Evolution of the genetic code; Evidence from serine codon use disparity in Escherichia coli.

Among the 20 amino acids, three of them-leucine (Leu), arginine (Arg), and serine (Ser)-are encoded by six different codons. In comparison, all of the other 17 amino acids are encoded by either 4, 3, 2, or 1 codon. Peculiarly, Ser is separated into two disparate Ser codon boxes, differing by at least two-base substitutions, in contrast to Leu and Arg, of which codons are mutually exchangeable by a single-base substitution. We propose that these two different Ser codons independently emerged during evolution. In this hypothesis, at the time of the origin of life there were only seven primordial amino acids: Valine (coded by GUX [X = U, C, A or G]), alanine (coded by GCX), aspartic acid (coded by GAY [Y = U or C]), glutamic acid (coded by GAZ [Z = A or G]), glycine (coded by GGX), Ser (coded by AGY), and Arg (coded by CGX and AGZ). All of these were derived from GGX for glycine by single-base substitutions. Later in evolution, another class of Ser codons, UCX, were derived from alanine codons, GCX, distinctly different from the other primordial Ser codon, AGY. From the analysis of the Escherichia coli genome, we find extensive disparities in the usage of these two Ser codons, as some genes use only AGY for Ser in their genes. In contrast, others use only UCX, pointing to distinct differences in their origins, consistent with our hypothesis.

Assessment of prediction methods for protein structures determined by NMR in CASP14: Impact of AlphaFold2.

NMR studies can provide unique information about protein conformations in solution. In CASP14, three reference structures provided by solution NMR methods were available (T1027, T1029, and T1055), as well as a fourth data set of NMR-derived contacts for an integral membrane protein (T1088). For the three targets with NMR-based structures, the best prediction results ranged from very good (GDT_TS = 0.90, for T1055) to poor (GDT_TS = 0.47, for T1029). We explored the basis of these results by comparing all CASP14 prediction models against experimental NMR data. For T1027, NMR data reveal extensive internal dynamics, presenting a unique challenge for protein structure prediction methods. The analysis of T1029 motivated exploration of a novel method of "inverse structure determination," in which an AlphaFold2 model was used to guide NMR data analysis. NMR data provided to CASP predictor groups for target T1088, a 238-residue integral membrane porin, was also used to assess several NMR-assisted prediction methods. Most groups involved in this exercise generated similar beta-barrel models, with good agreement with the experimental data. However, as was also observed in CASP13, some pure prediction groups that did not use any NMR data generated models for T1088 that better fit the NMR data than the models generated using these experimental data. These results demonstrate the remarkable power of modern methods to predict structures of proteins with accuracies rivaling solution NMR structures, and that it is now possible to reliably use prediction models to guide and complement experimental NMR data analysis.

Structural basis of mRNA recognition and cleavage by toxin MazF and its regulation by antitoxin MazE in Bacillus subtilis.

MazF is an mRNA interferase, which, upon activation during stress conditions, cleaves mRNAs in a sequence-specific manner, resulting in cellular growth arrest. During normal growth conditions, the MazF toxin is inactivated through binding to its cognate antitoxin, MazE. How MazF specifically recognizes its mRNA target and carries out cleavage and how the formation of the MazE-MazF complex inactivates MazF remain unclear. We present crystal structures of MazF in complex with mRNA substrate and antitoxin MazE in Bacillus subtilis. The structure of MazF in complex with uncleavable UUdUACAUAA RNA substrate defines the molecular basis underlying the sequence-specific recognition of UACAU and the role of residues involved in the cleavage through site-specific mutational studies. The structure of the heterohexameric (MazF)2-(MazE)2-(MazF)2 complex in Bacillus subtilis, supplemented by mutational data, demonstrates that the positioning of the C-terminal helical segment of MazE within the RNA-binding channel of the MazF dimer prevents mRNA binding and cleavage by MazF.

Protein structure prediction assisted with sparse NMR data in CASP13.

