Serine Codons Research Articles

Comparative Study on Codon Usage Patterns across Chloroplast Genomes of Eighteen Taraxacum Species

This study investigates codon usage bias within the chloroplast genomes of 18 Taraxacum species, focusing on the base composition and various metrics including GC content, Relative Synonymous Codon Usage (RSCU), Effective Number of Codons (ENc), and GC3s. Our analysis revealed a pronounced preference for A/T-ending codons across Taraxacum species, with GC content across the first, second, and third positions of the codons (GC1, GC2, GC3) and the average GC content consistently below 50%. A detailed examination using the RSCU metric identified 29 commonly preferred A/T-ending codons, indicating a strong codon usage bias towards these endings. Specifically, the codon for leucine (UUA) emerged as highly preferred, while the codon for serine (AGC) was least favored. Through the ENc–GC3s plot analysis, we explored the forces shaping this bias, finding evidence that both mutation pressure and natural selection significantly influence codon preference, with most coding sequences showing weak bias. The PR2 plot analysis further confirmed the role of these factors by demonstrating a higher frequency of T over A and C over G at the third codon position, pointing towards a mutation bias complemented by natural selection. Collectively, our findings highlight a consistent pattern of codon usage bias in the chloroplast genomes of Taraxacum species, influenced by a combination of mutation pressure, natural selection, and possibly other unidentified factors.

Clinical Manifestation and Phylogenetic Analysis of Peste des Petits Ruminants in Local Iraqi Breed Sheep in Al-Diwaniyah Province.

Peste des petits ruminants (PPR), a contagious virus that infects sheep and goats, damages livestock globally. This study examined the clinical features and phylogenetic analysis of the PPR virus in Iraqi breed sheep from Al-Diwaniyah province. A clinical trial of 610 sheep from different flocks found 150 oral lesions. Special primers for RT-PCR and Mega11 for phylogenetic analysis were used to study the PPR virus nucleoprotein (N) gene. The PPR infection rate was 44.6% in 4-12 month olds (n = 33/131) and 4.8% in 36-48 month olds (n = 3/75). A 608-bp PPR virus partial N gene sequence was found in 49.3% of samples by RT-PCR. In leucine, isoleucine, proline, glycine, alanine, glutamine, asparagine, threonine, serine, arginine, and lysine codons, 25 amino acid alterations were found. The protein codon 56 alanine-valine alteration was most significant. Moving from a smaller hydrophobic amino acid to one with a bigger side chain may reduce protein stability. Steric hindrance or protein shape change from Valine's extended side chain may impact folding, stability, functionality, and interactions with other molecules. Furthermore, phylogenetic analysis showed that the Nigerian strain (MN271586) was most similar to our Iraqi strain, with 100% identity and coverage. This study found the Peste des Petits Ruminants (PPR) virus in sheep flocks in Al-Diwaniyah Governorate, Iraq, which is genetically similar to neighboring countries. PPR virus strains must be monitored and genetically characterized since N gene alterations can affect infection and propagation.

The genome of Salmacisia buchloëana, the parasitic puppet master pulling strings of sexual phenotypic monstrosities in buffalograss.

To complete its parasitic lifecycle, Salmacisia buchloëana, a biotrophic fungus, manipulates reproductive organ development, meristem determinacy, and resource allocation in its dioecious plant host, buffalograss (Bouteloua dactyloides; Poaceae). To gain insight into S. buchloëana's ability to manipulate its host, we sequenced and assembled the 20.1 Mb genome of S. buchloëana into 22 chromosome-level pseudomolecules. Phylogenetic analysis suggests that S. buchloëana is nested within the genus Tilletia and diverged from Tilletia caries and Tilletia walkeri ∼40 MYA. We find that S. buchloëana contains a novel chromosome arm with no syntenic relationship to other publicly available Tilletia genomes, and that genes on the novel arm are upregulated upon infection, suggesting that this unique chromosomal segment may have played a critical role in S. buchloëana's evolution and host specificity. Salmacisia buchloëana has one of the largest fractions of serine peptidases (1.53% of the proteome) and one of the highest GC contents (62.3%) in all classified fungi. Analysis of codon base composition indicated that GC content is controlled more by selective constraints than directional mutation, and that S. buchloëana has a unique bias for the serine codon UCG. Finally, we identify 3 inteins within the S. buchloëana genome, 2 of which are located in a gene often used in fungal taxonomy. The genomic and transcriptomic resources generated here will aid plant pathologists and breeders by providing insight into the extracellular components contributing to sex determination in dioecious grasses.

