Role of a ribosomal RNA phosphate oxygen during the EF-G-triggered hydrolysis

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. (1) In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

The ribosome-in-pieces: Binding of elongation factor EF-G to oligoribonucleotides that mimic the sarcin/ricin and thiostrepton domains of 23S ribosomal RNA

An oligoribonucleotide (a 27-mer) that mimics the sarcin/ricin (S/R) domain of Escherichia coli 23S rRNA binds elongation factor EF-G; the Kd is 6.9 μM, whereas for binding to ribosomes it is 0.7 μM. Binding saturates when EF-G and the S/R RNA are equimolar; at saturation 70% of the input RNA is in complexes with EF-G. Binding of EF-G to S/R RNA does not require GTP but is inhibited by GDP; the inhibition by GDP is overcome by GTP. The effects of mutations of the S/R domain nucleotides G2655, A2660, and G2661 suggest that EF-G recognizes the conformation of the RNA rather than the identity of the nucleotides. EF-G also binds to an oligoribonucleotide (an 84-mer) that has the thiostrepton region of 23S rRNA; however, EF-G binds independently to S/R and thiostrepton oligoribonucleotides.

The EF-hand Ca2+-binding Protein p22 Associates with Microtubules in an N-Myristoylation–dependent Manner

Proteins containing the EF-hand Ca2+-binding motif, such as calmodulin and calcineurin B, function as regulators of various cellular processes. Here we focus on p22, an N-myristoylated, widely expressed EF-hand Ca2+-binding protein conserved throughout evolution, which was shown previously to be required for membrane traffic. Immunofluorescence studies show that p22 distributes along microtubules during interphase and mitosis in various cell lines. Moreover, we report that p22 associates with the microtubule cytoskeleton indirectly via a cytosolic microtubule-binding factor. Gel filtration studies indicate that the p22–microtubule-binding activity behaves as a 70- to 30-kDa globular protein. Our results indicate that p22 associates with microtubules via a novel N-myristoylation–dependent mechanism that does not involve classic microtubule-associated proteins and motor proteins. The association of p22 with microtubules requires the N-myristoylation of p22 but does not involve p22’s Ca2+-binding activity, suggesting that the p22–microtubule association and the role of p22 in membrane traffic are functionally related, because N-myristoylation is required for both events. Therefore, p22 is an excellent candidate for a protein that can mediate interactions between the microtubule cytoskeleton and membrane traffic.

The Relationship between Photosynthesis and a Mastoparan-Induced Hypersensitive Response in Isolated Mesophyll Cells1

The G-protein activator mastoparan (MP) was found to elicit the hypersensitive response (HR) in isolated Asparagus sprengeri mesophyll cells at micromolar concentrations. The HR was characterized by cell death, extracellular alkalinization, and an oxidative burst, indicated by the reduction of molecular O2 to O2⋅−. To our knowledge, this study was the first to monitor photosynthesis during the HR. MP had rapid and dramatic effects on photosynthetic electron transport and excitation energy transfer as determined by variable chlorophyll a fluorescence measurements. A large increase in nonphotochemical quenching of chlorophyll a fluorescence accompanied the initial stages of the oxidative burst. The minimal level of fluorescence was also quenched, which suggests the origin of this nonphotochemical quenching to be a decrease in the antenna size of photosystem II. In contrast, photochemical quenching of fluorescence decreased dramatically during the latter stages of the oxidative burst, indicating a somewhat slower inhibition of photosystem II electron transport. The net consumption of O2 and the initial rate of O2 uptake, elicited by MP, were higher in the light than in the dark. These data indicate that light enhances the oxidative burst and suggest a complex relationship between photosynthesis and the HR.

Role of essential light chain EF hand domains in calcium binding and regulation of scallop myosin.

