Mechanism for the disassembly of the posttermination complex inferred from cryo-EM studies

Ribosome recycling, the disassembly of the posttermination complex after each round of protein synthesis, is an essential step in mRNA translation, but its mechanism has remained obscure. In eubacteria, recycling is catalyzed by RRF (ribosome recycling factor) and EF-G (elongation factor G). By using cryo-electron microscopy, we have obtained two density maps, one of the RRF bound posttermination complex and one of the 50S subunit bound with both EF-G and RRF. Comparing the two maps, we found domain I of RRF to be in the same orientation, while domain II in the EF-G-containing 50S subunit is extensively rotated (approximately 60 degrees) compared to its orientation in the 70S complex. Mapping the 50S conformation of RRF onto the 70S posttermination complex suggests that it can disrupt the intersubunit bridges B2a and B3, and thus effect a separation of the two subunits. These observations provide the structural basis for the mechanism by which the posttermination complex is split into subunits by the joint action of RRF and EF-G.     

In vitro evolution of styrene monooxygenase from Pseudomonas putida CA-3 for improved epoxide synthesis

The styAB genes from Pseudomonas putida CA-3, which encode styrene monooxygenase, were subjected to three rounds of in vitro evolution using error-prone polymerase chain reaction with a view to improving the rate of styrene oxide and indene oxide formation. Improvements in styrene monooxygenase activity were monitored using an indole to indigo conversion assay. Each round of random mutagenesis generated variants improved in indigo formation with third round variants improved nine- to 12-fold over the wild type enzyme. Each round of in vitro evolution resulted in two to three amino acid substitutions in styrene monooxygenase. While the majority of mutations occurred in styA (oxygenase), mutations were also observed in styB (reductase). A mutation resulting in the substitution of valine with isoleucine at amino acid residue 303 occurred near the styrene and flavin adenine dinucleotide binding site of styrene monooxygenase. One mutation caused a shift in the reading frame in styA and resulted in a StyA variant that is 19 amino acids longer than the wild-type protein. Whole cells expressing the best styrene monooxygenase variants (round 3) exhibited eight- and 12-fold improvements in styrene and indene oxidation rates compared to the wild-type enzyme. In all cases, a single enantiomer, (S)-styrene oxide, was formed from styrene while (1S,2R)-indene oxide was the predominant enantiomer (e.e. 97%) formed from indene. The average yield of styrene oxide and indene oxide from their respective alkene substrates was 65% and 90%, respectively.     

Interaction of λ Cro repressor and its V55C mutant S-S dimer with symmetrical and asymmetrical DNA

Interaction of phage λ repressor Cro and its mutant form CroV55C, an S-S-bonded dimer that arises spontaneously as a result of substitution of Val55 with Cys, with synthetic oligonucleotide duplexes was studied using competition with the antibiotic distamycin A. The equilibrium binding constants of Cro with phage λ operator O_R3 and various oduplexes containing right and left O_R3 half-operators with single-basepair deletions and insertions in the center of duplexes were calculated. The highest binding constant was obtained when Cro was adsorbed on the symmetrical 17-bp complex O_S, composed of two left half-operators. Deletion of the central GC pair or insertion of an additional GC in O_S decreased the binding constant by a factor of 4–5. The Cro binding constant for the symmetrical 17-bp duplex O_D consisting of two right half-operators and its analog with an additional central GC was five times lower than that for O_S. The thermodynamic binding characteristics of some oligonucleotide duplexes with CroV55C were determined. The binding constant for CroV55C with O_R3 was lower by an order of magnitude than that of Cro, whereas with O_D the CroV55C and Cro binding constants were much the same. For CroV55C and a symmetrical analog of O_S with the central GC deleted, the binding constant increased by an order of magnitude over that for CroV55C and O_R3.     

