pseudomonas fluorescens and Serratia marcescens. The broad substrate spectrum and high thermostability of this endolysin makes it a good candidate for use as an antimicrobial agent to combat Gram-negative pathogens.     

High temperature-induced thermotolerance in pollen tubes of tradescantia and heat-shock proteins

Growing pollen tubes of Tradescantia paludosa are protected from inhibition of growth at 41°C by a prior exposure to gradually increasing temperatures. Heat shock proteins (hsps) are not synthesized by pollen tubes as determined by labeling with [³⁵S]methionine and two-dimensional gel electrophoresis, during either a heat shock at 41°C or a gradual temperature increase to 41°C. A comparison after two-dimensional electrophoresis of silver-stained spots and radioactive spots after autoradiography of an extract of ungerminated pollen mixed with a trace amount of [³⁵S]methionine-labeled vegetative tissue heat shocked at 41°C to act as a hsps marker, indicates that the majority, if not all, of the major hsps are not present in the pollen grain at anthesis. The type of thermotolerance seen with pollen tubes can thus be achieved without the presence or the new synthesis of the hsps.     

Studies on the function of the non-primer tRNAs associated with the 70 S RNA of avian myeloblastosis virus

Significant amounts of three tRNAs are associated with the 70 S RNA of avian myeloblastosis virus (AMV). The temperatures at which they are half dissociated from the 70 S RNA in 50 mM NaCl and their respective quantities relative to 35 S RNA are: tRNA^Arg, 51°C, 1.6; tRNA^Lys, 57°C, 0.7 and tRNA^Trp, 76°C, 1.0. Possible functions for the non-primer tRNAs (tRNA^Arg and tRNA^Lys) were evaluated by determining the effect of their thermal dissociation on: (a) conversion of 70 S to 35 S RNA, (b) capacity of 70 S and/or 35 S RNA to be translated in vitro, and (c) capacity of 70 S and/or 35 S RNA to be reverse transcribed in vitro. Conversion of 70 S to 35 S RNA occurred with a t_m of 56°C and is consistent with the hypothesis that tRNA^Lys might be involved in joining two 35 S RNA subunits to form the 70 S RNA complex. There was no indication that the association of either tRNA^Arg or tRNA^Lys influenced the rate or quality of translation of 70 S or 35 S RNA. A decrease in the rate at which 70 S RNA is transcribed occurs in parallel with the dissociation of tRNA^Arg and tRNA^Lys.     

Identification and Characterisation of Heat Shock Protein 70 in Thermal Stressed Blastocystis sp

Protistan parasites in order to ensure their viability and demonstrate successful progression in their life cycle need to respond towards various environmental stressors. Blastocystis sp. is known to be the most commonly found intestinal protistan parasite in any human stool surveys and has been incriminated to be responsible for diarrhea and bloating stomach. The present study demonstrates for the first time the presence of HSP70 in subtypes of Blastocystis sp. when the cultures were subjected to temperature of 39 and 41°C where the growth of parasites was reduced to a minimum to majority being granular forms. The growth of parasites exposed to higher temperatures however doubled compared to the controls when the parasites were re-cultured back at 37°C. Upon thermal stress at 41°C, subtype 3 and subtype 5 isolates'' growth reached up to 2.97×10⁶ and 3.05×10⁶ cells/ml compared to their respective controlled culture tubes at 37°C which peaked only at 1.34×10⁶ and 1.70×10⁶ cells/ml respectively. The designed primer set that amplified Blastocystis sp. subtype 7 HSP70 gene in subtypes 1, 3 and 5 was against a conserved region. The gene was amplified at 318 bp. The multiple sequence alignment showed that the targeted sequence length ranges from 291–295 bp. The pair wise alignment result showed that the sequence identity among the four sequence ranges from 88% to 96%. These findings were further evidenced by the up regulation of HSP70 gene in thermal stressed isolates of subtype 3 and 5 at 41°C. Higher number of granular forms was significantly found in thermal stressed isolates of subtype 3 and 5 which implicates that this life cycle stage has a role in responding to thermal stress.     

Greening under High Light or Cold Temperature Affects the Level of Xanthophyll-Cycle Pigments, Early Light-Inducible Proteins, and Light-Harvesting Polypeptides in Wild-Type Barley and the Chlorina f2 Mutant

