The terminal inverted repeats of the linear plasmid SCP1 of Streptomyces coelicolor A3(2) possess a truncated copy of the transposon Tn 4811 of Streptomyces lividans 66

Streptomyces coelicolor A3(2), the best genetically studied streptomycete and Streptomyces lividans 66 are very closely related strains. This is further emphasized by our finding that a truncated copy of Tn4811 of S. lividans is present in the terminal inverted repeats of the S. coelicolor giant linear plasmid SCP1. The copy of Tn4811 in SCP1 lacks the first 1276 bp and shows only minor changes in the nucleotide sequence of the remaining 4.12 kb. Tn4811 exists in both ends of SCP1.     

Random circular permutation leading to chain disruption within and near alpha helices in the catalytic chains of aspartate transcarbamoylase: effects on assembly, stability, and function

A collection of circularly permuted catalytic chains of aspartate transcarbamoylase (ATCase) has been generated by random circular permutation of the pyrB gene. From the library of ATCases containing permuted polypeptide chains, we have chosen for further investigation nine ATCase variants whose catalytic chains have termini located within or close to an alpha helix. All of the variants fold and assemble into dodecameric holoenzymes with similar sedimentation coefficients and slightly reduced thermal stabilities. Those variants disrupted within three different helical regions in the wild-type structure show no detectable enzyme activity and no apparent binding of the bisubstrate analog N:-phosphonacetyl-L-aspartate. In contrast, two variants whose termini are just within or adjacent to other alpha helices are catalytically active and allosteric. As expected, helical disruptions are more destabilizing than loop disruptions. Nonetheless, some catalytic chains lacking continuity within helical regions can assemble into stable holoenzymes comprising six catalytic and six regulatory chains. For seven of the variants, continuity within the helices in the catalytic chains is important for enzyme activity but not necessary for proper folding, assembly, and stability of the holoenzyme.     

U-box protein carboxyl terminus of Hsc70-interacting protein (CHIP) mediates poly-ubiquitylation preferentially on four-repeat Tau and is involved in neurodegeneration of tauopathy

Neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated and ubiquitylated tau, are exhibited at regions where neuronal loss occurs in neurodegenerative diseases; however, the mechanisms of NFT formation remain unknown. Molecular studies of frontotemporal dementia with parkinsonism-17 demonstrated that increasing the ratio of tau with exon 10 insertion induced fibrillar tau accumulation. Here, we show that carboxyl terminus of Hsc70-interacting protein (CHIP), a U-box protein, recognizes the microtubule-binding repeat region of tau and preferentially ubiquitylates four-repeat tau compared with three-repeat tau. Overexpression of CHIP induced the prompt degradation of tau, reduced the formation of detergent-insoluble tau and inhibited proteasome inhibitor-induced cell death. NFT bearing neurons in progressive supranuclear palsy, in which four-repeat tau is a component, showed the accumulation of CHIP. Thus, CHIP is a ubiquitin ligase for four-repeat tau and maintains neuronal survival by regulating the quality control of tau in neurons.     

N-terminal acetyltransferases and sequence requirements for N-terminal acetylation of eukaryotic proteins

N(alpha)-terminal acetylation occurs in the yeast Saccharomyces cerevisiae by any of three N-terminal acetyltransferases (NAT), NatA, NatB, and NatC, which contain Ard1p, Nat3p and Mak3p catalytic subunits, respectively. The N-terminal sequences required for N-terminal acetylation, i.e. the NatA, NatB, and NatC substrates, were evaluated by considering over 450 yeast proteins previously examined in numerous studies, and were compared to the N-terminal sequences of more than 300 acetylated mammalian proteins. In addition, acetylated sequences of eukaryotic proteins were compared to the N termini of 810 eubacterial and 175 archaeal proteins, which are rarely acetylated. Protein orthologs of Ard1p, Nat3p and Mak3p were identified with the eukaryotic genomes of the sequences of model organisms, including Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, Mus musculus and Homo sapiens. Those and other putative acetyltransferases were assigned by phylogenetic analysis to the following six protein families: Ard1p; Nat3p; Mak3p; CAM; BAA; and Nat5p. The first three families correspond to the catalytic subunits of three major yeast NATs; these orthologous proteins were identified in eukaryotes, but not in prokaryotes; the CAM family include mammalian orthologs of the recently described Camello1 and Camello2 proteins whose substrates are unknown; the BAA family comprise bacterial and archaeal putative acetyltransferases whose biochemical activity have not been characterized; and the new Nat5p family assignment was on the basis of putative yeast NAT, Nat5p (YOR253W). Overall patterns of N-terminal acetylated proteins and the orthologous genes possibly encoding NATs suggest that yeast and higher eukaryotes have the same systems for N-terminal acetylation.     

