Vinyl Sulfones as Antiparasitic Agents and a Structural Basis for Drug Design

Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs.Sleeping sickness (African trypanosomiasis), caused by Trypanosoma brucei, and malaria, caused by Plasmodium falciparum, are significant, parasitic diseases of sub-Saharan Africa (1). Chagas'' disease (South American trypanosomiasis), caused by Trypanosoma cruzi, affects approximately, 16–18 million people in South and Central America. For all three of these protozoan diseases, resistance and toxicity to current therapies makes treatment increasingly problematic, and thus the development of new drugs is an important priority (2–4).T. cruzi, T. brucei, and P. falciparum produce an array of potential target enzymes implicated in pathogenesis and host cell invasion, including a number of essential and closely related papain-family cysteine proteases (5, 6). Inhibitors of cruzain and rhodesain, major cathepsin L-like papain-family cysteine proteases of T. cruzi and T. brucei rhodesiense (7–10) display considerable antitrypanosomal activity (11, 12), and some classes have been shown to cure T. cruzi infection in mouse models (11, 13, 14).In P. falciparum, the papain-family cysteine proteases falcipain-2 (FP-2)⁶ and falcipain-3 (FP-3) are known to catalyze the proteolysis of host hemoglobin, a process that is essential for the development of erythrocytic parasites (15–17). Specific inhibitors, targeted to both enzymes, display antiplasmodial activity (18). However, although the abnormal phenotype of FP-2 knock-outs is “rescued” during later stages of trophozoite development (17), FP-3 has proved recalcitrant to gene knock-out (16) suggesting a critical function for this enzyme and underscoring its potential as a drug target.Sequence analyses and substrate profiling identify cruzain, rhodesain, and FP-3 as cathepsin L-like, and several studies describe classes of small molecule inhibitors that target multiple cathepsin L-like cysteine proteases, some with overlapping antiparasitic activity (19–22). Among these small molecules, vinyl sulfones have been shown to be effective inhibitors of a number of papain family-like cysteine proteases (19, 23–27). Vinyl sulfones have many desirable attributes, including selectivity for cysteine proteases over serine proteases, stable inactivation of the target enzyme, and relative inertness in the absence of the protease target active site (25). This class has also been shown to have desirable pharmacokinetic and safety profiles in rodents, dogs, and primates (28, 29). We have determined the crystal structures of cruzain, rhodesain, and FP-3 bound to vinyl sulfone inhibitors and performed inhibition kinetics for each enzyme. Our results highlight key areas of interaction between proteases and inhibitors. These results help validate the vinyl sulfones as a class of antiparasitic drugs and provide structural insights to facilitate the design or modification of other small molecule inhibitor scaffolds.     

DYn-2 Based Identification of Arabidopsis Sulfenomes

Identifying the sulfenylation state of stressed cells is emerging as a strategic approach for the detection of key reactive oxygen species signaling proteins. Here, we optimized an in vivo trapping method for cysteine sulfenic acids in hydrogen peroxide (H₂O₂) stressed plant cells using a dimedone based DYn-2 probe. We demonstrated that DYn-2 specifically detects sulfenylation events in an H₂O₂ dose- and time-dependent way. With mass spectrometry, we identified 226 sulfenylated proteins after H₂O₂ treatment of Arabidopsis cells, residing in the cytoplasm (123); plastid (68); mitochondria (14); nucleus (10); endoplasmic reticulum, Golgi and plasma membrane (7) and peroxisomes (4). Of these, 123 sulfenylated proteins have never been reported before to undergo cysteine oxidative post-translational modifications in plants. All in all, with this DYn-2 approach, we have identified new sulfenylated proteins, and gave a first glance on the locations of the sulfenomes of Arabidopsis thaliana.Among the different amino acids, the sulfur containing amino acids like cysteine are particularly susceptible to oxidation by reactive oxygen species (ROS)¹ (1, 2). Recent studies suggest that the sulfenome, the initial oxidation products of cysteine residues, functions as an intermediate state of redox signaling (3 –5). Thus, identifying the sulfenome under oxidative stress is a way to detect potential redox sensors (6, 7).This central role of the sulfenome in redox signaling provoked chemical biologists to develop strategies for sensitive detection and identification of sulfenylated proteins. The in situ trapping of the sulfenome is challenging because of two major factors: (1) the highly reactive, transient nature of sulfenic acids, which might be over-oxidized in excess of ROS, unless immediately protected by disulfide formation (7); (2) the intracellular compartmentalization of the redox state that might be disrupted during extraction procedures, resulting in artificial non-native protein oxidations (8, 9). Having a sulfur oxidation state of zero, sulfenic acids can react as both electrophile and nucleophile, however, direct detection methods are based on the electrophilic character of sulfenic acid (10). In 1974, Allison and coworkers reported a condensation reaction between the electrophilic sulfenic acid and the nucleophile dimedone (5,5-dimethyl-1,3-cyclohexanedione), producing a corresponding thioether derivative (11). This chemistry is highly selective and, since then, has been exploited to detect dimedone modified sulfenic acids using mass spectrometry (12). However, dimedone has limited applications for cellular sulfenome identification because of the lack of a functional group to enrich the dimedone tagged sulfenic acids. Later, dimedone-biotin/fluorophores conjugates have been developed, which allowed sensitive detection and enrichment of sulfenic acid modified proteins (13 –15). This approach, however, was not always compatible with in vivo cellular sulfenome analysis, because the biotin/fluorophores-conjugated dimedone is membrane impermeable (9) and endogenous biotinylated proteins might appear as false positives.More recently, the Carroll lab has developed DYn-2, a sulfenic acid specific chemical probe. This chemical probe consists of two functional units: a dimedone scaffold for sulfenic acid recognition and an alkyne chemical handle for enrichment of labeled proteins (9). Once the sulfenic acids are tagged with the DYn-2 probe, they can be biotinylated through click chemistry (16). The click reaction used here is a copper (I)-catalyzed azide-alkyne cycloaddition reaction (17), also known as azide-alkyne Huisgen cycloaddition (16). With this chemistry, a complex is formed between the alkyne functionalized DYn-2 and the azide functionalized biotin. This biotin functional group facilitates downstream detection, enrichment, and mass spectrometry based identification (Fig. 1). In an evaluation experiment, DYn-2 was found to efficiently detect H₂O₂-dependent sulfenic acid modifications in recombinant glutathione peroxidase 3 (Gpx3) of budding yeast (18). Moreover, it was reported that DYn-2 is membrane permeable, non-toxic, and a non-influencer of the intracellular redox balance (17, 18). Therefore, DYn-2 has been suggested as a global sulfenome reader in living cells (17, 18), and has been applied to investigate epidermal growth factor (EGF) mediated protein sulfenylation in a human epidermoid carcinoma A431 cell line and to identify intracellular protein targets of H₂O₂ during cell signaling (17).Open in a separate windowFig. 1.Schematic views of the molecular mechanism of the DYn-2 probe and the strategy to identify DYn-2 trapped sulfenylated proteins. A, DYn-2 specifically detects sulfenic acid modifications, but no other thiol modifications. B, Biotinylation of the DYn-2 tagged proteins by click reaction. C, Once DYn-2 tagged proteins are biotinylated, a streptavidin-HRP (Strep-HRP) blot visualizes sulfenylation, or alternatively, after enrichment on avidin beads, proteins are identified by mass spectrometry analysis.Here, we selected the DYn-2 probe to identify the sulfenome in plant cells under oxidative stress. Through a combination of biochemical, immunoblot and mass spectrometry techniques, and TAIR10 database and SUBA3-software predictions, we can claim that DYn-2 is able to detect sulfenic acids on proteins located in different subcellular compartments of plant cells. We identified 226 sulfenylated proteins in response to an H₂O₂ treatment of Arabidopsis cell suspensions, of which 123 proteins are new candidates for cysteine oxidative post-translational modification (PTM) events.     

