Start Codon Research Articles

A seamless connection from the burst sequence to the start codon is essential for polyhedrin hyperexpression in alphabaculoviruses

Alphabaculoviruses produce a large number of occlusion bodies (OBs) in host cells during the late stage of infection. OBs are mainly composed of polyhedrin (POLH), and high-level transcription of the polh gene has been exploited to express foreign proteins in insect cells. While making Bombyx mori nucleopolyhedrovirus (BmNPV) polh mutants using a conventional transfer vector-based method, we noticed that a virus with a short sequence insertion just before the polh start codon produces fewer very small OBs. Detailed analysis of several BmNPV mutants revealed that insertions between the burst sequence and start codon markedly decrease POLH accumulation and polh transcription. We further confirmed this decrease using recombinant viruses expressing a reporter gene driven by the polh promoter. These findings underscore the critical importance of a seamless connection from the burst sequence to the start codon for baculovirus polh hyperexpression.

Increased levels of eIF2A inhibit translation by sequestering 40S ribosomal subunits.

eIF2A was the first eukaryotic initiator tRNA carrier discovered but its exact function has remained enigmatic. Uncharacteristic of translation initiation factors, eIF2A is reported to be non-cytosolic in multiple human cancer cell lines. Attempts to study eIF2A mechanistically have been limited by the inability to achieve high yield of soluble recombinant protein. Here, we developed a purification paradigm that yields ∼360-fold and ∼6000-fold more recombinant human eIF2A from Escherichia coli and insect cells, respectively, than previous reports. Using a mammalian in vitro translation system, we found that increased levels of recombinant human eIF2A inhibit translation of multiple reporter mRNAs, including those that are translated by cognate and near-cognate start codons, and does so prior to start codon recognition. eIF2A also inhibited translation directed by all four types of cap-independent viral IRESs, including the CrPV IGR IRES that does not require initiation factors or initiator tRNA, suggesting excess eIF2A sequesters 40S subunits. Supplementation with additional 40S subunits prevented eIF2A-mediated inhibition and pull-down assays demonstrated direct binding between recombinant eIF2A and purified 40S subunits. These data support a model that eIF2A must be kept away from the translation machinery to avoid sequestering 40S ribosomal subunits.

Mitochondrial polymorphism m.3017C>T of SHLP6 relates to heterothermy.

Heterothermic thermoregulation requires intricate regulation of metabolic rate and activation of pro-survival factors. Eliciting these responses and coordinating the necessary energy shifts likely involves retrograde signalling by mitochondrial-derived peptides (MDPs). Members of the group were suggested before to play a role in heterothermic physiology, a key component of hibernation and daily torpor. Here we studied the mitochondrial single-nucleotide polymorphism (SNP) m.3017C>T that resides in the evolutionarily conserved gene MT-SHLP6. The substitution occurring in several mammalian orders causes truncation of SHLP6 peptide size from twenty to nine amino acids. Public mass spectrometric (MS) data of human SHLP6 indicated a canonical size of 20 amino acids, but not the use of alternative translation initiation codons that would expand the peptide. The shorter isoform of SHLP6 was found in heterothermic rodents at higher frequency compared to homeothermic rodents (p < 0.001). In heterothermic mammals it was associated with lower minimal body temperature (T b, p < 0.001). In the thirteen-lined ground squirrel, brown adipose tissue-a key organ required for hibernation, showed dynamic changes of the steady-state transcript level of mt-Shlp6. The level was significantly higher before hibernation and during interbout arousal and lower during torpor and after hibernation. Our finding argues to further explore the mode of action of SHLP6 size isoforms with respect to mammalian thermoregulation and possibly mitochondrial retrograde signalling.

HELZ2: a new, interferon-regulated, human 3'-5' exoribonuclease of the RNB family is expressed from a non-canonical initiation codon.