CASP13 has investigated the impact of sparse NMR data on the accuracy of protein structure prediction. NOESY and 15 N-1 H residual dipolar coupling data, typical of that obtained for 15 N,13 C-enriched, perdeuterated proteins up to about 40 kDa, were simulated for 11 CASP13 targets ranging in size from 80 to 326 residues. For several targets, two prediction groups generated models that are more accurate than those produced using baseline methods. Real NMR data collected for a de novo designed protein were also provided to predictors, including one data set in which only backbone resonance assignments were available. Some NMR-assisted prediction groups also did very well with these data. CASP13 also assessed whether incorporation of sparse NMR data improves the accuracy of protein structure prediction relative to nonassisted regular methods. In most cases, incorporation of sparse, noisy NMR data results in models with higher accuracy. The best NMR-assisted models were also compared with the best regular predictions of any CASP13 group for the same target. For six of 13 targets, the most accurate model provided by any NMR-assisted prediction group was more accurate than the most accurate model provided by any regular prediction group; however, for the remaining seven targets, one or more regular prediction method provided a more accurate model than even the best NMR-assisted model. These results suggest a novel approach for protein structure determination, in which advanced prediction methods are first used to generate structural models, and sparse NMR data is then used to validate and/or refine these models.

A CUGGU/UUGGU-specific MazF homologue from Methanohalobium evestigatum.

MazF is a sequence-specific endoribonuclease or mRNA interferase, which cleaves RNA at a specific sequence. Since the expression of a specific gene or a group of specific genes can be regulated by MazF, expanding the repertoire of recognition sequences by MazF mRNA interferases is highly desirable for biotechnological and medical applications. Here, we identified a gene for a MazF homologue (MazFme) from Methanohalobium evestigatum, an extremely halophilic archaeon. In order to suppress the toxicity of MazFme to the E. coli cells, the C-terminal half of the cognate antitoxin MazEme was fused to the N-terminal end of MazFme. Since the fusion of the C-terminal half of MazEme to MazFme was able to neutralize MazFme toxicity, the MazEme-MazFme fusion protein was expressed in a large amount without any toxic effects. After purification of the MazEme, the free MazFme RNA cleavage specificity was determined by primer extension and synthetic ribonucleotides, revealing that MazFme is a CUGGU/UUGGU-specific endoribonuclease.

Toxin-antitoxin systems in bacteria and archaea.

Almost all bacteria and many archaea contain genes whose expression inhibits cell growth and may lead to cell death when overproduced, reminiscent of apoptotic genes in higher systems. The cellular targets of these toxins are quite diverse and include DNA replication, mRNA stability, protein synthesis, cell-wall biosynthesis, and ATP synthesis. These toxins are co-expressed and neutralized with their cognate antitoxins from a TA (toxin-antitoxin) operon in normally growing cells. Antitoxins are more labile than toxins and are readily degraded under stress conditions, allowing the toxins to exert their toxic effect. Presence of at least 33 TA systems in Escherichia coli and more than 60 TA systems in Mycobacterium tuberculosis suggests that the TA systems are involved not only in normal bacterial physiology but also in pathogenicity of bacteria. The elucidation of their cellular function and regulation is thus crucial for our understanding of bacterial physiology under various stress conditions.

Genome-Wide Screening for Identification of Novel Toxin-Antitoxin Systems in Staphylococcus aureus.

Toxin-antitoxin (TA) systems consist of toxin-inhibiting diverse cellular functions (e.g., DNA replication, transcription, and translation) and a noncoding RNA or protein antitoxin. TA systems are associated with various cellular events, such as stress responses, programmed cell death, and bacterial pathogenicity. Recent advances in genome sequencing and bioinformatics research have demonstrated that most bacteria harbor various kinds of TA modules on their chromosomes; however, there is little understanding of chromosomally encoded TA systems in the Gram-positive pathogen Staphylococcus aureus Here, we report on newly discovered S. aureus TA systems, each of which is composed of two proteins. Manual search and gene operon prediction analysis identified eight 2-gene operons as potential candidates for TA systems. Subsequently, using an Escherichia coli host killing and rescue assay, we demonstrated that four of the eight candidates worked as TA systems, designated tsaAT, tsbAT, tscAT, and tsdAT Moreover, the TsaT, TsbT, TscT, and TsdT toxins inhibited S. aureus growth, and the toxicity of TsbT was neutralized by coexpressing the tsbA gene in the native host, S. aureus Further, the bioinformatics analysis of the gene clusters revealed that TsaAT, TsbAT, TscAT, and TsdAT did not exhibit sequence similarity to known bacterial TA systems, and their homologues were present only within Staphylococcus species and not among any other bacteria. Our results further advance not only the understanding of S. aureus TA systems but also the study of unannotated TA systems in various bacterial species.IMPORTANCE Recent advances in genome sequencing and bioinformatics research have demonstrated that most pathogenic bacteria harbor a large number of chromosomally encoded toxin-antitoxin (TA) modules. However, little is known about the TA systems in S. aureus Here, we newly identified four S. aureus TA systems using a combination of manual base-by-base screening and functional analysis in E. coli Moreover, all toxins of the identified TA systems caused growth inhibition in the native host S. aureus Although the newly identified TA systems did not exhibit sequence similarity with known bacterial TA systems, their orthologues were conserved only among other Staphylococcus species, indicating their uniqueness to staphylococci. Our approach opens the possibility for studying unannotated TA systems in various bacterial species.