Etiolated seedlings of wild type and the chlorina f2 mutant of barley (Hordeum vulgare) were exposed to greening at either 5°C or 20°C and continuous illumination varying from 50 to 800 μmol m⁻² s⁻¹. Exposure to either moderate temperature and high light or low temperature and moderate light inhibited chlorophyll a and b accumulation in the wild type and in the f2 mutant. Continuous illumination under these greening conditions resulted in transient accumulations of zeaxanthin, concomitant transient decreases in violaxanthin, and fluctuations in the epoxidation state of the xanthophyll pool. Photoinhibition-induced xanthophyll-cycle activity was detectable after only 3 h of greening at 20°C and 250 μmol m⁻² s⁻¹. Immunoblot analyses of the accumulation of the 14-kD early light-inducible protein but not the major (Lhcb2) or minor (Lhcb5) light-harvesting polypeptides demonstrated transient kinetics similar to those observed for zeaxanthin accumulation during greening at either 5°C or 20°C for both the wild type and the f2 mutant. Furthermore, greening of the f2 mutant at either 5°C or 20°C indicated that Lhcb2 is not essential for the regulation of the xanthophyll cycle in barley. These results are consistent with the thesis that early light-inducible proteins may bind zeaxanthin as well as other xanthophylls and dissipate excess light energy to protect the developing photosynthetic apparatus from excess excitation. We discuss the role of energy balance and photosystem II excitation pressure in the regulation of the xanthophyll cycle during chloroplast biogenesis in wild-type barley and the f2 mutant.     

Highly conserved modified nucleosides influence Mg2+-dependent tRNA folding

Transfer RNA structure involves complex folding interactions of the TΨC domain with the D domain. However, the role of the highly conserved nucleoside modifications in the TΨC domain, rT₅₄, Ψ₅₅ and m⁵C₄₉, in tertiary folding is not understood. To determine whether these modified nucleosides have a role in tRNA folding, the association of variously modified yeast tRNA^Phe T-half molecules (nucleosides 40–72) with the corresponding unmodified D-half molecule (nucleosides 1–30) was detected and quantified using a native polyacrylamide gel mobility shift assay. Mg²⁺ was required for formation and maintenance of all complexes. The modified T-half folding interactions with the D-half resulted in K_ds (rT₅₄ = 6 ± 2, m⁵C₄₉ = 11 ± 2, Ψ₅₅ = 14 ± 5, and rT₅₄,Ψ₅₅ = 11 ± 3 µM) significantly lower than that of the unmodified T-half (40 ± 10 µM). However, the global folds of the unmodified and modified complexes were comparable to each other and to that of an unmodified yeast tRNA^Phe and native yeast tRNA^Phe, as determined by lead cleavage patterns at U₁₇ and nucleoside substitutions disrupting the Levitt base pair. Thus, conserved modifications of tRNA’s TΨC domain enhanced the affinity between the two half-molecules without altering the global conformation indicating an enhanced stability to the complex and/or an altered folding pathway.     

Effects of low temperature and respiratory inhibitors on calcium flux in plant mitochondria

The effects of low temperature on uptake and release of ⁴⁵Ca²⁺ were studied with sound, well-coupled mitochondria extracted at room temperature from avocado (Persea americana Mill, cv Fuerte) fruits. Low Ca²⁺ concentrations (10 micromolar) were employed to simulate physiological conditions. At 25°C, the rate of Ca²⁺ uptake decreased with time, whereas at 5°C the initial rate, though lower, remained linear. As a consequence total uptake at 5°C was substantially greater than at 25°C for periods greater than 5 min. Preincubation of mitochondria at 5°C enhanced subsequent Ca²⁺ uptake at 25°C. Ca²⁺ uptake was inhibited by carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and by ruthenium red, but neither KCN nor salicylhydroxamic acid separately or together had any major inhibitory effect. Preloaded mitochondria held for 60 min in a Ca-free medium lost little Ca²⁺ at 25°C and none at 5°C, except in the presence of ruthenium red or CCCP.     

N7-Methylguanine at position 46 (m7G46) in tRNA from Thermus thermophilus is required for cell viability at high temperatures through a tRNA modification network

N⁷-methylguanine at position 46 (m⁷G46) in tRNA is produced by tRNA (m⁷G46) methyltransferase (TrmB). To clarify the role of this modification, we made a trmB gene disruptant (ΔtrmB) of Thermus thermophilus, an extreme thermophilic eubacterium. The absence of TrmB activity in cell extract from the ΔtrmB strain and the lack of the m⁷G46 modification in tRNA^Phe were confirmed by enzyme assay, nucleoside analysis and RNA sequencing. When the ΔtrmB strain was cultured at high temperatures, several modified nucleotides in tRNA were hypo-modified in addition to the lack of the m⁷G46 modification. Assays with tRNA modification enzymes revealed hypo-modifications of Gm18 and m¹G37, suggesting that the m⁷G46 positively affects their formations. Although the lack of the m⁷G46 modification and the hypo-modifications do not affect the Phe charging activity of tRNA^Phe, they cause a decrease in melting temperature of class I tRNA and degradation of tRNA^Phe and tRNA^Ile. ³⁵S-Met incorporation into proteins revealed that protein synthesis in ΔtrmB cells is depressed above 70°C. At 80°C, the ΔtrmB strain exhibits a severe growth defect. Thus, the m⁷G46 modification is required for cell viability at high temperatures via a tRNA modification network, in which the m⁷G46 modification supports introduction of other modifications.     