Statistical fluctuations of the level of operator filling by repressor determine the level of noise of reporter gene expression

The regulation of the reporter gene activity in a single bacterial cell by means of lambda-phage C1 repressor has been described by the methods of statistical thermodynamics. The equations for calculation of the mean production rate of the reporter protein and its standard deviation as a function of C1 repressor concentration in the cell have been obtained. The stochastic nature of C1 repressor binding with O_R1 and O_R2 operator sites becomes apparent when both repressor molecules and operators are present in the bacterial cell in a small number of copies. In this case, the number of repressor molecules that bind to O_R1 and O_R2 sites fluctuates considerably. The in vitro binding of C1 repressor to O_R1 and O_R2 sites, their mutant forms, and nonspecific DNA regions has been well studied. Using the binding constants of in vitro binding of C1 repressor to O_R1, O_R2, and nonspecific DNA regions and also the value of the cooperativity parameter for C1 repressor binding to O_R1 and O_R2 sites, we calculated the mean rate of synthesis of the reporter protein and its standard deviation as a function of repressor concentration in the cell. The theoretical relations fit well the experimental results. The results of calculations confirm the assumption that gene expression noise in a single cell at a repressor concentration exceeding 100 nM is related to the stochastic nature of binding of repressor dimers to O_R1 and O_R2 sites. Other mechanisms of the generation of gene expression noise (for example, monomer-dimer balance) make a significant contribution at concentrations less than 100 nM.     

The critical role of the constant region in thermal stability and aggregation of amyloidogenic immunoglobulin light chain

Light chain (LC) amyloidosis (AL) is a fatal disease in which immunoglobulin LC deposit as fibrils. Although the LC amyloid-forming propensity is attributed primarily to the variable region, fibrils also contain full-length LC comprised of variable-joining (V(L)) and constant (C(L)) regions. To assess the role of C(L) in fibrillogenesis, we compared the thermal stability of full-length LC and corresponding V(L) and C(L) fragments. Protein unfolding and aggregation were monitored by circular dichroism and light scattering. A full-length λ6 LC purified from urine of a patient with AL amyloidosis showed irreversible unfolding coupled to aggregation. The transition temperature decreased at slower heating rates, indicating kinetic effects. Next, we studied five recombinant λ6 proteins: full-length amyloidogenic LC, its V(L), germline LC, germline V(L), and C(L). Amyloidogenic and germline proteins showed similar rank order of stability, V(L) < LC < C(L); hence, in the full-length LC, V(L) destabilizes C(L). Amyloidogenic proteins were less stable than their germline counterparts, suggesting that reduction in V(L) stability destabilizes the full-length LC. Thermal unfolding of the full-length amyloidogenic and germline LC required high activation energy and involved irreversible aggregation, yet the unfolding of the isolated V(L) and C(L) fragments was partially reversible. Therefore, compared to their fragments, full-length LCs are more likely to initiate aggregation during unfolding and provide a template for the V(L) deposition. The kinetic barrier for this aggregation is regulated by the stability of the V(L) region. This represents a paradigm shift in AL fibrillogenesis and suggests C(L) region as a potential therapeutic target.     

Sociality in the spectral tarsier, Tarsius spectrum

Recent studies indicate that many of the nocturnal prosimian primates are gregarious rather than solitary. This paper shows that the spectral tarsier is gregarious during its nightly activity period as well as in its sleeping tree. Using mist nets and radiotelemetry, focal follows were conducted on six groups at Tangkoko Nature Reserve in Sulawesi, Indonesia. During 442 focal follows, 1072 encounters between a focal adult group member and another adult were observed. The number of encounters ranged from as few as 0 to as many as 18 encounters per night. Intragroup encounters lasted from less than 1 min to as long as 3 hr 12 min. Nearly one-half of all social behavior occurred between adult females and males. There were also substantial rates of social interaction between the two adult females in one group, and between sub-adults of the opposite sex in neighboring groups.     

Maize endosperm ADP-glucose pyrophosphorylase SHRUNKEN2 and BRITTLE2 subunit interactions

ADP-glucose pyrophosphorylase (AGP) represents a key regulatory step in polysaccharide synthesis in organisms ranging from bacteria to plants. Higher plant AGPs are complex in nature and are heterotetramers consisting of two similar but distinct subunits. How the subunits are assembled into enzymatically active polymers is not yet understood. Here, we address this issue by using naturally occurring null mutants of the Shrunken2 (Sh2) and Brittle2 (Bt2) loci of maize as well as the yeast two-hybrid expression system. In the absence of the maize endosperm large AGP subunit (SH2), the BT2 subunit remains as a monomer in the developing endosperm. In contrast, the SH2 protein, in the absence of BT2, is found in a complex of 100 kD. A direct interaction between SH2 and BT2 was proven when they were both expressed in yeast. Several motifs are essential for SH2:BT2 interaction because truncations removing the N or C terminus of either subunit eliminate SH2:BT2 interactions. Analysis of subunit interaction mutants (sim) also identified motifs essential for protein interactions.     