A full repertoire of Hemiptera genomes reveals a multi-step evolutionary trajectory of auto-RNA editing site in insect Adar gene

ABSTRACT Adenosine-to-inosine (A-to-I) RNA editing, mediated by metazoan ADAR enzymes, is a prevalent post-transcriptional modification that diversifies the proteome and promotes adaptive evolution of organisms. The Drosophila Adar gene has an auto-recoding site (termed S>G site) that forms a negative-feedback loop and stabilizes the global editing activity. However, the evolutionary trajectory of Adar S>G site in many other insects remains largely unknown, preventing us from a deeper understanding on the significance of this auto-editing mechanism. In this study, we retrieved the well-annotated genomes of 375 arthropod species including the five major insect orders (Lepidoptera, Diptera, Coleoptera, Hymenoptera and Hemiptera) and several outgroup species. We performed comparative genomic analysis on the Adar auto-recoding S>G site. We found that the ancestral state of insect S>G site was an uneditable serine codon (unSer) and that this state was largely maintained in Hymenoptera. The editable serine codon (edSer) appeared in the common ancestor of Lepidoptera, Diptera and Coleoptera and was almost fixed in the three orders. Interestingly, Hemiptera species possessed comparable numbers of unSer and edSer codons, and a few ‘intermediate codons’, demonstrating a multi-step evolutionary trace from unSer-to-edSer with non-synchronized mutations at three codon positions. We argue that the evolution of Adar S>G site is the best genomic evidence supporting the ‘proteomic diversifying hypothesis’ of RNA editing. Our work deepens our understanding on the evolutionary significance of Adar auto-recoding site which stabilizes the global editing activity and controls transcriptomic diversity.

Self-similarity and the maximum entropy principle in the genetic code.

This paper addresses the relationship between information and structure of the genetic code. The code has two puzzling anomalies: First, when viewed as 64 sub-cubes of a [Formula: see text] cube, the codons for serine (S) are not contiguous, and there are amino acid codons with zero redundancy, which goes counter to the objective of error correction. To make sense of this, the paper shows that the genetic code must be viewed not only on stereochemical, co-evolution, and error-correction considerations, but also on two additional factors of significance to natural systems, that of an information-theoretic dimensionality of the code data, and the principle of maximum entropy. One implication of non-integer dimensionality associated with data dimensions is self-similarity to different scales, and it is shown that the genetic code does satisfy this property, and it is further shown that the maximum entropy principle operates through the scrambling of the elements in the sense of maximum algorithmic information complexity, generated by an appropriate exponentiation mapping. It is shown that the new considerations and the use of maximum entropy transformation create new constraints that are likely the reasons for the non-uniform codon groups and codons with no redundancy.

A swapped genetic code prevents viral infections and gene transfer.

