Structural Basis for the Calmodulin-Mediated Activation of eEF-2K

Translation is a highly energy consumptive process tightly regulated for optimal protein quality and adaptation to energy and nutrient availability. A key facilitator of this process is the α-kinase eEF-2K that specifically phosphorylates the GTP-dependent translocase eEF-2, thereby reducing its affinity for the ribosome and suppressing the elongation phase of protein synthesis. eEF-2K activation requires calmodulin binding and auto-phosphorylation at the primary stimulatory site, T348. Biochemical studies have predicted that calmodulin activates eEF-2K through a unique allosteric process mechanistically distinct from other calmodulin-dependent kinases. Here we resolve the atomic details of this mechanism through a 2.3 Å crystal structure of the heterodimeric complex of calmodulin with the functional core of eEF-2K (eEF-2KTR). This structure, which represents the activated T348-phosphorylated state of eEF-2KTR, highlights how through an intimate association with the calmodulin C-lobe, the kinase creates a spine that extends from its N-terminal calmodulin-targeting motif through a conserved regulatory element to its active site. Modification of key spine residues has deleterious functional consequences.

Utilization of a Sugarcane100K Single Nucleotide Polymorphisms Microarray-Derived High-Density Genetic Map in Quantitative Trait Loci Mapping and Function Role Prediction of Genes Related to Chlorophyll Content in Sugarcane

Chlorophyll is the most important pigment for plant photosynthesis that plays an important role in crop growth and production. In this study, the chlorophyll content trait was explored to improve sugarcane yield. Two hundred and eighty-five F1 progenies from the cross YT93-159 × ROC22 with significantly different chlorophyll contents were included as test materials. The chlorophyll content of the +1 leaves during elongation phase was measured using a SPAD-502 meter through a three-crop cycle (plant cane, first ratoon, and second ratoon). Linkage analysis was conducted on a high-density genetic map constructed based on the sugarcane 100K SNP chip. In addition, Fv/Fm, plant height, stalk diameter, brix data were collected on plant cane during the elongation and maturation phases. The results showed that the +1 leaf SPAD values, which can be used as an important reference to evaluate the growth potential of sugarcane, were significantly and positively correlated with the Fv/Fm during elongation phase, as well as with plant height, stalk diameter, and brix during maturity phase (P < 0.01). The broad sense heritability (H2) of the chlorophyll content trait was 0.66 for plant cane crop, 0.67 for first ratoon crop, and 0.73 for second ratoon crop, respectively, indicating that this trait was mainly controlled by genetic factors. Thirty-one quantitative trait loci (QTL) were detected by QTL mapping. Among them, a major QTL, qCC-R1, could account for 12.95% of phenotypic variation explained (PVE), and the other 30 minor QTLs explained 2.37–7.99% PVE. Twenty candidate genes related to chlorophyll content were identified in the QTLs plus a 200-Kb extension region within either sides, of which four were homologous genes involved in the chlorophyll synthesis process and the remaining 16 played a certain role in chlorophyll catabolic pathway, chloroplast organization, or photosynthesis. These results provide a theoretical reference for analyzing the genetic mechanism of chlorophyll synthesis and subsequent improvement of photosynthetic characteristics in sugarcane.

Promoter-proximal elongation regulates transcription in archaea

Recruitment of RNA polymerase and initiation factors to the promoter is the only known target for transcription activation and repression in archaea. Whether any of the subsequent steps towards productive transcription elongation are involved in regulation is not known. We characterised how the basal transcription machinery is distributed along genes in the archaeon Saccharolobus solfataricus. We discovered a distinct early elongation phase where RNA polymerases sequentially recruit the elongation factors Spt4/5 and Elf1 to form the transcription elongation complex (TEC) before the TEC escapes into productive transcription. TEC escape is rate-limiting for transcription output during exponential growth. Oxidative stress causes changes in TEC escape that correlate with changes in the transcriptome. Our results thus establish that TEC escape contributes to the basal promoter strength and facilitates transcription regulation. Impaired TEC escape coincides with the accumulation of initiation factors at the promoter and recruitment of termination factor aCPSF1 to the early TEC. This suggests two possible mechanisms for how TEC escape limits transcription, physically blocking upstream RNA polymerases during transcription initiation and premature termination of early TECs.

The RNA chaperone protein CspA stimulates translation during cold acclimation by promoting the progression of the ribosomes

CspA is an RNA binding protein expressed during cold-shock in Escherichia coli, capable of stimulating translation of several mRNAs - including its own - at low temperature. We used reconstituted translation systems to monitor the effects of CspA on the different steps of the translation process and probing experiments to analyze the interactions with its target mRNAs. We specifically focused on cspA mRNA which adopts a cold-induced secondary structure at temperatures below 20°C and a more closed conformation at 37°C. We show that at low temperature CspA specifically promotes the translation of the mRNA folded in the conformation less accessible to the ribosome (37°C form). CspA interacts with its mRNA without inducing large structural rearrangement, does not bind the ribosomal subunits and is not able to stimulate the formation of the translation initiation complexes. On the other hand, CspA promotes the progression of the ribosomes during translation of its mRNA at low temperature and this stimulation is mRNA structure-dependent. A similar structure-dependent mechanism may be responsible for the CspA-dependent translation stimulation observed with other probed mRNAs, for which the transition to the elongation phase is progressively facilitated during cold acclimation with the accumulation of CspA.

