Activation and inhibition of GTPase translation factors on the prokaryotic ribosome

Throughout all domains of life, each protein in a cell is synthesized by a remarkable biomolecular machine called the ribosome, in a process referred to as translation. This process is regulated by proteins called translation factors, several of which belong to the GTPase superfamily of enzymes which require the binding and subsequent hydrolysis of guanosine 5\u27-triphosphate (GTP) to execute their function. In contrast to the regulatory role of translation factors, protein biosynthesis is inhibited by several naturally occurring antibiotics. While our understanding of translation has been revolutionized by the recent elucidation of atomic-resolution x-ray crystal structures of the ribosome trapped in various intermediate conformations, several crucial aspects of protein biosynthesis remain poorly understood, such as the identity of the molecular component of the ribosome which stimulates the activation of the translational GTPases, as well as the mechanism by which several antibiotics inhibit translation. The major aims of this work are twofold. First, investigations directed towards the elucidation of the ribosomal element responsible for GTPase activation are described. It is demonstrated that the depletion of a specific protein from the ribosome which is part of the GTPase binding site, L12, results in significant attenuation of ribosome-dependent GTP hydrolysis activity by translational GTPases IF2, EF-G, LepA, and RF3, and this lost activity is fully restored by preincubating L12-depleted ribosomes with purified L12 protein. However, L12 alone does not stimulate GTP hydrolysis by these GTPases, in contrast to a previous report (Savelsbergh et al., 2000). In fact, it is shown that none of the isolated rRNA or protein components which comprise the ribosomal GTPase binding region stimulate GTP hydrolysis by the translational GTPases, implying that the peripheral ribosomal architecture is needed for correct positioning of the GTPase-activating element of the ribosome. A second major goal of this work was to investigate the inhibitory mechanism of the antibiotic thiostrepton, which is known to interfere with the function of elongation factor EF-G, and has been recently shown to inhibit the growth of the malarial parasite Plasmodium falciparum. Many lines of evidence reported herein contradict the current predominantly accepted model of thiostrepton action. It is shown that thiostrepton strongly inhibits ribosome-dependent GTP hydrolysis by EF-G and a closely related GTPase LepA, and this is explained by results which indicate that thiostrepton obstructs the binding of these factors to the ribosome. Interestingly, an engineered mutant of EF-G lacking domains IV and V is insensitive to thiostrepton, which is in agreement with recent structural evidence which suggests that thiostrepton interferes with the interaction between domain V of EF-G and the ribosome

Ecology of an Isolated Muskrat Population During Regional Population Declines

Evidence indicating a decline in Ondatra zibethicus (Muskrat) populations in the United States during the past 40 years has led to speculation regarding factors influencing Muskrat survival. In order to understand population dynamics and survival, it is important to first define the ecology of local populations. We investigated the dwelling structure use, movements, home range, and survival of radio-tagged Muskrats (n = 14) in an urban wetland complex in central Pennsylvania. We used locations collected from intensive radio-telemetry monitoring to determine number of lodging structures used, hourly movement, and size and percent area overlap of home ranges. Muskrats shared an average of 9 lodging structures, and on average, 68% of a Muskrat’s home range overlapped home ranges of other Muskrats. We used 4 home-range estimators (kernel density estimator [KDE]href, KDEad hoc, KDEplug-in, and local convex hull estimator) to assess the ability of each estimator to represent Muskrat home ranges. The KDEplug-in that constrained the estimate of home range to habitat boundaries provided the most appropriate home-range size for Muskrats in a linear–non-linear habitat matrix. We also calculated overwinter survival estimates using known-fate models. Our top model indicated a positive effect of the average weekly precipitation on survival, with an overwinter survival estimate of 0.59 (SE = 0.16). The main cause of Muskrat mortality was predation by Neovison vison (American Mink; n = 6). The small sample size and uncertainty surrounding our model selection led to weak estimates of survival; however, our model suggests that snowfall may be an important factor in Muskrat survival. Our study provides novel data on Muskrat ecology in Pennsylvania as well as preliminary evidence for future investigations of factors affecting Muskrat survival during the winter months

Expression, Purification, and Analysis of Unknown Translation Factors from Escherichia coli: A Synthesis Approach

New approaches are currently being developed to expose biochemistry and molecular biology undergraduates to a more interactive learning environment. Here, we propose a unique project-based laboratory module, which incorporates exposure to biophysical chemistry approaches to address problems in protein chemistry. Each of the experiments described herein contributes to the stepwise process of isolating, identifying, and analyzing a protein involved in a central biological process, prokaryotic translation. Students are provided with expression plasmids that harbor an unknown translation factor, and it is their charge to complete a series of experiments that will allow them to develop hypotheses for discovering the identity of their unknown (from a list of potential candidates). Subsequent to the identification of their unknown translation factor, a series of protein unfolding exercises are performed employing circular dichroism and fluorescence spectroscopies, allowing students to directly calculate thermodynamic parameters centered around determining the equilibrium constant for unfolding as a function of denaturant (temperature or chemical). The conclusion of this multi-part laboratory exercise consists of both oral and written presentations, emphasizing synthesis of the roles of each translation factor during the stepwise process of translation

Radar Detection of High Concentrations of Ice Particles - Methodology and Preliminary Flight Test Results

High Ice Water Content (HIWC) has been identified as a primary causal factor in numerous engine events over the past two decades. Previous attempts to develop a remote detection process utilizing modern commercial radars have failed to produce reliable results. This paper discusses the reasons for previous failures and describes a new technique that has shown very encouraging accuracy and range performance without the need for any hardware modifications to industrys current radar designs. The performance of this new process was evaluated during the joint NASA/FAA HIWC RADAR II Flight Campaign in August of 2018. Results from that evaluation are discussed, along with the potential for commercial application, and development of minimum operational performance standards for a future commercial radar product

Infrared surface plasmons on heavily doped silicon

Conductors with infrared plasma frequencies are potentially useful hosts of surface plasmon polaritons (SPP) with sub-wavelength mode confinement for sensing applications. A challenge is to identify such a conductor that also has sharp SPP excitation resonances and the capability to be functionalized for biosensor applications. In this paper we present experimental and theoretical investigations of IR SPPs on doped silicon and their excitation resonances on doped-silicon gratings. The measured complex permittivity spectra for p-type silicon with carrier concentration 6 x 10(19) and 1 x 10(20) cm(-3) show that these materials should support SPPs beyond 11 and 6 mu m wavelengths, respectively. The permittivity spectra were used to calculate SPP mode heights above the silicon surface and SPP propagation lengths. Reasonable merit criteria applied to these quantities suggest that only the heaviest doped material has sensor potential, and then mainly within the wavelength range 6 to 10 mu m. Photon-to-plasmon coupling resonances, a necessary condition for sensing, were demonstrated near 10 mu m wavelength for this material. The shape and position of these resonances agree well with simple analytic calculations based on the theory of Hessel and Oliner (1965)

Summary of the High Ice Water Content (HIWC) RADAR Flight Campaigns

NASA and the FAA conducted two flight campaigns to quantify onboard weather radar measurements with in-situ measurements of high concentrations of ice crystals found in deep convective storms. The ultimate goal of this research was to improve the understanding and develop onboard weather radar processing to detect regions of high ice water content ahead of an aircraft and enable tactical avoidance of the potentially hazardous conditions. Both High Ice Water Content (HIWC) RADAR campaigns utilized the NASA DC-8 Airborne Science Laboratory which was equipped with a Honeywell RDR-4000 weather radar and icing instruments to characterize the ice crystal clouds. The purpose of this paper is to summarize how these campaigns were conducted and highlight key results