Engineering the genetic code of an organism has been proposed to provide a firewall from natural ecosystems by preventing viral infections and gene transfer1-6. However, numerous viruses and mobile genetic elements encode parts of the translational apparatus7-9, potentially rendering a genetic-code-based firewall ineffective. Here we show that such mobile transfer RNAs (tRNAs) enable gene transfer and allow viral replication in Escherichia coli despite the genome-wide removal of 3 of the 64 codons and the previously essential cognate tRNA and release factor genes. We then establish a genetic firewall by discovering viral tRNAs that provide exceptionally efficient codon reassignment allowing us to develop cells bearing an amino acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation. This amino acid-swapped genetic code renders cells resistant to viral infections by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. As these cells may have a selective advantage over wild organisms due to virus resistance, we also repurpose a third codon to biocontain this virus-resistant host through dependence on an amino acid not found in nature10. Our results may provide the basis for a general strategy to make any organism safely resistant to all natural viruses and prevent genetic information flow into and out of genetically modified organisms.

Attenuation of Avian Flavivirus by Rewiring the Leucine and Serine Codons of Its E-NS1 Protein toward Stop Mutation To Redirect Virus Evolution.

Recently, a new strategy for attenuating RNA viruses by redirecting their evolution in sequence space was confirmed for Enterovirus and Influenza viruses. Using avian flavivirus as a model, the 69 serine and 53 leucine codons on the E-NS1 genes were modified to change evolutionary direction of the viral sequence space. This means that all codons encoding serine or leucine residues were substituted with codons that are only one base different from the three stop codons, resulting in the initial position of the virus genome in sequence space being closer to the detrimental areas to achieve attenuation by reducing viral adaptability. The growth curve and plaque size of CQW1-one-to-stop (CQW1-OTS) were similar to those of CQW1-wild type (CQW1-WT) in vitro, but attenuated proliferation was detected when treated with a mutagenic reagent (ribavirin). However, comparably high CQW1-OTS and CQW1-WT lethality rates were detected in 9-day-old duck embryos and 5-day-old ducklings, suggesting that this strategy works but with limitations. With that in mind, homologous hosts in nonsensitive age (25-day-old ducks) and heterologous hosts (3-week-old Kunming mice) were employed to investigate if CQW1-OTS was attenuated under host selection pressure. Minimal attenuation of CQW1-OTS in elder ducks and apparent attenuation in mice were reported, providing reduced viral titers, mild clinical signs, and lower specific infectivity. Collectively, we experimentally demonstrate that the attenuation strategy of redirecting virus evolution in sequence space works for flavivirus. Redirection of the virus is attenuated only under some outside pressure, such as heterologous hosts or antiviral drugs treatment, limiting its usage in flaviviruses. IMPORTANCE Flaviviruses are medically important arboviruses that threaten public health, but no approved treatments are currently available. Vaccines prevent flavivirus infection. We employed duck Tembusu virus (TMUV), a mosquito-borne flavivirus, to evaluate virus redirection. TMUV is native to birds and could infect mice by intracerebral injection, making it an experimental animal model to study flavivirus characteristics in vivo. The 69 serine and 53 leucine codons on the E-NS1 proteins of CQW1 were synonymously substituted to change evolutionary direction of the virus in sequence space. In vitro mutagen reagent treatment suppressed CQW1-OTS viral multiplication, but in vivo attenuation depended on host selective pressure. CQW1-OTS viral attenuation was observed in older ducks but not sensitive ducklings; considerable attenuation was also observed in heterogenous host (mice), which provides more selective pressure on viruses. Collectively, these data indicated that there are very important preconditions for application of evaluating whether this strategy shows application prospects in novel flavivirus vaccine development.

Phosphorylation of serine residues S252, S268/S269, and S879 in p120 catenin activates migration of presomitic mesoderm in gastrulating zebrafish embryos.