Proteins containing a RNB domain, originally identified in Escherichia coli RNase II, are widely present throughout the tree of life. Many RNB proteins have 3'-5' exoribonucleolytic activity but some have lost catalytic activity during evolution. Database searches identified a new RNB domain-containing protein in human: HELZ2. Analysis of genomic and expression data combined with evolutionary information suggested that the human HELZ2 protein is produced from an unforeseen non-canonical initiation codon in Hominidae. This unusual property was confirmed experimentally, extending the human protein by 247 residues. Human HELZ2 was further shown to be an active ribonuclease despite the substitution of a key residue in its catalytic center. HELZ2 RNase activity is lost in cells from some cancer patients as a result of somatic mutations. HELZ2 harbors also two RNA helicase domains and several zinc fingers and its expression is induced by interferon treatment. We demonstrate that HELZ2 is able to degrade structured RNAs through the coordinated ATP-dependent displacement of duplex RNA mediated by its RNA helicase domains and its 3'-5' ribonucleolytic action. The expression characteristics and biochemical properties of HELZ2 support a role for this factor in response to viruses and/or mobile elements.

On translational control by ribosome speed in S. cerevisiae

Introduction: In addition to the widespread and well documented control of protein synthesis by translation initiation, recent evidence suggests that translation elongation can also control protein synthesis rates. One of the proposed mechanisms leading to elongation control is the interference of slow ribosome movement around the start codon with efficient translation initiation. Here we estimate the frequency with which this mode of control occurs in baker’s yeast growing in rich medium.Methods: We interrogate published genome-wide datasets for evidence of transcripts associated with queueing small ribosomal subunits, and confirm results from these surveys using additional experimental work.Results: Our analyses reveal that transcripts from around 20% of yeast genes show evidence of queueing ribosomes, which may be indicative of translation elongation control. Moreover, this subset of transcripts is sensitive to distinct regulatory signals compared to initiation-controlled mRNAs, and such distinct regulation occurs, for example, during the response to osmotic stress.Discussion: Our analyses provide a first quantitative estimate for the prevalence of translational control exerted via the elongation stage in a commonly used model organism, and suggest that transcript under elongation control form a separately addressable RNA regulon.

Assessment of genetic relation for Aegilops and Triticum possessing different genome-bearing species using start codon targeted marker

Assessment of genetic relation for Aegilops and Triticum possessing different genome-bearing species using start codon targeted marker

Molecular phylogeny of plant pathogenic fungi based on start codon targeted (SCoT) polymorphism.

A number of molecular marker systems have been developed to assess genetic diversity, carry out phylogenetic analysis, and diagnose and discriminate plant pathogenic fungi. The start codon targeted (SCoT) markers system is a novel approach used here to investigate intra and interspecific polymorphisms of phytopathogenic fungi. This study assessed genetic variability between and within 96 isolates of ten fungal species associated with a variety of plant species using 36 SCoT primers. The six primers generated 331 distinct and reproducible banding patterns, of which 322 were polymorphic (97.28%), resulting in 53.67 polymorphic bands per primer. All primers produced informative amplification profiles that distinguished all fungal species. With a resolving power of 10.65, SCoT primer 12 showed the highest polymorphism among species, followed by primer 33 and primer 29. Polymorphic loci (PPL), Nei's diversity index (h), and Shannon index (I) percentages were 6.25, 0.018, and 0.028, respectively. UPGMA analysis separated all isolates based on morphological classification and revealed significant genetic variation among fungal isolates at the intraspecific level. PCoA analysis strongly supported fungal species discrimination and genetic variation. The other parameters of evaluation proved that SCoT markers are at least as effective as other DNA markers. SCoT markers were effective in identifying plant pathogenic fungi and were a powerful tool for estimating genetic variation and population structure of different fungi species.

Association study between some cultivated species and their wild relatives from Apiaceae, Asteraceae and Brassicaceae families based on molecular and DNA barcoding in Egypt

Concerns about food security worldwide have been raised due to the adverse impacts of climate change on agricultural productivity. Utilizing wild relatives of cultivated plants in breeding schemes is a potential solution to address the abovementioned challenges. Wild relatives refer to indigenous plant species that share genetic ancestry with recently cultivated plants. The capacity of plants to withstand abiotic stresses and enhance crop yield stability in cold and arid environments renders them a valuable prospective resource for breeding programs. Molecular markers are employed to assess the genetic variability of cultivated plants and their wild relatives under investigation. This study employed a set of five primers derived from ISSR (Inter Simple Sequence Repeats) and SCoT (Start Codon Targeted) to assess comparative research between cultivated species and their wild relatives from three families: Apiaceae (Apium gravolens L., A. leptophyllum (Pers.) F.Muell., Daucus carota L.& D. litoralis Sm.), Asteraceae (Lactuca sativa L. & L. serriola L.) and Brassicaceae (B. oleracea L., Brassica tourneforttii Gouan., Raphanus sativus L. & R. raphanistrum L.). The ISSR technique yields approximately 206 bands, whereas the SCoT primers generate approximately 207 bands. The ISSR primers yielded 169 polymorphic bands, exhibiting a polymorphism rate of 82.04%, whereas the SCoT primers produced 169 polymorphic bands with a polymorphism rate of 81.64%. The Brassicaceae has been found to exhibit the highest resolving power (RP) of 48.25, as determined by ISSR. The primary objective of DNA barcoding is to create a communal database of DNA sequences that can be utilized to identify organisms and clarify their taxonomic classification. The findings indicate that the internal transcribed spacer (ITS) regions exhibit high efficacy as a DNA barcoding tool. Overall, the results obtained from two molecular markers and DNA barcoding analyses suggest a significant degree of genetic diversity and relatedness among the cultivated taxa and their wild relatives examined in this study.

