Eliminating the requirement of an essential gene product in an already very small virus: scaffolding protein B-free øX174, B-free

Unlike most viral assembly systems, two scaffolding proteins, B and D, mediate bacteriophage ?X174 morphogenesis. The external scaffolding protein D is highly ordered in the atomic structure and proper function is very sensitive to mutation. In contrast, the internal scaffolding protein B is relatively unordered and extensive alterations do not eliminate function. Despite this genetic laxity, protein B is absolutely required for virus assembly. Thus, this system, with its complex arrangements of overlapping reading frames, can be regarded as an example of "irreducible complexity." To address the biochemical functions of a dual scaffolding protein system and the evolution of complexity, progressive and targeted genetic selections were employed to lessen and finally eliminate B protein-dependence. The biochemical and genetic bases of adaptation were characterized throughout the analysis that led to the sextuple mutant with a B-independent phenotype, as evaluated by plaque formation in wild-type cells. The primary adaptation appears to be the over-expression of a mutant external scaffolding protein. Progeny production was followed in lysis-resistant cells. The ability to produce infectious virions does not require all six mutations. However, the lag phase before progeny production is shortened as mutations accumulate. The results suggest that the primary function of the internal scaffolding protein may be to lower the critical concentration of the external scaffolding protein needed to nucleate procapsid formation. Moreover, they demonstrate a novel mechanism by which a stringently required gene product can be bypassed, even in a system encoding only eight strictly essential proteins.     

RNA-dependent RNA polymerase of hepatitis C virus: Study on inhibition by α,γ-diketo acid derivatives

It is supposed that α,γ-diketo acids (DKAs) inhibit the activity of hepatitis C virus RNA-dependent RNA poly-merase (RdRP HCV) via chelation of catalytic magnesium ions in the active center of the enzyme. However, DKAs display noncompetitive mode of inhibition with respect to NTP substrate, which contradicts the proposed mechanism. We have examined the NTP substrate entry channel and the active site of RdRP HCV for their possible interaction with DKAs. The substitutions R48A, K51A, and R222A greatly facilitated RdRP inhibition by DKAs and simultaneously increased K _m values for UTP substrate. Interestingly, C223A was the only one of a number of substitutions that decreased K _m(UTP) but facilitated the inhibitory action of DKAs. The findings allowed us to model an enzyme-inhibitor complex. According to the proposed model, DKAs introduce an additional Mg²⁺ ion into the active site of the enzyme at a stage of phosphodiester bond formation, which results in displacement of the NTP substrate triphosphate moiety to a catalytically inactive binding mode. This mechanism, in contrast to the currently adopted one, explains the noncompetitive mode of inhibition.     

The 3′ end of Turnip crinkle virus contains a highly interactive structure including a translational enhancer that is disrupted by binding to the RNA-dependent RNA polymerase

Precise temporal control is needed for RNA viral genomes to translate sufficient replication-required products before clearing ribosomes and initiating replication. A 3′ translational enhancer in Turnip crinkle virus (TCV) overlaps an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits. The higher-order structure in the region was examined through alteration of critical sequences revealing novel interactions between an H-type pseudoknot and upstream residues, and between the TSS and internal and terminal loops of an upstream hairpin. Our results suggest that the TSS forms a stable scaffold that allows for simultaneous interactions with external sequences through base pairings on both sides of its large internal symmetrical loop. Binding of TCV RNA-dependent RNA polymerase (RdRp) to the region potentiates a widespread conformational shift with substantial rearrangement of the TSS region, including the element required for efficient ribosome binding. Degrading the RdRp caused the RNA to resume its original conformation, suggesting that the initial conformation is thermodynamically favored. These results suggest that the 3′ end of TCV folds into a compact, highly interactive structure allowing RdRp access to multiple elements including the 3′ end, which causes structural changes that potentiate the shift between translation and replication.     