Determination of alpha-helix N1 energies after addition of N1, N2, and N3 preferences to helix/coil theory

Surveys of protein crystal structures have revealed that amino acids show unique structural preferences for the N1, N2, and N3 positions in the first turn of the alpha-helix. We have therefore extended helix-coil theory to include statistical weights for these locations. The helix content of a peptide in this model is a function of N-cap, C-cap, N1, N2, N3, C1, and helix interior (N4 to C2) preferences. The partition function for the system is calculated using a matrix incorporating the weights of the fourth residue in a hexamer of amino acids and is implemented using a FORTRAN program. We have applied the model to calculate the N1 preferences of Gln, Val, Ile, Ala, Met, Pro, Leu, Thr, Gly, Ser, and Asn, using our previous data on helix contents of peptides Ac-XAKAAAAKAAGY-CONH2. We find that Ala has the highest preference for the N1 position. Asn is the most unfavorable, destabilizing a helix at N1 by at least 1.4 kcal mol(-1) compared to Ala. The remaining amino acids all have similar preferences, 0.5 kcal mol(-1) less than Ala. Gln, Asn, and Ser, therefore, do not stabilize the helix when at N1.     

Role of the N terminus in enzyme activity, stability and specificity in thermophilic esterases belonging to the HSL family

A superposition between the structures of Alicyclobacillus acidocaldarius esterase 2 (EST2) and Burkholderia cepacia lipase, the latter complexed with a phosphonate inhibitor, allowed us to hypothesize for the EST2 N terminus a role in restricting the access to the active site and therefore in modulating substrate specificity. In order to test this hypothesis we generated by site-directed mutagenesis some truncated versions of EST2 and its double mutant M211S/R215L (S/L) at the N terminus. In parallel, an analysis of the Sulfolobus solfataricus P2 genome allowed us to identify a gene coding for a putative esterase of the HSL family having a natural deletion of the corresponding region. The product of this gene and the above-mentioned EST2 mutants were expressed in Escherichia coli, purified and characterised. These studies support the notion that the N terminus affects substrate specificity other than several other enzyme parameters. Although the deletions afforded a tenfold and 550-fold decrease in catalytic efficiency towards the best substrate pNP-hexanoate at 50 degrees C for EST2 and S/L, respectively, the analysis of the specific activities with different triacylglycerols with respect to pNP-hexanoate showed that their ratios were higher for deleted versus non-deleted enzymes, on all tested substrates. In particular, the above ratios for glyceryl tridecanoate were 30-fold and 14-fold higher in S/L and EST2 deleted forms, respectively, compared with their full-length versions. This behaviour was confirmed by the analysis of the S.solfataricus esterase, which showed similar specific activities on pNP-hexanoate and triacylglycerols; in addition, higher activities on the latter substrates were observed in comparison with EST2, S/L and their deleted forms. Finally, a dramatic effect on thermophilicity and thermostability in the EST2 deleted forms was observed. This is the first report highlighting the importance of the "cap" domain in the HSL family, since the N terminus partly contributes to the building up of this structure.     