Cadherin-associated protein p120 catenin regulates cell adhesion and migration in cell cultures and is required for axial elongation in embryos. Its roles in adhesion and cell migration are regulated by phosphorylation. We determined the effects of phosphorylation of six serine and three threonine residues in p120 catenin during zebrafish (Danio rerio) embryogenesis. We knocked down endogenous p120 catenin-δ1 with an antisense RNA-splice-site morpholino (Sp-MO) causing defects in axis elongation. These defects were rescued by co-injections of mRNAs for wildtype mouse p120 catenin-δ1-3A or various mutated forms. Several mRNAs containing serine or threonine codons singly or doubly mutated to phosphomimetic glutamic acid rescued, and some nonphosphorylatable mutants did not. We discovered that phosphorylation of serine residue S252 or S879 is required for convergent extension of zebrafish embryos, since rescue occurred only when these residues were mutated to glutamic acid. In addition, the phosphorylation of either S268 or S269 is required, not both, consistent with the presence of only a single one of these residues in two isoforms of zebrafish and Xenopus laevis. In summary, phosphorylation of multiple serine and threonine residues of p120 catenin activates migration of presomitic mesoderm of zebrafish embryos facilitating elongation of the dorsal axis.

No evidence for widespread positive selection on double substitutions within codons in primates and yeasts.

Nucleotide substitutions in protein-coding genes can be divided into synonymous (S) and non-synonymous (N) ones that alter amino acids (including nonsense mutations causing stop codons). The S substitutions are expected to have little effect on function. The N substitutions almost always are affected by strong purifying selection that eliminates them from evolving populations. However, additional mutations of nearby bases can modulate the deleterious effect of single N substitutions and, thus, could be subjected to the positive selection. This effect has been demonstrated for mutations in the serine codons, stop codons and double N substitutions in prokaryotes. In all abovementioned cases, a novel technique was applied that allows elucidating the effects of selection on double substitutions considering mutational biases. Here, we applied the same technique to study double N substitutions in eukaryotic lineages of primates and yeast. We identified markedly fewer cases of purifying selection relative to prokaryotes and no evidence of codon double substitutions under positive selection. This is consistent with previous studies of serine codons in primates and yeast. In general, the obtained results strongly suggest that there are major differences between studied pro- and eukaryotes; double substitutions in primates and yeasts largely reflect mutational biases and are not hallmarks of selection. This is especially important in the context of detection of positive selection in codons because it has been suggested that multiple mutations in codons cause false inferences of lineage-specific site positive selection. It is likely that this concern is applicable to previously studied prokaryotes but not to primates and yeasts where markedly fewer double substitutions are affected by positive selection.

Next-Generation Sequencing Revealed a Distinct Immunoglobulin Repertoire with Specific Mutation Hotspots in Acute Myeloid Leukemia

Simple SummaryIdentifying new molecular targets is of great importance for prognosis prediction and target therapy of acute myeloid leukemia (AML). We previously reported on frequent expression of immunoglobulin (Ig) in myeloblasts. In this study, we investigated the clinical significance of Ig expression in sorted myeloblasts from 59 AML patients. We found that a higher level of AML-derived Ig expression correlated with a significantly shorter disease-free survival. Furthermore, we performed a comprehensive analysis of AML-derived Ig repertoire by next-generation sequencing (NGS) in 16 patients. The transcripts of AML-derived Ig shared some features with B cell-derived Ig, such as a typical V(D)J recombination and high mutation rates. However, they also showed distinct features. In contrast to the huge diversity of classical Ig, the VH-D-JH rearrangements used by AML-derived Ig were biased in each AML patient. In particularly, the Vκ-Jκ rearrangements were skewed in both AML blasts and normal peripheral blood mononucleated cells (PBMCs). However, AML-derived IGK showed high somatic mutation rates (>2%), while IGK in normal PBMCs rarely displayed hypermutation (<2%). More importantly, we identified five mutation hotspots at serine codons of IGKV3-20 in AML blasts, which may be involved in leukemogenesis and serve as a novel marker for disease monitoring and target therapy.Immunoglobulin (Ig) is known as a hallmark of B-lymphocytes exerting antibody functions. However, our previous studies demonstrated that myeloblasts from acute myeloid leukemia (AML) patients could also express Ig with distinct roles. Here, we quantified Ig (IGHG and IGK) transcripts by real-time PCR and performed a comprehensive analysis of Ig repertoire (both heavy chains and light chains) in AML blasts. We found that Ig was frequently expressed by AML blasts. A higher level of AML-derived IGHG expression correlated with a significantly shorter disease-free survival. Next-generation sequencing revealed dysregulated transcripts of all five Ig classes (IGHA, IGHD, IGHE, IGHG, and IGHM) and two Ig types (IGK and IGL) in AML. VH-D-JH rearrangements in myeloblasts were biased with individual specificity rather than generally diverse as in B-cells. Compared to AML-derived IgH, AML-derived IGK was more conserved among different AML samples. The frequently shared Vκ-Jκ patterns were IGKV3-20*01/IGKJ1*01, IGKV2D-28*01/IGKJ1*01, and IGKV4-1*01/IGKJ1*01. Moreover, AML-derived IGK was different from classical IGK in B-cells for the high mutation rates and special mutation hotspots at serine codons. Findings of the distinct Ig repertoire in myeloblasts may facilitate the discovery of a new molecular marker for disease monitoring and target therapy.