Characterization and Gene Mapping of an Open-Glume Oryza sativa L. Mutant.

Floral organ development determines agricultural productivity by affecting seed development, seed quality, and final yield. In this study, we described the novel ogl mutant in rice (Oryza sativa L.), which is characterized by an open-glume phenotype, increased pistil number, reduced stamen number, decreased seed setting rate, and smaller rice grains. Genetic analysis showed that the open-glume phenotype might be controlled by a recessive qualitative trait locus. Employing bulked segregant analysis (BSA), one candidate region was identified on rice chromosome 1. The glume opening phenotype cosegregated with SNP (Chr1:1522703), which was located at the start codon of one transcript of OsJAG, resulting in partial loss of OsJAG function. cDNA analysis revealed that OsJAG encodes two transcript variants. Compared to normal plants, the expression of OsJAG.1 was upregulated in open-glume plants. When investigating the glume phenotype, we found that the expression of genes related to floral development changed greatly in open-glume plants. Taken together, this work increases our understanding of the developmental role of OsJAG in rice floral development.

Defective airway intraflagellar transport underlies a combined motile and primary ciliopathy syndrome caused by IFT74 mutations.

Ciliopathies are inherited disorders caused by defective cilia. Mutations affecting motile cilia usually cause the chronic muco-obstructive sinopulmonary disease primary ciliary dyskinesia (PCD) and are associated with laterality defects, while a broad spectrum of early developmental as well as degenerative syndromes arise from mutations affecting signalling of primary (non-motile) cilia. Cilia assembly and functioning requires intraflagellar transport (IFT) of cargos assisted by IFT-B and IFT-A adaptor complexes. Within IFT-B, the N-termini of partner proteins IFT74 and IFT81 govern tubulin transport to build the ciliary microtubular cytoskeleton. We detected a homozygous 3-kb intragenic IFT74 deletion removing the exon 2 initiation codon and 40N-terminal amino acids in two affected siblings. Both had clinical features of PCD with bronchiectasis, but no laterality defects. They also had retinal dysplasia and abnormal bone growth, with a narrowed thorax and short ribs, shortened long bones and digits, and abnormal skull shape. This resembles short-rib thoracic dysplasia, a skeletal ciliopathy previously linked to IFT defects in primary cilia, not motile cilia. Ciliated nasal epithelial cells collected from affected individuals had reduced numbers of shortened motile cilia with disarranged microtubules, some misorientation of the basal feet, and disrupted cilia structural and IFT protein distributions. No full-length IFT74 was expressed, only truncated forms that were consistent with N-terminal deletion and inframe translation from downstream initiation codons. In affinity purification mass spectrometry, exon 2-deleted IFT74 initiated from the nearest inframe downstream methionine 41 still interacts as part of the IFT-B complex, but only with reduced interaction levels and not with all its usual IFT-B partners. We propose that this is a hypomorphic mutation with some residual protein function retained, which gives rise to a primary skeletal ciliopathy combined with defective motile cilia and PCD.

The transcription factor EMB1444-like affects tomato fruit ripening by regulating YELLOW-FRUITED TOMATO 1, a core component of ethylene signaling transduction.