Preferential use of minor codons in the translation initiation region of human genes.

More than 31,000 protein-coding sequences (CCDS) have been identified in the human genome. Here, we analyzed codon usage in all human CCDS and found that there is a preferential usage of minor codons for Ala (CGC), Pro (CCG), Ser (UCG), and Thr (ACG) in the initial 50-codon sequences of the CCDS. These codons, with consensus XCG sequences, are most infrequently used among their synonymous codons. Thus, the tRNA concentrations per codon are considered to be highest for the minor codons for Ala, Pro, Ser and Thr in comparison with other synonymous codons for each of them to enhance the translation efficiency. This suggests that human genes are regulated at the level of translation by preferentially using minor codons within the first 50 codons of the CCDS. This hypothesis was experimentally confirmed by comparing the expression of the luciferase gene encoded by minor codons with that encoded by major codons.

The role of the loop 1 region in MazFbs mRNA interferase from Bacillus subtilis in recognition of the 3' end of the RNA substrate.

MazFbs is an mRNA interferase from Bacillus subtilis specifically recognizing UACAU. The X-ray structure of its complex with an RNA substrate has been also solved. When its amino acid sequence is compared with that of MazFhw, an mRNA interferase from a highly halophilic archaeon, recognizing UUACUCA, the 9-residue loop-1 region is highly homologous except that the V16V17 sequence in MazFbs is replaced with TK in MazFhw. Thus, we examined the role of the VV sequence in RNA substrate recognition by replacing it with TK, GG, AA or LL. The substitution mutants thus constructed showed significant differences in cleavage specificity using MS2 phage RNA. The primer extension analysis of the cleavage sites revealed that the VV sequence plays an important role in the recognition of the 3'-end base of the RNA substrate.

Designing of a single gene encoding four functional proteins.

In the genomes of some organisms such as bacteriophages and bacteria, a DNA sequence is able to encode two different proteins, indicating that genetic information is compacted in DNA twice denser than in usual DNA. In theory, a DNA sequence has a maximal capacity to produce six different mRNAs, however, it is an intriguing question how many of these mRNAs are able to synthesize functional proteins. Here, we design a DNA sequence encoding four collagen-like proteins, two, (Gly-Arg-Pro)n and (Gly-Ala-Pro)n, from a sense mRNA and the other two, also (Gly-Arg-Pro)n and (Gly-Ala-Pro)n from its antisense mRNA, all of which are expected to form triple-helical structures unique to collagens. Other designs such as the combination of (Gly-Arg-Pro)n, (Gly-Val-Pro)n, (Gly-Thr-Pro)n and (Gly-Arg-Pro)n are also possible. The proposed DNA sequence is considered to contain the most compact genetic information ever created.

An endogenous protein inhibitor, YjhX (TopAI), for topoisomerase I from Escherichia coli.