Determinants of the temperature adaptation of mRNA degradation.

The rate of chemical reactions increases proportionally with temperature, but the interplay of biochemical reactions permits deviations from this relation and adaptation. The degradation of individual mRNAs in yeast increased to varying degrees with temperature. We examined how these variations are influenced by the translation and codon composition of mRNAs. We developed a method that revealed the existence of a neutral half-life above which mRNAs are stabilized by translation but below which they are destabilized. The proportion of these two mRNA subpopulations remained relatively constant under different conditions, even with slow cell growth due to nutrient limitation, but heat shock reduced the proportion of translationally stabilized mRNAs. At the same time, the degradation of these mRNAs was partially temperature-compensated through Upf1, the mediator of nonsense-mediated decay. Compensation was also promoted by some asparagine and serine codons, whereas tyrosine codons promote temperature sensitization. These codons play an important role in the degradation of mRNAs encoding key cell membrane and cell wall proteins, which promote cell integrity.

Proteogenomics analysis of CUG codon translation in the human pathogen Candida albicans

BackgroundYeasts of the CTG-clade lineage, which includes the human-infecting Candida albicans, Candida parapsilosis and Candida tropicalis species, are characterized by an altered genetic code. Instead of translating CUG codons as leucine, as happens in most eukaryotes, these yeasts, whose ancestors are thought to have lost the relevant leucine-tRNA gene, translate CUG codons as serine using a serine-tRNA with a mutated anticodon, {mathrm{tRNA}}_{mathrm{CAG}}^{mathrm{Ser}} . Previously reported experiments have suggested that 3–5% of the CTG-clade CUG codons are mistranslated as leucine due to mischarging of the {mathrm{tRNA}}_{mathrm{CAG}}^{mathrm{Ser}} . The mistranslation was suggested to result in variable surface proteins explaining fast host adaptation and pathogenicity.ResultsIn this study, we reassess this potential mistranslation by high-resolution mass spectrometry-based proteogenomics of multiple CTG-clade yeasts, including various C. albicans strains, isolated from colonized and from infected human body sites, and C. albicans grown in yeast and hyphal forms. Our data do not support a bias towards CUG codon mistranslation as leucine. Instead, our data suggest that (i) CUG codons are mistranslated at a frequency corresponding to the normal extent of ribosomal mistranslation with no preference for specific amino acids, (ii) CUG codons are as unambiguous (or ambiguous) as the related CUU leucine and UCC serine codons, (iii) tRNA anticodon loop variation across the CTG-clade yeasts does not result in any difference of the mistranslation level, and (iv) CUG codon unambiguity is independent of C. albicans’ strain pathogenicity or growth form.ConclusionsOur findings imply that C. albicans does not decode CUG ambiguously. This suggests that the proposed misleucylation of the {mathrm{tRNA}}_{mathrm{CAG}}^{mathrm{Ser}} might be as prevalent as every other misacylation or mistranslation event and, if at all, be just one of many reasons causing phenotypic diversity.

