Exploration of the transition state for tertiary structure formation between an RNA helix and a large structured RNA

Docking of the P1 duplex into the pre-folded core of the Tetrahymena group I ribozyme exemplifies the formation of tertiary interactions in the context of a complex, structured RNA. We have applied Phi-analysis to P1 docking, which compares the effects of modifications on the rate constant for docking (k(dock)) with the effects on the docking equilibrium (K(dock)). To accomplish this we used a single molecule fluorescence resonance energy transfer assay that allows direct determination of the rate constants for formation of thermodynamically favorable, as well as unfavorable, states. Modification of the eight groups of the P1 duplex that make tertiary interactions with the core and changes in solution conditions decrease K(dock) up to 500-fold, whereas k(dock) changes by 相似文献   

The carboxyl-terminal domain of bacteriophage T7 single-stranded DNA-binding protein modulates DNA binding and interaction with T7 DNA polymerase

Gene 2.5 of bacteriophage T7 is an essential gene that encodes a single-stranded DNA-binding protein (gp2.5). Previous studies have demonstrated that the acidic carboxyl terminus of the protein is essential and that it mediates multiple protein-protein interactions. A screen for lethal mutations in gene 2.5 uncovered a variety of essential amino acids, among which was a single amino acid substitution, F232L, at the carboxyl-terminal residue. gp2.5-F232L exhibits a 3-fold increase in binding affinity for single-stranded DNA and a slightly lower affinity for T7 DNA polymerase when compared with wild type gp2.5. gp2.5-F232L stimulates the activity of T7 DNA polymerase and, in contrast to wild-type gp2.5, promotes strand displacement DNA synthesis by T7 DNA polymerase. A carboxyl-terminal truncation of gene 2.5 protein, gp2.5-Delta 26C, binds single-stranded DNA 40-fold more tightly than the wild-type protein and cannot physically interact with T7 DNA polymerase. gp2.5-Delta 26C is inhibitory for DNA synthesis catalyzed by T7 DNA polymerase on single-stranded DNA, and it does not stimulate strand displacement DNA synthesis at high concentration. The biochemical and genetic data support a model in which the carboxyl-terminal tail modulates DNA binding and mediates essential interactions with T7 DNA polymerase.     

TetL tetracycline efflux protein from Bacillus subtilis is a dimer in the membrane and in detergent solution

The TetL antiporter from the Bacillus subtilis inner membrane is a tetracycline-divalent cation efflux protein that is energized by the electrochemical proton gradient across the membrane. In this study, we expressed tetL in Escherichia coli and investigated the oligomeric state of TetL in the membrane and in detergent solution. Evidence for an oligomeric state of TetL emerged from SDS-PAGE and Western blot analysis of membrane samples as well as purified protein samples from cells that expressed two differently tagged TetL species. Furthermore, no formation or restoration of TetL oligomers occurred upon detergent solubilization of the membrane. Rather, oligomeric forms established in vivo persisted after solubilization. Mass spectrometry of the purified protein showed the absence of proteolysis and posttranslational modifications. Analytical size-exclusion chromatography of the purified protein revealed a dimeric TetL in dodecyl-maltoside solution. In addition, TetL dimers were found in a number of other detergents and over a wide pH range. It is therefore likely that the oligomeric form of the protein in the membrane is also a dimer.     

Prokaryote multidrug efflux proteins of the major facilitator superfamily: amplified expression, purification and characterisation

In bacterial genomes 3-12% of open reading frames are predicted to encode membrane transport proteins. These proteins can be vital for antibiotic efflux, protein/ toxin secretion, cell nutrition, environmental sensing, ATP synthesis, and other functions. Some, such as the multidrug efflux proteins, are potential targets for the development of new antibacterials and also for applications in biotechnology. In general membrane transport proteins are poorly understood, because of the technical difficulties involved in isolating sufficient protein for elucidation of their structure-activity relationships. We describe a general strategy for the amplified expression, purification and characterisation of prokaryotic multidrug efflux proteins of the 'Major facilitator superfamily' of transport proteins, using the Bacillus subtilis multidrug resistance protein, 'Bmr', as example.     