Almost all free-living bacteria contain toxin-antitoxin (TA) systems on their genomes and the targets of toxins are highly diverse. Here, we found a novel, previously unidentified TA system in Escherichia coli named yjhX-yjhQ. Induction of YjhX (85 amino acid residues) causes cell-growth arrest resulting in cell death, while YjhQ (181 residues) co-induction resumes cell growth. The primary cellular target of YjhX was found to be topoisomerase I (TopA), inhibiting both DNA replication and RNA synthesis. Notably, YjhX has no homology to any other toxins of the TA systems. YjhX was expressed well with an N-terminal protein S (PrS) tag in soluble forms. PrS-YjhX specifically interacts with the N-terminal region of TopA (TopA67) but not full-TopA in the absence of plasmid DNA, while PrS-YjhX binds to full-TopA in the presence of DNA. Notably, YjhX does not directly interact with DNA and RNA. YjhX inhibits only topoisomerase I but not topoisomerase III and IV in vitro. Hence, yjhX is renamed as the gene for the TopA inhibitor (the topAI gene). TopAI is the first endogenous protein inhibitor specific for topoisomerase I.

Highly efficient residue-selective labeling with isotope-labeled Ile, Leu, and Val using a new auxotrophic E. coli strain.

We recently developed a practical protocol for preparing proteins bearing stereo-selectively (13)C-methyl labeled leucines and valines, instead of the commonly used (13)C-methyl labeled precursors for these amino acids, by E. coli cellular expression. Using this protocol, proteins with any combinations of isotope-labeled or unlabeled Leu and Val residues were prepared, including some that could not be prepared by the precursor methods. However, there is still room for improvement in the labeling efficiencies for Val residues, using the methods with labeled precursors or Val itself. This is due to the fact that the biosynthesis of Val could not be sufficiently suppressed, even by the addition of large amounts of Val or its precursors. In this study, we completely solved this problem by using a mutant strain derived from E. coli BL21(DE3), in which the metabolic pathways depending on two enzymes, dihydroxy acid dehydratase and ß-isopropylmalate dehydrogenase, are completely aborted by deleting the ilvD and leuB genes, which respectively encode these enzymes. The ΔilvD E. coli mutant terminates the conversion from α,ß-dihydroxyisovalerate to α-ketoisovalerate, and the conversion from α,ß-dihydroxy-α-methylvalerate to α-keto-ß-methylvalerate, which produce the preceding precursors for Val and Ile, respectively. By the further deletion of the leuB gene, the conversion from Val to Leu was also fully terminated. Taking advantage of the double-deletion mutant, ΔilvDΔleuB E. coli BL21(DE3), an efficient and residue-selective labeling method with various isotope-labeled Ile, Leu, and Val residues was established.

Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications.

A simple and cost effective method to independently and stereo-specifically incorporate [(1)H,(13)C]-methyls in Leu and Val in proteins is presented. Recombinant proteins for NMR studies are produced using a tailored set of auxotrophic E. coli strains. NMR active isotopes are routed to either Leu or Val methyl groups from the commercially available and scrambling-free precursors α-ketoisovalerate and acetolactate. The engineered strains produce deuterated proteins with stereospecific [(1)H,(13)C]-methyl labeling separately at Leu or Val amino acids. This is the first method that achieves Leu-specific stereospecific [(1)H,(13)C]-methyl labeling of proteins and scramble-free Val-specific labeling. Use of auxotrophs drastically decreases the amount of labeled precursor required for expression without impacting the yield. The concept is extended to Thr methyl labeling by means of a Thr-specific auxotroph that provides enhanced efficiency for use with the costly L-[4-(13)C,2,3-(2)H2,(15)N]-Thr reagent. The Thr-specific strain allows for the production of Thr-[(13)CH3](γ2) labeled protein with an optimal isotope incorporation using up to 50 % less labeled Thr than the traditional E. coli strain without the need for (2)H-glycine to prevent scrambling.

Structural analysis of the mechanism of phosphorylation of a critical autoregulatory tyrosine residue in FGFR1 kinase domain.

Receptor and nonreceptor tyrosine kinases are enzymes that play important roles in regulating signal transduction pathways in a variety of normal cellular process and in many pathological conditions. Ordered phosphorylation is required for receptor tyrosine kinase (RTK) activation, a process mediated by transient dimer formation of the kinase domains. This process is triggered by the tyrosine phosphorylation in the activation loop. Here, we report structural and biochemical analyses of the tyrosine kinase domain interaction of fibroblast growth factor receptor 1 (FGFR1) required for the initial phosphorylation step. On the basis of nuclear magnetic resonance (NMR) analysis and covalent cross-linking experiments, we propose a parallel symmetric dimer model where specific contacts are formed between the N-lobes and C-lobes, respectively, in the FGFR1 kinase domains. Moreover, assignment of the contact sites between two FGFR1 kinase domains are supported by a trans-phosphorylation assay and by mutational analyses. The present report shows the molecular mechanism underlying the control of trans-phosphorylation of a critical auto-regulatory site in FGF receptors' catalytic domain.