Harnessing the Genetic Plasticity of Porcine Circovirus Type 2 to Target Suicidal Replication.

Porcine circovirus type 2 (PCV2), the causative agent of a wasting disease in weanling piglets, has periodically evolved into several new subtypes since its discovery, indicating that the efficacy of current vaccines can be improved. Although a DNA virus, the mutation rates of PCV2 resemble RNA viruses. The hypothesis that recoding of selected serine and leucine codons in the PCV2b capsid gene could result in stop codons due to mutations occurring during viral replication and thus result in rapid attenuation was tested. Vaccination of weanling pigs with the suicidal vaccine constructs elicited strong virus-neutralizing antibody responses. Vaccination prevented lesions, body-weight loss, and viral replication on challenge with a heterologous PCV2d strain. The suicidal PCV2 vaccine construct was not detectable in the sera of vaccinated pigs at 14 days post-vaccination, indicating that the attenuated vaccine was very safe. Exposure of the modified virus to immune selection pressure with sub-neutralizing levels of antibodies resulted in 5 of the 22 target codons mutating to a stop signal. Thus, the described approach for the rapid attenuation of PCV2 was both effective and safe. It can be readily adapted to newly emerging viruses with high mutation rates to meet the current need for improved platforms for rapid-response vaccines.

Characteristics of Quinolone Resistance in Multidrug-Resistant Acinetobacter baumannii Strains Isolated from General Hospitals

Background: Acinetobacter baumannii is the causative agent in various types of hospital-acquired infections, including respiratory, urinary tract, and wound infections. Objectives: This study investigated the primary mechanisms underlying quinolone resistance in A. baumannii strains, isolated from samples collected from general hospitals. Methods: Ninety-eight strains of A. baumannii were isolated from clinical specimens from general hospitals from 2017 – 2019. Antimicrobial susceptibility, efflux pump inhibition tests, multilocus sequence typing (MLST), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses were conducted on 64 strains, and the blaoxa-51-like gene sequence was detected. Results: In the antimicrobial susceptibility test, 78.1% (n = 50) of the strains exhibited resistance to ciprofloxacin, a quinolone antibiotic, and 57.8% (n = 37) strains were multidrug resistant (MDR). For 18 strains, the minimum inhibitory concentration of ciprofloxacin reduced in presence of an efflux pump inhibitor. Sequence analysis revealed that in 50 strains of A. baumannii, the codon for serine (TCA) in gyrA was replaced by that for leucine (TTA), whereas in 43 strains, the codon for serine (TCG) in parC was replaced by that for leucine (TTG). Multilocus sequence typing analysis confirmed 18 sequence types, and allelic number analysis showed the presence of nine gyrB alleles, with gyrB3 showing the highest frequency (62.5%). Conclusions: The findings of this study will be useful in improving treatment efficiency and preventing the spread of A. baumannii (both MDR and non-MDR strains).

Extra base hits: Widespread empirical support for instantaneous multiple-nucleotide changes

Despite many attempts to introduce evolutionary models that permit substitutions to instantly alter more than one nucleotide in a codon, the prevailing wisdom remains that such changes are rare and generally negligible or are reflective of non-biological artifacts, such as alignment errors. Codon models continue to posit that only single nucleotide change have non-zero rates. Here, we develop and test a simple hierarchy of codon-substitution models with non-zero evolutionary rates for only one-nucleotide (1H), one- and two-nucleotide (2H), or any (3H) codon substitutions. Using over 42, 000 empirical alignments, we find widespread statistical support for multiple hits: 61% of alignments prefer models with 2H allowed, and 23%-with 3H allowed. Analyses of simulated data suggest that these results are not likely to be due to simple artifacts such as model misspecification or alignment errors. Further modeling reveals that synonymous codon island jumping among codons encoding serine, especially along short branches, contributes significantly to this 3H signal. While serine codons were prominently involved in multiple-hit substitutions, there were other common exchanges contributing to better model fit. It appears that a small subset of sites in most alignments have unusual evolutionary dynamics not well explained by existing model formalisms, and that commonly estimated quantities, such as dN/dS ratios may be biased by model misspecification. Our findings highlight the need for continued evaluation of assumptions underlying workhorse evolutionary models and subsequent evolutionary inference techniques. We provide a software implementation for evolutionary biologists to assess the potential impact of extra base hits in their data in the HyPhy package and in the Datamonkey.org server.