The fleshy fruit of tomato (Solanum lycopersicum) are climacteric and, as such, ethylene plays a pivotal role in their ripening and quality traits. In this study, a basic helix-loop-helix transcription factor, EMB1444-like, was found to induce the expression of YELLOW-FRUITED TOMATO 1 (YFT1), which encodes the SlEIN2 protein, a key element in the ethylene signaling pathway. Yeast one-hybrid and EMSA analyses revealed that EMB1444-like binds to the E-box motif (CACTTG, -1295 bp to -1290 bp upstream of the ATG start codon) of the YFT1 promoter (pYFT1). Suppression of EMB1444-like expression in tomato lines (sledl) using RNAi reduced ethylene production by lowering the expression of 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE 2/4 (ACS2/4) and ACC OXIDASE1 (ACO1) in a positive feedback loop. sledl tomato also showed differences in numerous quality traits related to fruit ripening, compared with the wild type, such as delayed chromoplast differentiation, a decrease in carotenoid accumulation, and delayed fruit ripening in an ethylene-independent manner, or at least upstream of ripening mediated by YFT1/SlEIN2. This study elucidates the regulatory framework of fruit ripening in tomato, providing information that may be used to breed tomato hybrid cultivars with an optimal balance of shelf-life, durability, and high quality.

The Use of Wheat Starch as Gelling Agent for In Vitro Proliferation of Blackberry (Rubus fruticosus L.) Cultivars and the Evaluation of Genetic Fidelity after Repeated Subcultures

Micropropagation has an important role in the large-scale production of blackberry plant material, given the high proliferation rates of this species. The aim of the present study was to evaluate the proliferative capacity of blackberry grown in vitro on wheat starch-gelled culture medium compared to classical agar-gelled medium and to assess the genetic fidelity between the proliferated shoots in starch-gelled culture medium and their mother plants. Six blackberry varieties (‘Čačanska Bestrna’, ‘Chester Thornless’, ‘Driscoll’s Victoria’, ‘Loch Ness’, ‘Polar’, and ‘Karaka Black’) were tested. For the in vitro shoots proliferation, Murashige and Skoog (MS) medium supplemented with 0.5 mg dm−3 6-benzyladenine (BA) was used. The conventional medium was gelled with 0.5% plant agar, and wheat starch was used as an alternative gelling agent in a concentration of 5%. The results showed that for all blackberry cultivars, the highest number of shoots/inoculum was obtained in wheat starch-gelled culture medium, with a maximum value of 54.42 ± 4.18 presented by ‘Karaka Black’. Considering the length of the proliferated shoots, all tested cultivars presented outstanding results on the culture medium gelled with 5% wheat starch. The highest values regarding shoots length were observed on the ‘Chester Thornless’ followed by ‘Čačanska Bestrna’, and ‘Loch Ness’ with values of 5.55 ± 0.04 cm, 5.46 ± 0.06 cm, and 5.37 ± 0.09 cm, respectively. The genetic uniformity of the micropropagated shoots in relation to their mother plants was confirmed by sequence-related amplified polymorphism (SRAP) and start codon targeted (SCoT) molecular markers.

Mitogenomic Characterization of Cameroonian Endemic Coptodon camerunensis (Cichliformes: Cichlidae) and Matrilineal Phylogeny of Old-World Cichlids.

The mitogenomic evolution of old-world cichlids is still largely incomplete in Western Africa. In this present study, the complete mitogenome of the Cameroon endemic cichlid, Coptodon camerunensis, was determined by next-generation sequencing. The mitogenome was 16,557 bp long and encoded with 37 genes (13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and a control region). The C. camerunensis mitogenome is AT-biased (52.63%), as exhibited in its congener, Coptodon zillii (52.76% and 53.04%). The majority of PCGs start with an ATG initiation codon, except COI, which starts with a GTG codon and five PCGs and ends with the TAA termination codon and except seven PCGs with an incomplete termination codon. In C. camerunensis mitogenome, most tRNAs showed classical cloverleaf secondary structures, except tRNA-serine with a lack of DHU stem. Comparative analyses of the conserved blocks of two Coptodonini species control regions revealed that the CSB-II block was longer than other blocks and contained highly variable sites. Using 13 concatenated PCGs, the mitogenome-based Bayesian phylogeny easily distinguished all the examined old-world cichlids. Except for Oreochromini and Coptodinini tribe members, the majority of the taxa exhibited monophyletic clustering within their respective lineages. C. camerunensis clustered closely with Heterotilapia buttikoferi (tribe Heterotilapiini) and had paraphyletic clustering with its congener, C. zillii. The Oreochromini species also displayed paraphyletic grouping, and the genus Oreochromis showed a close relationship with Coptodinini and Heterotilapiini species. In addition, illustrating the known distribution patterns of old-world cichlids, the present study is congruent with the previous hypothesis and proclaims that prehistoric geological evolution plays a key role in the hydroclimate of the African continent during Mesozoic, which simultaneously disperses and/or colonizes cichlids in different ichthyological provinces and Rift Lake systems in Africa. The present study suggests that further mitogenomes of cichlid species are required, especially from western Africa, to understand their unique evolution and adaptation.