In Vitro Propagation of the Amazonian Medicinal Plant Guayusa (Ilex Guayusa) and Effects of Light in the Growth and Development of this Shade Tolerant Plant

Guayusa ( Ilex guayusa ) is an endemic plant from the Amazon with potential medicinal applications. Indigenous people are familiar with such applications and use guayusa based on ancestral knowledge. There is a growing interest in guayusa-based products in urban areas of Ecuador and internationally. The supply cannot meet the demand. Currently, traditional practices are used for guayusa growth and the potential use of the protected forest is foreseen. This work describes a protocol for the in vitro propagation of guayusa, a sustainable solution to generate high quality plants in reduced space. Stakes obtained from stems were used as explants. Chemical sterilization with ethanol and sodium hypochlorite resulted in 100% surface-sterilized stakes. The growth medium mWPM resulted in favorable outcomes regarding shoot development and elongation, as well as rooting. Supplementation with activated charcoal resulted in reduced browning during the elongation phase. The majority of shoots were able to develop roots spontaneously. Medium supplementation with the auxin indole-3-butyric acid, IBA, may be considered when rooting does not occur spontaneously. Acclimatization was performed in soil. The protocol was tested under different light spectra, revealing that guayusa growth is affected by light quality. The photobiology of this shade tolerant plant requires further characterization, but the data uncovered a potential role for green and far-red light in root development.

Deep conservation of ribosome stall sites across RNA processing genes

The rate of translation can vary depending on the mRNA template. During the elongation phase the ribosome can transiently pause or permanently stall. A pause can provide the nascent protein with the time to fold or be transported, while stalling can serve as quality control and trigger degradation of aberrant mRNA and peptide. Ribosome profiling has allowed for the genome-wide detection of such pauses and stalls, but due to library-specific biases, these predictions are often unreliable. Here, we take advantage of the deep conservation of protein synthesis machinery, hypothesizing that similar conservation could exist for functionally important locations of ribosome slowdown, here collectively called stall sites. We analyze multiple ribosome profiling datasets from phylogenetically diverse eukaryotes: yeast, fruit fly, zebrafish, mouse and human to identify conserved stall sites. We find thousands of stall sites across multiple species, with the enrichment of proline, glycine and negatively charged amino acids around conserved stalling. Many of the sites are found in RNA processing genes, suggesting that stalling might have a conserved role in RNA metabolism. In summary, our results provide a rich resource for the study of conserved stalling and indicate possible roles of stalling in gene regulation.

Selenium Biofortification of Wheat as a Strategy to Improve Human Nutrition

This paper analyses the effects of soil and foliar fertilization with sodium selenate (VI) on the selenium content in spring wheat grain. The research was carried out at the Departmental Experimental Station of the Institute of Agriculture WULS in Skierniewice in 2018 and 2019. The dose of selenium used was 5.00 g Se·ha−1 in various development stages of spring wheat. The results showed that selenium fertilisation did not affect the size of the grain yield, but both soil and foliar fertilisation significantly increased the content of selenium in wheat grain compared to the control group. The highest Se content was obtained with the method of soil fertilisation combined with the foliar application with a total dose of 10.00 g·ha-1 Se in the stem elongation phase (S + F2), and in the tillering and stem elongation phase (S + F1 + F2), which resulted in the values of 0.615 and 0.719 mg·kg−1 Se in grain, respectively. On this basis, it was concluded that the best time to carry out foliar fertilisation treatment is in the stem elongation phase (BBCH 30–39). The results show that the greatest increase in selenium content in the grain is achieved with soil and foliar fertilisation combined.

Total synthesis and biological characterization of SR-A3, a ternatin-related eEF1A inhibitor with enhanced cellular residence time

Ternatin and related cyclic peptides inhibit the elongation phase of protein synthesis by targeting the eukaryotic elongation factor-1α (eEF1A), a potential therapeutic vulnerability in cancer and viral infections. The cyclic peptide natural product “A3” appears to be related to ternatin, but its complete structure is unknown and only 4 of its 11 stereocenters have been assigned. Hence, A3 could be any one of 128 possible stereoisomers. Guided by the stereochemistry of ternatin and more potent structural variants, we synthesized two A3 epimers, “SR-A3” and “SS-A3”. We found that synthetic SR-A3 is indistinguishable from naturally derived A3 and potently inhibits cancer cell proliferation. Relative to SS-A3 and previously characterized ternatin variants, SR-A3 exhibits a dramatically enhanced duration of action. This increase in cellular residence time is conferred, stereospecifically, by a single β-hydroxy group attached to N-methyl leucine. SR-A3 thus exemplifies a mechanism for enhancing the pharmacological potency of cyclic peptide natural products via side-chain hydroxylation.

Deep conservation of ribosome stall sites across RNA processing genes

The rate of translation can vary considerably depending on the mRNA template. During the elongation phase the ribosome can transiently pause or permanently stall. A pause can provide the nascent protein with the required time to fold or be transported, while stalling can serve as quality control and trigger degradation of aberrant mRNA and peptide. Ribosome profiling has allowed for the genome-wide detection of such pause and stall sites, but due to library-specific biases, these predictions are often unreliable.Here, we address this by taking advantage of the deep conservation of the protein synthesis machinery, hypothesizing that similar conservation could exist for functionally important positions of ribosome slowdown - here collectively called stall sites. We analyze multiple ribosome profiling datasets from a phylogenetically diverse group of eukaryotes: yeast, fruit fly, zebrafish, mouse, and human and identify conserved stall sites. We find thousands of stall sites across multiple species, with proline, glycine, and negatively charged amino acids being the main facilitators of stalling. Many of the sites are found in RNA processing genes, suggesting that stalling might have a conserved regulatory effect on RNA metabolism. In summary, our results provide a rich resource for the study of conserved stalling and indicate possible roles of stalling in gene regulation.