Replacement of all arginine residues with canavanine in MazF-bs mRNA interferase changes its specificity.

Replacement of a specific amino acid residue in a protein with nonnatural analogues is highly challenging because of their cellular toxicity. We demonstrate for the first time the replacement of all arginine (Arg) residues in a protein with canavanine (Can), a toxic Arg analogue. All Arg residues in the 5-base specific (UACAU) mRNA interferase from Bacillus subtilis (MazF-bs(arg)) were replaced with Can by using the single-protein production system in Escherichia coli. The resulting MazF-bs(can) gained a 6-base recognition sequence, UACAUA, for RNA cleavage instead of the 5-base sequence, UACAU, for MazF-bs(arg). Mass spectrometry analysis confirmed that all Arg residues were replaced with Can. The present system offers a novel approach to create new functional proteins by replacing a specific amino acid in a protein with its analogues.

Characterization of a mazEF toxin-antitoxin homologue from Staphylococcus equorum.

Toxin-antitoxin (TA) systems encoded in prokaryotic genomes fall into five types, typically composed of two distinct small molecules, an endotoxic protein and a cis-encoded antitoxin of ribonucleic or proteinaceous nature. In silico analysis revealed seven putative type I and three putative type II TA systems in the genome of the nonpathogenic species strain Staphylococcus equorum SE3. Among these, a MazEF system orthologue termed MazEF(seq) was further characterized. 5' rapid amplification of cDNA ends (RACE) revealed the expression and the transcriptional start site of mazE(seq), indicating an immediately upstream promoter. Heterologous expression of the putative toxin-encoding mazF(seq) gene imposed growth cessation but not cell death on Escherichia coli. In vivo and in vitro, MazF(seq) was shown to cleave at UACAU motifs, which are remarkably abundant in a number of putative metabolic and regulatory S. equorum gene transcripts. Specific interaction between MazF(seq) and the putative cognate antitoxin MazE(seq) was demonstrated by bacterial two-hybrid analyses. These data strongly suggest that MazEF(seq) represents the first characterized TA system in a nonpathogenic Staphylococcus species and indicate that MazEF modules in staphylococci may also control processes beyond pathogenicity.

Defining requirements for collagenase cleavage in collagen type III using a bacterial collagen system.

Degradation of fibrillar collagens is important in many physiological and pathological events. These collagens are resistant to most proteases due to the tightly packed triple-helical structure, but are readily cleaved at a specific site by collagenases, selected members of the matrix metalloproteinases (MMPs). To investigate the structural requirements for collagenolysis, varying numbers of GXY triplets from human type III collagen around the collagenase cleavage site were inserted between two triple helix domains of the Scl2 bacterial collagen protein. The original bacterial CL domain was not cleaved by MMP-1 (collagenase 1) or MMP-13 (collagenase 3). The minimum type III sequence necessary for cleavage by the two collagenases was 5 GXY triplets, including 4 residues before and 11 residues after the cleavage site (P4-P11'). Cleavage of these chimeric substrates was not achieved by the catalytic domain of MMP-1 or MMP-13, nor by full-length MMP-3. Kinetic analysis of the chimeras indicated that the rate of cleavage by MMP-1 of the chimera containing six triplets (P7-P11') of collagen III was similar to that of native collagen III. The collagenase-susceptible chimeras were cleaved very slowly by trypsin, a property also seen for native collagen III, supporting a local structural relaxation of the triple helix near the collagenase cleavage site. The recombinant bacterial-human collagen system characterized here is a good model to investigate the specificity and mechanism of action of collagenases.

Growth and translation inhibition through sequence-specific RNA binding by Mycobacterium tuberculosis VapC toxin.