Molecular evaluation of genetic diversity and relationships of Musa cultivars in Thailand using Start Codon Targeted (SCoT) markers

Abstract. Nitiworakarn S, Phae-Ngam W, Vanijajiva O. 2023. Molecular evaluation of genetic diversity and relationships of Musa cultivars in Thailand using Start Codon Targeted (SCoT) markers. Biodiversitas 24: 4060-4068. Musa cultivars are mostly obtained from crossing between wild diploid subspecies of M. acuminata (A genome) and M. balbisiana (B genome), and they reveal numerous levels of ploidy and genomic constitution. The present study, for the first time, investigated the genetic diversity of 90 Musa cultivars from 17 localities of Chai Badan district in Lopburi Province, Thailand, using start codon targeted (SCoT) markers. The SCoT technique has shown very high repeatability for characterizing Musa cultivars. The result indicated that DNA fingerprints from 30 SCoT primers generated 244 amplicons, among which 238 bands (97.54%) were polymorphic, with a mean of 7.93 polymorphic bands per primer. The average polymorphism information content (PIC) was high, ranging from 0.345 to 0.483, with an average of 0.432. Genetic diversity and its partitioning parameters were calculated and demonstrated that present Musa cultivars maintain relatively high genetic diversity. The unweighted pair-group method with an arithmetic cluster analysis, principal coordinate analysis and STRUCTURE analysis results were the same and showed the clear division of the genotypes into two distinct clusters, which were cluster-I consisted of only the AA genomes and cluster-II comprised of ABB and BB genomes. In conclusion, SCoT markers obtained here showed their manipulation in genetic diversity and the relationship of cultivated Musa, which would be practical for genetic preservation and continuing breeding programs in Thailand and other regions.

Meta-Topolin-induced regeneration and ameliorated rebaudioside-A production in genetically uniform candy-leaf plantlets (Stevia rebaudiana Bert.)

Present report ascertains the efficacy of meta-Topolin (mT) for improved in vitro mass propagation of stevia (Stevia rebaudiana Bert.) with high-frequency shoot initiation, multiplication, and proliferation over other cytokinins. Shoot tips were inoculated on Murashige and Skoog (MS) medium fortified with 6-benzyladenine, kinetin, mT, thidiazuron, and zeatin individually, at different concentrations ranged from 0.5—1.5 mg L−1. Among various combinations, MS medium + 1.5 mg L−1 mT developed the maximum number (15.33) and length (57.33 mm) of shoots with a greater number of leaves (38.67) after 4 weeks of culture. Elongated shoots were then transferred for root induction on MS medium fortified with a indole-3-butyric acid (IBA), indole-3-acetic acid, and α-naphthalene acetic acid individually, at concentrations ranged from 0.5—1.5 mg L−1. MS medium + 1 mg L−1 IBA was found as the superior rooting medium with the maximum number (10.67) and length (19.00 mm) of roots after next 4 weeks of culture. Rooted plantlets were acclimatized with 95% survival rate in sand followed by sand-soil (1:1; w w−1) mixture in next 8 weeks. Proper growth and development of acclimatized plantlets without any abnormalities was confirmed by micromorphological assessment (at cellular level), plant pigment analyses as well as leaf gas exchange measurement. Stability of ploidy level of the acclimatized plantlets was confirmed by flow cytometry. Furthermore, genetic fidelity of the acclimatized plantlets with their mother plant was confirmed by various molecular markers such as primers from directed amplification of minisatellite-region DNA, conserved DNA-derived polymorphism, inter simple sequence repeats, and start codon targeted polymorphism markers. High-performance liquid chromatography analysis indicated that acclimatized plantlets produced significantly higher (2.4-folds) rebaudioside-A (96.50 mg g−1 dry weight) in comparison to mother plant (40.20 mg g−1 dry weight). The current report, for first time, accounts the mT-mediated large-scale in vitro propagation of stevia ensuring genetic fidelity and simultaneously ameliorating the rebaudioside-A production.