cysTMTRAQ—An Integrative Method for Unbiased Thiol-based Redox Proteomics

Protein redox regulation plays important roles in many biological processes. Protein cysteine thiols are sensitive to redox changes and may function as redox switches, which turn signaling and metabolic pathways on or off to ensure speedy responses to environmental stimuli or stresses. Here we report a novel integrative proteomics method called cysTMTRAQ that combines two types of isobaric tags, cysteine tandem mass tags and isobaric tag for relative and absolute quantification, in one experiment. The method not only enables simultaneous analysis of cysteine redox changes and total protein level changes, but also allows the determination of bona fide redox modified cysteines in proteins through the correction of protein turnover. Overlooking the factor of protein-level changes in the course of protein posttranslational modification experiments could lead to misleading results. The capability to analyze protein posttranslational modification dynamics and protein-level changes in one experiment will advance proteomic studies in many areas of biology and medicine.Changes in the redox states of protein cysteine thiols serve as regulatory switches in diverse biological processes (1). The redox cycle is regulated by well-known factors such as the ferredoxin-thioredoxin and glutathione-glutaredoxin systems, which reduce oxidized cysteines. Other oxidoreductases and oxidants such as reactive oxygen species act primarily to oxidize cysteine thiol groups (2, 3). In order to map and quantify cysteine redox modifications on the proteome scale, several approaches and methods have been developed, mostly using thiol-specific reagents and isotope tags. Two-dimensional gel electrophoresis technology combined with fluorescent dye labeling (e.g. monobromobimane (4, 5) and cyanine dyes (6, 7)) and gel-free technology with isotope tagging (e.g. isotope-coded affinity tagging (6–8), cysTMT¹ (9), and iTRAQ labeling of enriched cysteine-containing peptides (10–14)) are often used to identify potential redox-sensitive cysteine residues and quantify redox changes.In addition to the well-known capabilities and limitations associated with two-dimensional gel electrophoresis–based and gel-free approaches (15), each method has its strengths and weaknesses in redox proteomics. For example, the two-dimensional gel electrophoresis methods allow the inspection of spot patterns related to redox and protein-level changes. However, spot-volume-based quantification becomes problematic, as each spot often contains more than one protein species from complex samples. In addition, the limited number of fluorescent reagents compromises multiplexing capability, and the use of cyanine dyes does not allow mapping of the modified cysteines (6, 7). Other thiol labeling approaches such as the use of N-ethylmaleimide, biotin-N-[6-(Biotinamido)hexyl]-3-(2-pyridyldithio) propionamide (16), and isotope-coded affinity tags allow specific enrichment of cysteine-containing peptides, mapping of cysteine modification sites, and duplex experiments in the case of isotope-coded affinity tags (6, 7). To enable multiplexing, 4- or 8-plex iTRAQ tags were recently used to label cysteine-containing peptides isolated from thiol-affinity chromatography (10, 11, 14, 16). Another multiplexing technology, cysTMT, was developed to specifically label cysteines with free thiol groups of proteins from six different samples (9). Although these multiplexing technologies have found utility, they do not address the issue of protein turnover in the course of experiments, and many researchers have overlooked this important factor that could lead to misleading results (8, 13, 14, 17). Only a small number of researchers have attempted to compare potential redox changes determined in proteomics experiments to total protein-level changes obtained from parallel or different studies (6, 7, 16). However, the success of this strategy is often low because many proteins quantified in redox experiments are either absent or not quantified with confidence in total proteomics experiments as a result of experimental variation and mass spectrometry stochastical sampling issues (18, 19).To overcome this challenge, we have developed a double-labeling strategy that uses iTRAQ and cysTMT in one experiment for the simultaneous determination of quantifiable cysteine redox changes and protein-level changes. This new strategy, named cysTMTRAQ, utilizes each of the tags for their specific chemical properties. cysTMT tags (m/z 126, 127, 128, 129, 130, and 131 for six samples) were used to label protein thiols responsive to a treatment, and iTRAQ tags (m/z 114, 115, 116, 117, 119, and 121 for six samples) were used to label the N termini of peptides for analysis of protein-level changes during the experiments. By taking advantage of the different mass tags and their labeling specificities, one can quantify changes in protein redox and total levels in the same experiment. As protein redox regulation is a ubiquitous process (1, 2, 12), the utility of this new integrative cysTMTRAQ method is expected to greatly advance redox proteomic studies in many fields of biology and medicine, and thus benefit a broad range of scientists.     