Replication intermediate of a hybrid plasmid carrying the replication terminus (ter) site of R6K as revealed by agarose gel electrophoresis

Summary A 4.32 kb DNA fragment, on which the DNA replication terminus (terR) site of plasmid R 6K was located, was inserted into the unique EcoRI site of plasmid pUC9. To detect replication intermediate molecules with a replication fork halted at the terR site, a cell DNA extract was digested with EcoRI, electrophoresed through an agarose gel and stained with ethidium bromide. In addition to two major bands, one derived from vector DNA and the other from the ter insert fragment, two extra minor bands were detected. Following DNA-DNA hybridization and electron microscopic observation we concluded that the two minor bands corresponded to the two Y-shaped molecules, produced from the -shaped intermediate molecules by EcoRI digestion.Abbreviations Ap ampicillin - kb kilobase pair(s) - EtBr ethidium bromide     

Distinct Requirements for 5′-Monophosphate-assisted RNA Cleavage by Escherichia coli RNase E and RNase G

RNase E and RNase G are homologous endonucleases that play important roles in RNA processing and decay in Escherichia coli and related bacterial species. Rapid mRNA degradation is facilitated by the preference of both enzymes for decay intermediates whose 5′ end is monophosphorylated. In this report we identify key characteristics of RNA that influence the rate of 5′-monophosphate-assisted cleavage by these two ribonucleases. In vitro, both require at least two and prefer three or more unpaired 5′-terminal nucleotides for such cleavage; however, RNase G is impeded more than RNase E when fewer than four unpaired nucleotides are present at the 5′ end. Each can tolerate any unpaired nucleotide (A, G, C, or U) at either of the first two positions, with only modest biases. The optimal spacing between the 5′ end and the scissile phosphate appears to be eight nucleotides for RNase E but only six for RNase G. 5′-Monophosphate-assisted cleavage also occurs, albeit more slowly, when that spacing is greater or at most one nucleotide shorter than the optimum, but there is no simple inverse relationship between increased spacing and the rate of cleavage. These properties are also manifested during 5′-end-dependent mRNA degradation in E. coli.     

Computational analysis of the CB1 carboxyl‐terminus in the receptor‐G protein complex

Despite the important role of the carboxyl‐terminus (Ct) of the activated brain cannabinoid receptor one (CB1) in the regulation of G protein signaling, a structural understanding of interactions with G proteins is lacking. This is largely due to the highly flexible nature of the CB1 Ct that dynamically adapts its conformation to the presence of G proteins. In the present study, we explored how the CB1 Ct can interact with the G protein by building on our prior modeling of the CB1‐Gi complex (Shim, Ahn, and Kendall, The Journal of Biological Chemistry 2013;288:32449–32465) to incorporate a complete CB1 Ct (Glu416^Ct–Leu472^Ct). Based on the structural constraints from NMR studies, we employed ROSETTA to predict tertiary folds, ZDOCK to predict docking orientation, and molecular dynamics (MD) simulations to obtain two distinct plausible models of CB1 Ct in the CB1‐Gi complex. The resulting models were consistent with the NMR‐determined helical structure (H9) in the middle region of the CB1 Ct. The CB1 Ct directly interacted with both Gα and Gβ and stabilized the receptor at the Gi interface. The results of site‐directed mutagenesis studies of Glu416^Ct, Asp423^Ct, Asp428^Ct, and Arg444^Ct of CB1 Ct suggested that the CB1 Ct can influence receptor‐G protein coupling by stabilizing the receptor at the Gi interface. This research provided, for the first time, models of the CB1 Ct in contact with the G protein. Proteins 2016; 84:532–543. © 2016 Wiley Periodicals, Inc.     

Characterization of the terminal inverted repeats and their neighboring tandem repeats in the Chlorella CVK1 virus genome

A unique group of large icosahedral viruses that infect a unicellular green alga (Chlorella sp. NC64A) were isolated from freshwater sources in Japan. These viruses contain a linear double-stranded DNA (dsDNA) genome with hairpin ends. A physical map was constructed for the genomic DNA of CVK1 (Chlorella virus isolated in Kyoto, no. 1) by pulsed-field gel electrophoresis of restriction fragments. The nucleotide sequences around both termini of the CVK1 DNA revealed the presence of inverted terminal repeats (ITR) of approximately 1.0 kb. Adjacent to the ITR, unique sequence elements of 10 to 20 by were directly repeated 20 to 30 times in tandem array. Several copies of these repeat elements were deleted in virus mutants that were occasionally generated from Chlorella cells that were in a putative CVK1 carrier state. These repeats might represent a hot spot of rearrangement in the CVK1 genome.