Gap junction-dependent and -independent EDHF-type relaxations may involve smooth muscle cAMP accumulation

We have compared the mechanisms that contribute to endothelium-derived hyperpolarizing factor (EDHF)-type responses induced by ACh and the Ca(2+) ionophore A-23187 in the rabbit iliac artery. Relaxations to both agents were associated with ~1.5-fold elevations in smooth muscle cAMP levels and were attenuated by the adenylyl cyclase inhibitor 2',5'-dideoxyadenosine (DDA) and potentiated by the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Mechanical responses were inhibited by coadministration of the Ca(2+)-activated K(+) channel blockers apamin and charybdotoxin, both in the absence and presence of IBMX, but were unaffected by blockade of ATP-sensitive K(+) channels with the sulphonylurea glibenclamide. Relaxations and elevations in cAMP evoked by ACh were abolished by 18alpha-glycyrrhetinic acid, which disrupts gap junction plaques, whereas the corresponding responses to A-23187 were unaffected by this agent. Consistently, in "sandwich" bioassay experiments, A-23187, but not ACh, elicited extracellular release of a factor that evoked relaxations that were inhibited by DDA and potentiated by IBMX. These findings provide evidence that EDHF-type relaxations of rabbit iliac arteries evoked by ACh and A-23187 depend on cAMP accumulation in smooth muscle, but involve signaling via myoendothelial gap junctions and the extracellular space, respectively.     

Chemiluminescent measurement of telomere DNA content in biopsies

Telomeres are nucleoprotein complexes that protect the ends of chromosomes from fusion and degradation. They are typically shorter in tumor cells than in paired normal cells, and shorter telomeres are associated with poor outcome in cancer. We previously described a slot blot-based methodfor measuring telomere DNA content, a proxy for telomere length. Although this method represented an improvement over existing methods, its 30-ng limit of sensitivity was insufficient for use with biopsy or other scant tissues. Here we describe a chemiluminescent slot blot assay for telomere DNA content that has the sensitivity required for use with biopsy materials. The results obtained with DNA derived from human placental, HeLa, human peripheral blood lymphocytes, sham-needle core prostate biopsies, and archival prostatectomy tissues demonstrated that telomere DNA content can be reliably and reproducibly measured in 5 ng, and sometimes as little as 2 ng, genomic DNA. Sham-needle core prostate biopsy and prostatectomy specimens processed in parallel produced comparable results. The contribution of truncated telomeres in admixtures containing as much as 75% normal placental DNA could be established. We also demonstrated that the treatment of tissue with formalin before DNA purification does not decrease the efficacy of the assay.     

Calcium/calmodulin-dependent protein kinase II phosphorylates and regulates the Drosophila eag potassium channel

Modulation of neuronal excitability is believed to be an important mechanism of plasticity in the nervous system. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been postulated to regulate the ether à go-go (eag) potassium channel in Drosophila. Inhibition of CaMKII and mutation of the eag gene both cause hyperexcitability at the larval neuromuscular junction (NMJ) and memory formation defects in the adult. In this study, we identify a single site, threonine 787, as the major CaMKII phosphorylation site in Eag. This site can be phosphorylated by CaMKII both in a heterologous cell system and in vivo at the larval NMJ. Expression of Eag in Xenopus oocytes was used to assess the function of phosphorylation. Injection of either a specific CaMKII inhibitor peptide or lavendustin C, another CaMKII inhibitor, reduced Eag current amplitude acutely. Mutation of threonine 787 to alanine also reduced amplitude. Moreover, both CaMKII inhibition and the alanine mutation accelerated inactivation. The reduction in current amplitudes and the accelerated inactivation of dephosphorylated Eag channels would result in decreased outward potassium currents and hyperexcitability at presynaptic terminals and, thus, are consistent with the NMJ phenotype observed when CaMKII is inhibited. These results show that Eag is a substrate of CaMKII and suggest that direct modulation of potassium channels may be an important function of this kinase.     

Extra pair paternity in birds: a review of interspecific variation and adaptive function

The application of molecular genetic techniques has revolutionized our view of avian mating systems. Contrary to prior expectations, birds are only very rarely sexually monogamous, with 'extra-pair offspring' found in approximately 90% of species. Even among socially monogamous species, over 11% of offspring are, on average, the result of extra-pair paternity (EPP). Based on over 150 molecular genetic studies of EPP in birds, we review two topical areas: (i) ecological explanations for interspecific variation in the rate of EPP; and (ii) evidence bearing on the adaptive function of EPP. We highlight the remaining challenges of understanding the relative roles of genes and ecology in determining variation between taxa in the rate of extra paternity, and testing for differences between extra-pair offspring and those sired within-pair.