Commitment and Effector Phases of the Physiological Cell Death Pathway Elucidated with Respect to Bcl-2, Caspase,and Cyclin-Dependent Kinase Activities

Physiological cell deaths occur ubiquitously throughout biology and have common attributes, including apoptotic morphology with mitosis-like chromatin condensation and prelytic genome digestion. The fundamental question is whether a common mechanism of dying underlies these common hallmarks of death. Here we describe evidence of such a conserved mechanism in different cells induced by distinct stimuli to undergo physiological cell death. Our genetic and quantitative biochemical analyses of T- and B-cell deaths reveal a conserved pattern of requisite components. We have dissected the role of cysteine proteases (caspases) in cell death to reflect two obligate classes of cytoplasmic activities functioning in an amplifying cascade, with upstream interleukin-1β-converting enzyme-like proteases activating downstream caspase 3-like caspases. Bcl-2 spares cells from death by punctuating this cascade, preventing the activation of downstream caspases while leaving upstream activity undisturbed. This observation permits an operational definition of the stages of the cell death process. Upstream steps, which are necessary but not themselves lethal, are modulators of the death process. Downstream steps are effectors of, and not dissociable from, actual death; the irreversible commitment to cell death reflects the initiation of this downstream phase. In addition to caspase 3-like proteases, the effector phase of death involves the activation in the nucleus of cell cycle kinases of the cyclin-dependent kinase (Cdk) family. Nuclear recruitment and activation of Cdk components is dependent on the caspase cascade, suggesting that catastrophic Cdk activity may be the actual effector of cell death. The conservation of the cell death mechanism is not reflected in the molecular identity of its individual components, however. For example, we have detected different cyclin-Cdk pairs in different instances of cell death. The ordered course of events that we have observed in distinct cases reflects essential thematic elements of a conserved sequence of modulatory and effector activities comprising a common pathway of physiological cell death.Although interest in the process of physiological cell death has grown enormously in recent years, the mechanism of death has remained enigmatic. While the induction of physiological death in diverse cell types is effected by a wide variety of stimuli, a common morphology, described as apoptosis, ensues in all cases. The commonality of morphology has led to the belief that disparate inducers trigger distinct signaling events which ultimately converge in a common biochemical pathway of death. This hypothesis suggests a division of the biochemical process into upstream events that are specific for individual inducers and downstream steps, comprising the common pathway, which bring about the actual demise of the cell.Since most cell deaths in the nematode Caenorhabditis elegans are induced in a lineage-determined program, the simple pathway of death elucidated in that species (17) is likely to be revealing of downstream steps. Cell death in C. elegans is dependent on the activation of Ced3, a cysteine protease (77, 79), and is inhibited by Ced9 (27). In mammalian cells, a group of Ced3 homologs, termed caspases (1), appears to play a role in virtually all of the physiological cell deaths studied to date. These enzymes cleave on the carboxyl-terminal side of aspartate residues within distinct recognition motifs. Each caspase is synthesized as a proenzyme and activated by cleavage at internal sites, potentially by the same or another caspase class (66, 77). This leads to the notion that caspases function in an ordered cascade, with members of one family activating members of the next. Data consistent with this pattern have been obtained from studies in vitro (41, 60, 65).Of the large family of mammalian caspases, caspase 3 is closely homologous to Ced3 and appears to be involved widely in cell deaths (50, 65). Nonetheless, specific caspases seem not to be associated uniquely with distinct cases of death, and gene-targeting experiments reveal that the absence of a single caspase has extremely limited consequences for cell death responsiveness (38, 39).Similarly, a