The Mycobacterium tuberculosis genome harbors an unusually large number of toxin-antitoxin (TA) modules. Curiously, over half of these are VapBC (virulence-associated protein) family members. Nonetheless, the cellular target, precise mode of action, and physiological role of the VapC toxins in this important pathogen remain unclear. To better understand the function of this toxin family, we studied the features and biochemical properties of a prototype M. tuberculosis VapBC TA system, vapBC-mt4 (Rv0596c-Rv0595c). VapC-mt4 expression resulted in growth arrest, a hallmark of all TA toxins, in Escherichia coli, Mycobacterium smegmatis, and M. tuberculosis. Its expression led to translation inhibition accompanied by a gradual decrease in the steady-state levels of several mRNAs. VapC-mt4 exhibited sequence-specific endoribonuclease activity on mRNA templates at ACGC and AC(A/U)GC sequences. However, the cleavage activity of VapC-mt4 was comparatively weak relative to the TA toxin MazF-mt1 (Rv2801c). Unlike other TA toxins, translation inhibition and growth arrest preceded mRNA cleavage, suggesting that the RNA binding property of VapC-mt4, not RNA cleavage, initiates toxicity. In support of this hypothesis, expression of VapC-mt4 led to an increase in the recovery of total RNA with time in contrast to TA toxins that inhibit translation via direct mRNA cleavage. Additionally, VapC-mt4 exhibited stable, sequence-specific RNA binding in an electrophoretic mobility shift assay. Finally, VapC-mt4 inhibited protein synthesis in a cell-free system without cleaving the corresponding mRNA. Therefore, the activity of VapC-mt4 is mechanistically distinct from other TA toxins because it appears to primarily inhibit translation through selective, stable binding to RNA.

ACA-specific RNA sequence recognition is acquired via the loop 2 region of MazF mRNA interferase.

MazF is an mRNA interferase that cleaves mRNAs at a specific RNA sequence. MazF from E. coli (MazF-ec) cleaves RNA at A^CA. To date, a large number of MazF homologs that cleave RNA at specific three- to seven-base sequences have been identified from bacteria to archaea. MazF-ec forms a dimer, in which the interface between the two subunits is known to be the RNA substrate-binding site. Here, we investigated the role of the two loops in MazF-ec, which are closely associated with the interface of the MazF-ec dimer. We examined whether exchanging the loop regions of MazF-ec with those from other MazF homologs, such as MazF from Myxococcus xanthus (MazF-mx) and MazF from Mycobacterium tuberculosis (MazF-mt3), affects RNA cleavage specificity. We found that exchanging loop 2 of MazF-ec with loop 2 regions from either MazF-mx or MazF-mt3 created a new cleavage sequence at (A/U)(A/U)AA^C in addition to the original cleavage site, A^CA, whereas exchanging loop 1 did not alter cleavage specificity. Intriguingly, exchange of loop 2 with 8 or 12 consecutive Gly residues also resulted in a new RNA cleavage site at (A/U)(A/U)AA^C. The present study suggests a method for expanding the RNA cleavage repertoire of mRNA interferases, which is crucial for potential use in the regulation of specific gene expression and for biotechnological applications.

YeeU enhances the bundling of cytoskeletal polymers of MreB and FtsZ, antagonizing the CbtA (YeeV) toxicity in Escherichia coli.

All free-living bacteria carry the toxin-antitoxin (TA) systems controlling cell growth and death under stress conditions. YeeU-YeeV (CbtA) is one of the Escherichia coli TA systems, and the toxin, CbtA, has been reported to inhibit the polymerization of bacterial cytoskeletal proteins, MreB and FtsZ. Here, we demonstrate that the antitoxin, YeeU, is a novel type of antitoxin (type IV TA system), which does not form a complex with CbtA but functions as an antagonist for CbtA toxicity. Specifically, YeeU was found to directly interact with MreB and FtsZ, and enhance the bundling of their filamentous polymers in vitro. Surprisingly, YeeU neutralized not only the toxicity of CbtA but also the toxicity caused by other inhibitors of MreB and FtsZ, such as A22, SulA and MinC, indicating that YeeU-induced bundling of MreB and FtsZ has an intrinsic global stabilizing effect on their homeostasis. Here we propose to rename YeeU as CbeA for cytoskeleton bundling-enhancing factor A.