Chemolithotrophic acetogenic H2/CO2 utilization in Italian rice field soil

Acetate oxidation in Italian rice field at 50 °C is achieved by uncultured syntrophic acetate oxidizers. As these bacteria are closely related to acetogens, they may potentially also be able to synthesize acetate chemolithoautotrophically. Labeling studies using exogenous H₂ (80%) and ¹³CO₂ (20%), indeed demonstrated production of acetate as almost exclusive primary product not only at 50 °C but also at 15 °C. Small amounts of formate, propionate and butyrate were also produced from ¹³CO₂. At 50 °C, acetate was first produced but later on consumed with formation of CH₄. Acetate was also produced in the absence of exogenous H₂ albeit to lower concentrations. The acetogenic bacteria and methanogenic archaea were targeted by stable isotope probing of ribosomal RNA (rRNA). Using quantitative PCR, ¹³C-labeled bacterial rRNA was detected after 20 days of incubation with ¹³CO₂. In the heavy fractions at 15 °C, terminal restriction fragment length polymorphism, cloning and sequencing of 16S rRNA showed that Clostridium cluster I and uncultured Peptococcaceae assimilated ¹³CO₂ in the presence and absence of exogenous H₂, respectively. A similar experiment showed that Thermoanaerobacteriaceae and Acidobacteriaceae were dominant in the ¹³C treatment at 50 °C. Assimilation of ¹³CO₂ into archaeal rRNA was detected at 15 °C and 50 °C, mostly into Methanocellales, Methanobacteriales and rice cluster III. Acetoclastic methanogenic archaea were not detected. The above results showed the potential for acetogenesis in the presence and absence of exogenous H₂ at both 15 °C and 50 °C. However, syntrophic acetate oxidizers seemed to be only active at 50 °C, while other bacterial groups were active at 15 °C.     

The Effect of Temperature on the Uptake of Radiosulfate by Rat Renal Tissue from Radiosulfate-Containing Solutions in Vitro

Kidney cortex slices incubated in vitro at 0°C. accumulate radiosulfate from the incubation medium. This process differs from the previously described uptake of radiosulfate by renal tissue incubated at 38°C., for instance, in the lesser sensitivity of the uptake at 0°C. to differential effects of Na⁺ as compared with K⁺ ions, and of sucrose as compared with glucose. Phlorizin inhibits radiosulfate accumulation at 0°C., whereas it enhances the uptake at 38°C. Effects of the cations K⁺ and Na⁺ and of phlorizin at temperatures intermediate between 0° and 38°C. have been studied. Parallels have been noted between the accumulative processes for radiosulfate of kidney slices maintained at 0°C. and of mitochondria isolated from rat liver and kidney cortex. These similarities may be attributed to an important role of radiosulfate uptake by mitochondria in slice accumulation of radiosulfate in the cold.     

Iron Superoxide Dismutase Protects against Chilling Damage in the Cyanobacterium Synechococcus species PCC7942

A strain of Synechococcus sp. PCC7942 lacking functional Fe superoxide dismutase (SOD), designated sodB⁻, was characterized by its growth rate, photosynthetic pigments, inhibition of photosynthetic electron transport activity, and total SOD activity at 0°C, 10°C, 17°C, and 27°C in moderate light. At 27°C, the sodB⁻ and wild-type strains had similar growth rates, chlorophyll and carotenoid contents, and cyclic photosynthetic electron transport activity. The sodB⁻ strain was more sensitive to chilling stress at 17°C than the wild type, indicating a role for FeSOD in protection against photooxidative damage during moderate chilling in light. However, both the wild-type and sodB⁻ strains exhibited similar chilling damage at 0°C and 10°C, indicating that the FeSOD does not provide protection against severe chilling stress in light. Total SOD activity was lower in the sodB⁻ strain than in the wild type at 17°C and 27°C. Total SOD activity decreased with decreasing temperature in both strains but more so in the wild type. Total SOD activity was equal in the two strains when assayed at 0°C.     

Insertional Editing of Mitochondrial tRNAs of Physarum polycephalum and Didymium nigripes

tRNAs encoded on the mitochondrial DNA of Physarum polycephalum and Didymium nigripes require insertional editing for their maturation. Editing consists of the specific insertion of a single cytidine or uridine relative to the mitochondrial DNA sequence encoding the tRNA. Editing sites are at 14 different locations in nine tRNAs. Cytidine insertion sites can be located in any of the four stems of the tRNA cloverleaf and usually create a G·C base pair. Uridine insertions have been identified in the T loop of tRNA^Lys from Didymium and tRNA^Glu from Physarum. In both tRNAs, the insertion creates the GUUC sequence, which is converted to GTΨC (Ψ = pseudouridine) in most tRNAs. This type of tRNA editing is different from other, previously described types of tRNA editing and resembles the mRNA and rRNA editing in Physarum and Didymium. Analogous tRNAs in Physarum and Didymium have editing sites at different locations, indicating that editing sites have been lost, gained, or both since the divergence of Physarum and Didymium. Although cDNAs derived from single tRNAs are generally fully edited, cDNAs derived from unprocessed polycistronic tRNA precursors often lack some of the editing site insertions. This enrichment of partially edited sequences in unprocessed tRNAs may indicate that editing is required for tRNA processing or at least that RNA editing occurs as an early event in tRNA synthesis.