family of ced9-related death response modulatory genes exists in mammals; the most closely related homolog, bcl-2, is functionally interchangeable with ced9 in the worm (28, 73). These gene products do not function in all mammalian cell deaths (61, 72). Moreover, while the products of some bcl-2 gene family members have death-sparing activity (6, 7), others exert the opposite effect (52, 78).Several cellular proteins, among them poly(ADP-ribose) polymerase (PARP), nuclear lamins, fodrin, and DNA-dependent protein kinase (10, 16, 34), are targets for cleavage by various caspases. In cells spared from death, for example by Bcl-2, these proteolytic events do not occur (9, 13, 18). Still, the cleavage of none of these proteins has been shown to be essential for the cell death response (42, 54, 74). The specific consequences of caspase activation which are lethal are unknown.It may be that the consequence of protease activity is the specific activation of distinct death effectors. We have proposed that essential genes involved in cell division may be critically involved in cell death as well and that the difficulty in identifying distal effector steps genetically reflects the indispensable function of those gene products in cell life (67). Data from several groups have shown that cell cycle catastrophes, the precocious expression of mitosis-like cyclin-dependent histone kinases (Cdks), are associated with a variety of physiological cell deaths and that the inhibition of death by Bcl-2 is associated with alterations in the expression and localization of these Cdk proteins (22, 23, 29, 36, 40, 46, 47, 58, 59, 70).We have taken advantage of the death-sparing activities of Bcl-2 and two viral caspase inhibitors, CrmA and p35 (64, 77), to dissect the mechanism of cell death in two separate cellular paradigms. These studies allow us to draw a generalized skeletal pathway of the death-associated biochemical activities discussed above and demonstrate the requisite involvement of these different classes of activities in a conserved and ordered pathway by which cells die physiologically.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Tyrosine Phosphorylation of Growth Factor Receptor-bound Protein-7 by Focal Adhesion Kinase in the Regulation of Cell Migration, Proliferation, and Tumorigenesis

We have previously reported that growth factor receptor-bound protein-7 (Grb7), an Src-homology 2 (SH2)-containing adaptor protein, enables interaction with focal adhesion kinase (FAK) to regulate cell migration in response to integrin activation. To further elucidate the signaling events mediated by FAK·Grb7 complexes in promoting cell migration and other cellular functions, we firstly examined the phos pho ryl a ted tyrosine site(s) of Grb7 by FAK using an in vivo mutagenesis. We found that FAK was capable of phos pho rylating at least 2 of 12 tyrosine residues within Grb7, Tyr-188 and Tyr-338. Moreover, mutations converting the identified Tyr to Phe inhibited integrin-dependent cell migration as well as impaired cell proliferation but not survival compared with the wild-type control. Interestingly, the above inhibitory effects caused by the tyrosine phos pho ryl a tion-deficient mutants are probably attributed to their down-regulation of phospho-Tyr-397 of FAK, thereby implying a mechanism by competing with wild-type Grb7 for binding to FAK. Consequently, these tyrosine phos pho ryl a tion-deficient mutants evidently altered the phospho-Tyr-118 of paxillin and phos pho ryl a tion of ERK1/2 but less on phospho-Ser-473 of AKT, implying their involvement in the FAK·Grb7-mediated cellular functions. Additionally, we also illustrated that the formation of FAK·Grb7 complexes and Grb7 phos pho ryl a tion by FAK in an integrin-dependent manner were essential for cell migration, proliferation and anchorage-independent growth in A431 epidermal carcinoma cells, indicating the importance of FAK·Grb7 complexes in tumorigenesis. Our data provide a better understanding on the signal transduction event for FAK·Grb7-mediated cellular functions as well as to shed light on a potential therapeutic in cancers.Growth factor receptor bound protein-7 (Grb7)² is initially identified as a SH2 domain-containing adaptor protein bound to the activated EGF receptor (1). Grb7 is composed of an N-terminal proline-rich region, following a putative RA (Ras-associating) domain