Age as a Determinant for Dissemination of Seasonal and Pandemic Influenza: An Open Cohort Study of Influenza Outbreaks in ?sterg?tland County,Sweden

An understanding of the occurrence and comparative timing of influenza infections in different age groups is important for developing community response and disease control measures. This study uses data from a Scandinavian county (population 427.000) to investigate whether age was a determinant for being diagnosed with influenza 2005–2010 and to examine if age was associated with case timing during outbreaks. Aggregated demographic data were collected from Statistics Sweden, while influenza case data were collected from a county-wide electronic health record system. A logistic regression analysis was used to explore whether case risk was associated with age and outbreak. An analysis of variance was used to explore whether day for diagnosis was also associated to age and outbreak. The clinical case data were validated against case data from microbiological laboratories during one control year. The proportion of cases from the age groups 10–19 (p<0.001) and 20–29 years old (p<0.01) were found to be larger during the A pH1N1 outbreak in 2009 than during the seasonal outbreaks. An interaction between age and outbreak was observed (p<0.001) indicating a difference in age effects between circulating virus types; this interaction persisted for seasonal outbreaks only (p<0.001). The outbreaks also differed regarding when the age groups received their diagnosis (p<0.001). A post-hoc analysis showed a tendency for the young age groups, in particular the group 10–19 year olds, led outbreaks with influenza type A H1 circulating, while A H3N2 outbreaks displayed little variations in timing. The validation analysis showed a strong correlation (r = 0.625;p<0.001) between the recorded numbers of clinically and microbiologically defined influenza cases. Our findings demonstrate the complexity of age effects underlying the emergence of local influenza outbreaks. Disentangling these effects on the causal pathways will require an integrated information infrastructure for data collection and repeated studies of well-defined communities.     

Binding of bis-linked netropsin derivatives in the parallel-stranded hairpin form to DNA

Cis-diammine Pt(II)- bridged bis-netropsin and oligomethylene-bridged bis-netropsin in which two monomers are linked in a tail-to-tail manner bind to the DNA oligomer with the sequence 5'-CCTATATCC-3' in a parallel-stranded hairpin form with a stoichiometry 1:1. The difference circular dichroism (CD) spectra characteristic of binding of these ligands in the hairpin form are similar. They differ from CD patterns obtained for binding to the same duplex of another bis-netropsin in which two netropsin moieties were linked in a head-to-tail manner. This reflects the fact that tail-to-tail and head-to-tail bis-netropsins use parallel and antiparallel side-by-side motifs, respectively, for binding to DNA in the hairpin forms. The binding affinity of cis-diammine Pt(II)-bridged bis-netropsin in the hairpin form to DNA oligomers with nucleotide sequences 5'-CCTATATCC-3' (I), 5'-CCTTAATCC-3' (II), 5'-CCTTATTCC-3' (III), 5'-CCTTTTTCC-3' (IV) and 5'-CCAATTTCC-3' (V) decreases in the order I = II > III > IV > V . The binding of oligomethylene-bridged bis-netropsin in the hairpin form follows a similar hierarchy. An opposite order of sequence preferences is observed for partially bonded monodentate binding mode of the synthetic ligand.     

Sequence-specific recognition of double-stranded DNA by synthetic minor groove binder conjugates. toward the construction of artificial site-specific deoxyribonucleases

Bis-conjugates of hairpin N-methylpyrrole/N-methylimidazole oligocarboxamide minor groove binders (MGB) possessing enhanced affinity and sequence-specificity for dsDNA were synthesized. Two hairpin MGBs were connected by their N-termini via an aminodiacetate linker. The binding of bis-MGB conjugates to the target DNA was studied by gel mobility retardation, footprinting, and circular dichroism; their affinity and binding mode in the DNA minor groove were determined. In order to functionalize the bis-MGB conjugates, DNA-cleaving agents such as phenanthroline or bipyridine were attached. Effective site-specific cleavage of target DNA in the presence of Cu(2+) ions was observed.