Characterization of the oat1 gene of Penicillium chrysogenum encoding an ω-aminotransferase: induction by L-lysine, L-ornithine and L-arginine and repression by ammonium

The Penicillium chrysogenum oat1 gene, which encodes a class III omega-aminotransferase, was cloned and characterized. This enzyme converts lysine into 2-aminoadipic semialdehyde, and plays an important role in the biosynthesis of 2-aminoadipic acid, a precursor of penicillin and other beta-lactam antibiotics. The enzyme is related to ornithine-5-aminotransferases and to the lysine-6-aminotransferases encoded by the lat genes found in bacterial cephamycin gene clusters. Expression of oat1 is induced by lysine, ornithine and arginine, and repressed by ammonium ions. AreA-binding GATA and GATT sequences involved in regulation by ammonium, and an 8-bp direct repeat associated with arginine induction in Emericella (Aspergillus nidulans and Saccharomyces cerevisiae, were found in the oat1 promoter region. Deletion of the oat1 gene resulted in the loss of omega-aminotransferase activity. The null mutants were unable to grow on ornithine or arginine as sole nitrogen sources and showed reduced growth on lysine. Complementation of the null mutant with the oat1 gene restored normal levels of omega-aminotransferase activity and the ability to grow on ornithine, arginine and lysine. The role of the oat1 gene in the biosynthesis of 2-aminoadipic acid is discussed.     

Global effect of an RNA polymerase β-subunit mutation on gene expression in the radiation-resistant bacterium <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

The β-subunit of RNA polymerase, which is involved in rifampin binding, is highly conserved among prokaryotes, and Rifr mutants detected in many bacteria are the result of amino acid changes. Spontaneous rifampin resistance mutations resulting in amino acid replacement (L420R) and deletion (1258–66 9 bp deletion) have been previously isolated in the rpoB gene of Deinococcus radiodurans. In this study, a β-subunit mutation in D. radiodurans resulted in a unique effect on growth rate. We used DNA microarrays and biochemical assays to investigate how the Rifr mutation in the β-subunit led to changes in growth rate via altered regulation of multiple genes. The expression of genes with predicted functions in metabolism, cellular processes and signaling, and information storage and processing were significantly altered in the 9 bp-deletion rpoB mutant. The consensus promoter sequence of up-regulated genes in the 9 bp-deletion rpoB mutant was identified as an AT-rich sequence. Greater levels of reactive oxygen species accumulated in the L420R and 9 bp-deletion rpoB mutants compared with wild type. These results provide insight into the molecular mechanism of how the β-subunit Rifr mutation alters the regulation of multiple genes.     

Translational control of the expression of the β subunit gene of E., coli RNA polymerase

Using the plasmid pNF1337 as template, a mRNA preparation has been obtained that directs the $\text{in}\text{,}\text{vitro}$ synthesis of fMet-Val, the N-terminal dipeptide of the β subunit of RNA polymerase. RNA polymerase holoenzyme specifically inhibits the mRNA-directed synthesis of fMet-Val showing that the autoregulation by RNA polymerase of β,β′ synthesis is at the level of translation. L factor ($\text{nusA}$ gene product) stimulates the synthesis of fMet-Val from a DNA template but not from mRNA. Rifampicin has no effect on the mRNA-directed synthesis of fMet-Val or the ability of RNA polymerase to inhibit fMet-Val synthesis.     

Transcription of the human microsomal epoxide hydrolase gene (EPHX1) is regulated by an HNF-4α/CAR/RXR/PSF complex

Microsomal epoxide hydrolase (mEH) is a bifunctional protein that plays a central role in the metabolism of numerous xenobiotics as well as mediating the sodium-dependent transport of bile acids into hepatocytes where they are involved in cholesterol excretion and metabolism, lipid digestion and regulating numerous signaling pathways. Previous studies have demonstrated the critical role of GATA-4 and a C/EBPα–NF/Y complex in the regulation of the mEH gene (EPHX1). In this study we show that HNF-4α and CAR/RXR also bind to the proximal promoter region and regulate EPHX1 expression. Bile acids, which inhibit the expression of HNF-4α also decrease the expression of EPHX1. Studies also established that the binding of HNF-4α was essential for the activation of EPHX1 activity by CAR suggesting the formation of a complex between these adjacent factors. The nature of this regulatory complex was further explored using a biotinylated oligonucleotide of this region in conjunction with BioMag beads and mass spectrometric analysis which demonstrated the presence of an additional inhibitory factor (PSF), confirmed by co-immunoprecipitation and ChIP analyses, which interacted with DNA-bound CAR/RXR/HNF-4α forming a 4-component regulatory complex.     