Oocyte-specific Wee1-like protein kinase 2 is dispensable for fertility in mice.

Wee1-like protein kinase 2 (WEE2) is an oocyte-specific protein tyrosine kinase involved in the regulation of oocyte meiotic arrest in humans. As such, it has been proposed as a candidate for non-hormonal female contraception although pre-clinical models have not been reported. Therefore, we developed two novel knockout mouse models using CRISPR/Cas9 to test loss-of-function of Wee2 on female fertility. A frameshift mutation at the Wee2 translation start codon in exon 2 had no effect on litter size, litter production, or the ability of oocytes to maintain prophase I arrest. Because of the lack of a reproductive phenotype, we additionally generated a Wee2 allele with a large deletion by removing all coding exons. While there was no difference in the total number of litters produced, homozygous Wee2 female knockout mice with the larger deletion produced fewer pups than heterozygous littermates. Furthermore, there was no difference for key reproductive parameters measured in the mouse models, including ovarian weight, number of ovulated oocytes, or oocytes that underwent in vitro maturation. Therefore, as loss of Wee2 in mice shows only minor effects on overall fecundity, contraceptive development with WEE2 should consider exploiting alternative properties such as gain-of-function or protein-protein interactions, as Wee2 loss-of-function is likely complicated by biological redundancies with other proteins co-expressed in oocytes.

The DEAD-Box RNA Helicase Ded1 Is Associated with Translating Ribosomes.

DEAD-box RNA helicases are ATP-dependent RNA binding proteins and RNA-dependent ATPases that possess weak, nonprocessive unwinding activity in vitro, but they can form long-lived complexes on RNAs when the ATPase activity is inhibited. Ded1 is a yeast DEAD-box protein, the functional ortholog of mammalian DDX3, that is considered important for the scanning efficiency of the 48S pre-initiation complex ribosomes to the AUG start codon. We used a modified PAR-CLIP technique, which we call quicktime PAR-CLIP (qtPAR-CLIP), to crosslink Ded1 to 4-thiouridine-incorporated RNAs in vivo using UV light centered at 365 nm. The irradiation conditions are largely benign to the yeast cells and to Ded1, and we are able to obtain a high efficiency of crosslinking under physiological conditions. We find that Ded1 forms crosslinks on the open reading frames of many different mRNAs, but it forms the most extensive interactions on relatively few mRNAs, and particularly on mRNAs encoding certain ribosomal proteins and translation factors. Under glucose-depletion conditions, the crosslinking pattern shifts to mRNAs encoding metabolic and stress-related proteins, which reflects the altered translation. These data are consistent with Ded1 functioning in the regulation of translation elongation, perhaps by pausing or stabilizing the ribosomes through its ATP-dependent binding.

Domains and residues of the Saccharomyces cerevisiae hnRNP protein Hrp1 important for transcriptional autoregulation and noncoding RNA termination.

Proteins that bind the nascent transcript exiting RNA polymerase II can regulate transcription elongation. The essential Saccharomyces cerevisiae hnRNP protein Hrp1 is one such protein and participates in both cleavage and polyadenylation-coupled and Nrd1-Nab3-Sen1-dependent RNA polymerase II termination. Prior evidence that Hrp1 is a positive RNA polymerase II elongation factor suggests that its release from the elongation complex promotes termination. Here we report the effects of deletions and substitutions in Hrp1 on its autoregulation via an Nrd1-Nab3-Sen1-dependent transcription attenuator in the 5'-UTR of its mRNA and on the function of an Hrp1-dependent Nrd1-Nab3-Sen1 terminator in the SNR82 snoRNA gene. Deletion of either of two central RNA recognition motifs or either of the flanking low-sequence complexity domains is lethal. Smaller, viable deletions in the amino-terminal low-sequence complexity domain cause readthrough of both the HRP1 attenuator and SNR82 terminator. Substitutions that cause readthrough localized mostly to the RNA recognition motifs, although not always to the RNA-binding face. We found that autoregulation of Hrp1 mRNA synthesis is surprisingly robust, overcoming the expected lethal effects of the start codon and frameshift mutations via overexpression of the mRNA up to 40-fold. Our results suggest a model in which binding of attenuator or terminator elements in the nascent transcript by RNA recognition motifs 1 and 2 disrupts interactions between RNA recognition motif 2 and the RNA polymerase II elongation complex, increasing its susceptibility to termination.