The specific Ca2+ binding site that triggers contraction of molluscan muscle requires the presence of an essential light chain (ELC) from a Ca2+ binding myosin. Of the four EF hand-like domains in molluscan ELCs, only domain III has an amino acid sequence predicted to be capable of binding Ca2+. In this report, we have used mutant ELCs to locate the Ca2+ binding site in scallop myosin and to probe the role of the ELC in regulation. Point mutations in domain III of scallop ELC have no effect on Ca2+ binding. Interestingly, scallop and rat cardiac ELC chimeras support Ca2+ binding only if domain I is scallop. These results are nevertheless in agreement with structural studies on a proteolytic fragment of scallop myosin, the regulatory domain. Furthermore, Ca2+ sensitivity of the scallop myosin ATPase requires scallop ELC domain I: ELCs containing cardiac domain I convert scallop myosin to an unregulated molecule whose activity is no longer repressed in the absence of Ca2+. Despite its unusual EF hand domain sequence, our data indicate that the unique and required contribution of molluscan ELCs to Ca2+ binding and regulation of molluscan myosins resides exclusively in domain I.

Educación Física y su Didáctica. Tema 7. La evaluación en el área de EF

La evaluación en el área de educación física en primaria.

Traethawd ar y Sabboth: gyd ag amryw annogaethau a chyfarwyddiadau i'w sancteiddio ef.

Mode of access: Internet.

Hanes am y diweddar Barch.Richard Jones,Bala, wedi ei ysgrifenu ganddo ef ei hun;a sylwedd rhai o'i bregethau;y'nghyda sylwadau ar ei nodweddiad.

Mode of access: Internet.

Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation

The universally conserved translation elongation factor EF-Tu delivers aminoacyl(aa)-tRNA in the form of an aa-tRNA·EF-Tu·GTP ternary complex (TC) to the ribosome where it binds to the cognate mRNA codon within the ribosomal A-site, leading to formation of a pretranslocation (PRE) complex. Here we describe preparation of QSY9 and Cy5 derivatives of the variant E348C-EF-Tu that are functional in translation elongation. Together with fluorophore derivatives of aa-tRNA and of ribosomal protein L11, located within the GTPase associated center (GAC), these labeled EF-Tus allow development of two new FRET assays that permit the dynamics of distance changes between EF-Tu and both L11 (Tu-L11 assay) and aa-tRNA (Tu-tRNA assay) to be determined during the decoding process. We use these assays to examine: (i) the relative rates of EF-Tu movement away from the GAC and from aa-tRNA during decoding, (ii) the effects of the misreading-inducing antibiotics streptomycin and paromomycin on tRNA selection at the A-site, and (iii) how strengthening the binding of aa-tRNA to EF-Tu affects the rate of EF-Tu movement away from L11 on the ribosome. These FRET assays have the potential to be adapted for high throughput screening of ribosomal antibiotics.

Posterior Probability Modeling and Image Classification for Archaeological Site Prospection: Building a Survey Efficacy Model for Identifying Neolithic Felsite Workshops in the Shetland Islands

The application of custom classification techniques and posterior probability modeling (PPM) using Worldview-2 multispectral imagery to archaeological field survey is presented in this paper. Research is focused on the identification of Neolithic felsite stone tool workshops in the North Mavine region of the Shetland Islands in Northern Scotland. Sample data from known workshops surveyed using differential GPS are used alongside known non-sites to train a linear discriminant analysis (LDA) classifier based on a combination of datasets including Worldview-2 bands, band difference ratios (BDR) and topographical derivatives. Principal components analysis is further used to test and reduce dimensionality caused by redundant datasets. Probability models were generated by LDA using principal components and tested with sites identified through geological field survey. Testing shows the prospective ability of this technique and significance between 0.05 and 0.01, and gain statistics between 0.90 and 0.94, higher than those obtained using maximum likelihood and random forest classifiers. Results suggest that this approach is best suited to relatively homogenous site types, and performs better with correlated data sources. Finally, by combining posterior probability models and least-cost analysis, a survey least-cost efficacy model is generated showing the utility of such approaches to archaeological field survey.