and a central PH (pleckstrin homology) domain and a BPS motif (between PH and SH2 domains), and a C-terminal SH2 domain (2–6). Despite the lack of enzymatic activity, the presence of multiple protein-protein interaction domains allows Grb7 family adaptor proteins to participate in versatile signal transduction pathways and, therefore, to regulate many cellular functions (4–6). A number of signaling molecules has been reported to interact with these featured domains, although most of the identified Grb7 binding partners are mediated through its SH2 domain. For example, the SH2 domain of Grb7 has been demonstrated to be capable of binding to the phospho-tyrosine sites of EGF receptor (1), ErbB2 (7), ErbB3 and ErbB4 (8), Ret (9), platelet-derived growth factor receptor (10), insulin receptor (11), SHPTP2 (12), Tek/Tie2 (13), caveolin (14), c-Kit (15), EphB1 (16), G6f immunoreceptor protein (17), Rnd1 (18), Shc (7), FAK (19), and so on. The proceeding α-helix of the PH domain of Grb7 is the calmodulin-binding domain responsible for recruiting Grb7 to plasma membrane in a Ca²⁺-dependent manner (20), and the association between the PH domain of Grb7 and phosphoinositides is required for the phosphorylation by FAK (21). Two additional proteins, NIK (nuclear factor κB-inducing kinase) and FHL2 (four and half lim domains isoform 2), in association with the GM region (Grb and Mig homology region) of Grb7 are also reported, although the physiological functions for these interactions remain unknown (22, 23). Recently, other novel roles in translational controls and stress responses through the N terminus of Grb7 are implicated for the findings of Grb7 interacting with the 5′-untranslated region of capped targeted KOR (kappa opioid receptor) mRNA and the Hu antigen R of stress granules in an FAK-mediated phosphorylation manner (24, 25).Unlike its member proteins Grb10 and Grb14, the role of Grb7 in cell migration is unambiguous and well documented. This is supported by a series of studies. Firstly, Grb7 family members share a significantly conserved molecular architecture with the Caenorhabditis elegans Mig-10 protein, which is involved in neuronal cell migration during embryonic development (4, 5, 26), suggesting that Grb7 may play a role in cell migration. Moreover, Grb7 is often co-amplified with Her2/ErbB2 in certain human cancers and tumor cell lines (7, 27, 28), and its overexpression resulted in invasive and metastatic consequences of various cancers and tumor cells (23, 29–33). On the contrary, knocking down Grb7 by RNA interference conferred to an inhibitory outcome of the breast cancer motility (34). Furthermore, interaction of Grb7 with autophosphorylated FAK at Tyr-397 could promote integrin-mediated cell migration in NIH 3T3 and CHO cells, whereas overexpression of its SH2 domain, an dominant negative mutant of Grb7, inhibited cell migration (19, 35). Recruitment and phosphorylation of Grb7 by EphB1 receptors enhanced cell migration in an ephrin-dependent manner (16). Recently, G7–18NATE, a selective Grb7-SH2 domain affinity cyclic peptide, was demonstrated to efficiently block cell migration of tumor cells (32, 36). In addition to cell migration, Grb7 has been shown to play a role in a variety of physiological and pathological events, for instance, kidney development (37), tumorigenesis (7, 14, 38–41), angiogenic activity (20), proliferation (34, 42, 43), anti-apoptosis (44), gene expression regulation (24), Silver-Russell syndrome (45), rheumatoid arthritis (46), atopic dermatitis (47), and T-cell activation (17, 48). Nevertheless, it remains largely unknown regarding the downstream signaling events of Grb7-mediated various functions. In particular, given the role of Grb7 as an adaptor molecule and its SH2 domain mainly interacting with upstream regulators, it will be interesting to identify potential downstream effectors through interacting with the functional GM region or N-terminal proline-rich region.In this report, we identified two tyrosine phosphorylated sites of Grb7 by FAK and deciphered the signaling targets downstream through these phosphorylated tyrosine sites to regulate various cellular functions such as cell migration, proliferation, and survival. In addition, our study sheds light on tyrosine phosphorylation of Grb7 by FAK involved in tumorigenesis.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]