Escherichia Coli-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles.

Tandem-core hepatitis B core antigen (HBcAg) virus-like particles (VLPs), in which two HBcAg monomers are joined together by a peptide linker, can be used to display two different antigens on the VLP surface. We produced universal influenza vaccine candidates that use this scaffold in an Escherichia coli-based cell-free protein synthesis (CFPS) platform. We then used the CFPS system to rapidly test modifications to the arginine-rich region typically found in wild-type HBcAg, the peptide linkers around the influenza antigen inserts, and the plasmid vector backbone to improve titer and quality. Using a minimal plasmid vector backbone designed for CFPS improved titers by at least 1.4-fold over the original constructs. When the linker lengths for the influenza inserts were more consistent in length and a greater variety of codons for glycine and serine were utilized, titers were further increased to over 70 μg/mL (4.0-fold greater than the original construct) and the presence of lower molecular weight product-related impurities was significantly reduced, although improvements in particle assembly were not seen. Furthermore, any constructs with the C-terminal arginine-rich region removed resulted in asymmetric particles of poor quality. This demonstrates the potential for CFPS as a screening platform for VLPs.

Kinetic properties of recombinant phosphomimic mutant of Zea mays phosphoenolpyruvate carboxylase (ZmPEPCS15D)

Phosphoenolpyruvate carboxylase (PEPC) is the key enzyme for fixing atmospheric carbon dioxide in C4 and CAM plants, and in anaplerotic metabolism in most plants. It is regulated by reversible phosphorylation, and allosteric activation or inhibition. The regulatory role of phosphorylation at N-terminal serine residue (S15) is documented earlier. In this study PEPC was cloned from maize and engineered to generate phosphomimic mutant enzyme by conversion of the codon for Serine 15 to aspartate (PEPCS15D). In vitro study with recombinant wild-type PEPC and phosphomimic PEPCS15D showed that both the enzymes are active catalytically. Kinetics analysis showed that PEPCS15D enzyme has comparatively lower Km value over WT PEPC, thus implying higher affinity towards its substrate PEP than the wild type PEPC. However, both WT PEPC and mutant PEPCS15D showed similar regulation with the allosteric activator (Glycine) and allosteric inhibitors (l-Aspartate or l-Malate). Thus, the engineered phosphomimic mutant PEPCS15D with high affinity for PEP will be useful to engineer rice and other C3 plants for enhancing photosynthesis and minimizing respiratory CO2 loss.

The mechanism of β-N-methylamino-l-alanine inhibition of tRNA aminoacylation and its impact on misincorporation.

β-N-methylamino-l-alanine (BMAA) is a nonproteinogenic amino acid that has been associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). BMAA has been found in human protein extracts; however, the mechanism by which it enters the proteome is still unclear. It has been suggested that BMAA is misincorporated at serine codons during protein synthesis, but direct evidence of its cotranslational incorporation is currently lacking. Here, using LC-MS-purified BMAA and several biochemical assays, we sought to determine whether any aminoacyl-tRNA synthetase (aaRS) utilizes BMAA as a substrate for aminoacylation. Despite BMAA's previously predicted misincorporation at serine codons, following a screen for amino acid activation in ATP/PPi exchange assays, we observed that BMAA is not a substrate for human seryl-tRNA synthetase (SerRS). Instead, we observed that BMAA is a substrate for human alanyl-tRNA synthetase (AlaRS) and can form BMAA-tRNAAla by escaping from the intrinsic AlaRS proofreading activity. Furthermore, we found that BMAA inhibits both the cognate amino acid activation and the editing functions of AlaRS. Our results reveal that, in addition to being misincorporated during translation, BMAA may be able to disrupt the integrity of protein synthesis through multiple different mechanisms.