NlCDK1 gene is required for the ovarian development and survival of the brown planthopper, Nilaparvata lugens Stål,as revealed by RNA interference

CDK1 is a kind of cyclin-dependent kinases (CDKs) involved in diverse biological processes besides cell cycle regulation in various species. However, little is known about CDK1 in Nilaparvata lugens Stål (brown planthopper, BPH). In this study, the full-length cDNA of NlCDK1, a CDK1 gene in BPH, was obtained using rapid-amplification of cDNA ends (RACE) technique. RT-qPCR revealed that the expression of NlCDK1 was relatively low and stable at nymph stages from 1st star to 5th star, but increased to higher levels in gravid females 3?day to 5?day post-eclosion. Feeding-based RNA interference (RNAi) with 0.5?μg/μL dsNlCDK1 achieved an efficient knockdown of target gene expression in females after being fed with dsRNA for 6?days, and the mRNA level of NlCDK1 decreased to 19.9% in comparison with that of the dsGFP control. As a result, RNA inerference with dsNlCDK1 caused a significant decrease of the BPH survival rate to 28.0% of that for the control. RNAi of NlCDK1 also suppressed the ovarian development and significantly affected the fecundity of the females, leading to a 70.2% reduction to the number of the offspring. These data suggest that NlCDK1 is required for the ovarian development and survival of the brown planthopper.     

Synthesis of the penicillin precursor, δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV), by an immobilized enzyme preparation

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase activity has been partially-purified from cell-free extracts of Streptomyces clavuligerus by ammonium sulfate precipitation. The salt precipitated enzyme was immobilized on an anion exchange resin and synthesis of ACV was observed by exposing the immobilized enzyme preparation to a reaction mixture containing l--aminoadipic acid, l-valine and l-cysteine in the presence of appropriate cofactors. Reaction mixtures containing l--aminobutyric acid(aB) in place of l-valine synthesized the ACV analog ACaB. Immobilized ACV synthetase can be reused, and after six cycles of reaction, 28.9% of original activity remains.     

ASB2α, an E3 Ubiquitin Ligase Specificity Subunit,Regulates Cell Spreading and Triggers Proteasomal Degradation of Filamins by Targeting the Filamin Calponin Homology 1 Domain

Filamins are actin-binding and cross-linking proteins that organize the actin cytoskeleton and anchor transmembrane proteins to the cytoskeleton and scaffold signaling pathways. During hematopoietic cell differentiation, transient expression of ASB2α, the specificity subunit of an E3-ubiquitin ligase complex, triggers acute proteasomal degradation of filamins. This led to the proposal that ASB2α regulates hematopoietic cell differentiation by modulating cell adhesion, spreading, and actin remodeling through targeted degradation of filamins. Here, we show that the calponin homology domain 1 (CH1), within the filamin A (FLNa) actin-binding domain, is the minimal fragment sufficient for ASB2α-mediated degradation. Combining an in-depth flow cytometry analysis with mutagenesis of lysine residues within CH1, we find that arginine substitution at each of a cluster of three lysines (Lys-42, Lys-43, and Lys-135) renders FLNa resistant to ASB2α-mediated degradation without altering ASB2α binding. These lysines lie within previously predicted actin-binding sites, and the ASB2α-resistant filamin mutant is defective in targeting to F-actin-rich structures in cells. However, by swapping CH1 with that of α-actinin1, which is resistant to ASB2α-mediated degradation, we generated an ASB2α-resistant chimeric FLNa with normal subcellular localization. Notably, this chimera fully rescues the impaired cell spreading induced by ASB2α expression. Our data therefore reveal ubiquitin acceptor sites in FLNa and establish that ASB2α-mediated effects on cell spreading are due to loss of filamins.     

Hydrolysis of di-substituted hydantoins,by an enzyme preparation from lentil (Lens esculenta) seeds,for the synthesis of α,α-dialkylated amino acids with linear and cyclic substituents

Hydrolysis of 5,5-di-substituted hydantoins with linear and cyclic side chains, to produce the corresponding ,-dialkylated amino acids and 1-aminocycloalkane-1-carboxylic acids, using an enzyme preparation from lentil (Lens esculenta) seeds is reported for the first time. This method could be developed on a large scale and should be of interest to the chemical and pharmaceutical industries.R. Rai and V. Taneja are with the Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi-221 005, India; R. Balaji Rao is with the Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi-221 005, India.