Metabolic engineering of Corynebacterium glutamicum for fatty alcohol production from glucose and wheat straw hydrolysate

BackgroundFatty acid-derived products such as fatty alcohols (FAL) find growing application in cosmetic products, lubricants, or biofuels. So far, FAL are primarily produced petrochemically or through chemical conversion of bio-based feedstock. Besides the well-known negative environmental impact of using fossil resources, utilization of bio-based first-generation feedstock such as palm oil is known to contribute to the loss of habitat and biodiversity. Thus, the microbial production of industrially relevant chemicals such as FAL from second-generation feedstock is desirable.ResultsTo engineer Corynebacterium glutamicum for FAL production, we deregulated fatty acid biosynthesis by deleting the transcriptional regulator gene fasR, overexpressing a fatty acyl-CoA reductase (FAR) gene of Marinobacter hydrocarbonoclasticus VT8 and attenuating the native thioesterase expression by exchange of the ATG to a weaker TTG start codon. C. glutamicum ∆fasR cg2692TTG (pEKEx2-maqu2220) produced in shaking flasks 0.54 ± 0.02 gFAL L−1 from 20 g glucose L−1 with a product yield of 0.054 ± 0.001 Cmol Cmol−1. To enable xylose utilization, we integrated xylA encoding the xylose isomerase from Xanthomonas campestris and xylB encoding the native xylulose kinase into the locus of actA. This approach enabled growth on xylose. However, adaptive laboratory evolution (ALE) was required to improve the growth rate threefold to 0.11 ± 0.00 h−1. The genome of the evolved strain C. glutamicum gX was re-sequenced, and the evolved genetic module was introduced into C. glutamicum ∆fasR cg2692TTG (pEKEx2-maqu2220) which allowed efficient growth and FAL production on wheat straw hydrolysate. FAL biosynthesis was further optimized by overexpression of the pntAB genes encoding the membrane-bound transhydrogenase of E. coli. The best-performing strain C. glutamicum ∆fasR cg2692TTG CgLP12::(Ptac-pntAB-TrrnB) gX (pEKEx2-maqu2220) produced 2.45 ± 0.09 gFAL L−1 with a product yield of 0.054 ± 0.005 Cmol Cmol−1 and a volumetric productivity of 0.109 ± 0.005 gFAL L−1 h−1 in a pulsed fed-batch cultivation using wheat straw hydrolysate.ConclusionThe combination of targeted metabolic engineering and ALE enabled efficient FAL production in C. glutamicum from wheat straw hydrolysate for the first time. Therefore, this study provides useful metabolic engineering principles to tailor this bacterium for other products from this second-generation feedstock.

SmAMPsTK: a toolkit to unravel the smORFome encoding AMPs of plant species

The pervasive repertoire of plant molecules with the potential to serve as a substitute for conventional antibiotics has led to obtaining better insights into plant-derived antimicrobial peptides (AMPs). The massive distribution of Small Open Reading Frames (smORFs) throughout eukaryotic genomes with proven extensive biological functions reflects their practicality as antimicrobials. Here, we have developed a pipeline named smAMPsTK to unveil the underlying hidden smORFs encoding AMPs for plant species. By applying this pipeline, we have elicited AMPs of various functional activity of lengths ranging from 5 to 100 aa by employing publicly available transcriptome data of five different angiosperms. Later, we studied the coding potential of AMPs-smORFs, the inclusion of diverse translation initiation start codons, and amino acid frequency. Codon usage study signifies no such codon usage biases for smORFs encoding AMPs. Majorly three start codons are prominent in generating AMPs. The evolutionary and conservational study proclaimed the widespread distribution of AMPs encoding genes throughout the plant kingdom. Domain analysis revealed that nearly all AMPs have chitin-binding ability, establishing their role as antifungal agents. The current study includes a developed methodology to characterize smORFs encoding AMPs, and their implications as antimicrobial, antibacterial, antifungal, or antiviral provided by SVM score and prediction status calculated by machine learning-based prediction models. The pipeline, complete package, and the results derived for five angiosperms are freely available at https://github.com/skbinfo/smAMPsTK. Communicated by Ramaswamy H. Sarma