Effect of cysteine/serine changes to glucose-6-phosphate dehydrogenase of Synechocystis sp. PCC6803 in cell-free extracts

ABSTRACTIn cyanobacteria and other photosynthetic organisms, the mechanism of glucose-6-phosphate dehydrogenase (G6PDH) regulation has long been hypothesized to be a disulphide bond formation/reduction between –SH groups of two internal cysteine residues. In this study, four cysteine codons in the zwf gene of Synechocystis sp. PCC6803 were changed to serine codons, and then each mutation was separately transferred to the chromosome, yielding four cysteine-mutant strains designated ZWFCS101, ZWFCS187, ZWFCS265 and ZWFCS445. Thus, cysteine-mutant G6PDH was analysed in cyanobacteria. Cell-free extracts of the other three mutants were used as an enzyme solution. The mutant ZWFCS187, located close to the active site, completely lost all enzyme activity. To test whether the redox sensitivity of the mutant enzymes was changed, reduced dithiothreitol (DTTred) was added to the enzyme solution immediately prior to the assay. Three of the mutant G6PDHs were found to still be sensitive. This result is inconsistent with other reports where the cysteine-mutant G6PDH lost redox sensitivity. The results herein indicate that redox regulation is not the sole mechanism for the regulation of G6PDH activity, and the regulation most probably involves a sum of interactions at the cellular level, as well as additional contributing factors.

A Decrease in Serine Levels during Growth Transition Triggers Biofilm Formation in Bacillus subtilis.

Biofilm development in Bacillus subtilis is regulated at multiple levels. While a number of known signals that trigger biofilm formation do so through the activation of one or more sensory histidine kinases, it was discovered that biofilm activation is also coordinated by sensing intracellular metabolic signals, including serine starvation. Serine starvation causes ribosomes to pause on specific serine codons, leading to a decrease in the translation rate of sinR, which encodes a master repressor for biofilm matrix genes and ultimately triggers biofilm induction. How serine levels change in different growth stages, how B. subtilis regulates intracellular serine levels, and how serine starvation triggers ribosomes to pause on selective serine codons remain unknown. Here, we show that serine levels decrease as cells enter stationary phase and that unlike most other amino acid biosynthesis genes, expression of serine biosynthesis genes decreases upon the transition into stationary phase. The deletion of the gene for a serine deaminase responsible for converting serine to pyruvate led to a delay in biofilm formation, further supporting the idea that serine levels are a critical intracellular signal for biofilm activation. Finally, we show that levels of all five serine tRNA isoacceptors are decreased in stationary phase compared with exponential phase. However, the three isoacceptors recognizing UCN serine codons are reduced to a much greater extent than the two that recognize AGC and AGU serine codons. Our findings provide evidence for a link between serine homeostasis and biofilm development in B. subtilisIMPORTANCE In Bacillus subtilis, biofilm formation is triggered in response to environmental and cellular signals. It was proposed that serine limitation acts as a proxy for nutrient status and triggers biofilm formation at the onset of biofilm entry through a novel signaling mechanism caused by global ribosome pausing on selective serine codons. In this study, we reveal that serine levels decrease at the biofilm entry due to catabolite control and a serine shunt mechanism. We also show that levels of five serine tRNA isoacceptors are differentially decreased in stationary phase compared with exponential phase; three isoacceptors recognizing UCN serine codons are reduced much more than the two recognizing AGC and AGU codons. This finding indicates a possible mechanism for selective ribosome pausing.