guanosine-triphosphate and cyanine-dye-3

Release factors RF1 and RF2 promote hydrolysis of peptidyl-tRNA during translation termination. The GTPase RF3 promotes recycling of RF1 and RF2. Using single molecule FRET and biochemical assays, we show that ribosome termination complexes that carry two factors, RF1-RF3 or RF2-RF3, are dynamic and fluctuate between non-rotated and rotated states, whereas each factor alone has its distinct signature on ribosome dynamics and conformation. Dissociation of RF1 depends on peptide release and the presence of RF3, whereas RF2 can dissociate spontaneously. RF3 binds in the GTP-bound state and can rapidly dissociate without GTP hydrolysis from termination complex carrying RF1. In the absence of RF1, RF3 is stalled on ribosomes if GTP hydrolysis is blocked. Our data suggest how the assembly of the ribosome-RF1-RF3-GTP complex, peptide release, and ribosome fluctuations promote termination of protein synthesis and recycling of the release factors.

Topics: Carbocyanines; Escherichia coli; Escherichia coli Proteins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Guanosine Triphosphate; Hydrolysis; Kinetics; Peptide Chain Termination, Translational; Peptide Termination Factors; Protein Binding; Protein Conformation; Ribosomes; RNA, Transfer, Amino Acyl; Single Molecule Imaging; Thermodynamics

Fluorescent imaging of cytoskeletal structures permits studies of both organization within the cell and dynamic reorganization of the cytoskeleton itself. Traditional fluorescent labels of microtubules, part of the cytoskeleton, have been used to study microtubule localization, structure, and dynamics, both in vivo and in vitro. However, shortcomings of existing labels make imaging of microtubules with high precision light microscopy difficult. In this paper, we report a new fluorescent labeling technique for microtubules, which involves a GTP analog modified with a bright, organic fluorophore (TAMRA, Cy3, or Cy5). This fluorescent GTP binds to a specific site, the exchangeable site, on tubulin in solution with a dissociation constant of 1.0±0.4 µM. Furthermore, the label becomes permanently incorporated into the microtubule lattice once tubulin polymerizes. We show that this label is usable as a single molecule fluorescence probe with nanometer precision and expect it to be useful for modern subdiffraction optical microscopy of microtubules and the cytoskeleton.

Topics: Animals; Binding Sites; Carbocyanines; Cattle; Fluorescent Dyes; Guanosine Triphosphate; Microtubules; Rhodamines; Staining and Labeling; Tubulin

Recent structural data have revealed two distinct conformations of the ribosome during initiation. We employed single-molecule fluorescence methods to probe the dynamic relation of these ribosomal conformations in real time. In the absence of initiation factors, the ribosome assembles in two distinct conformations. The initiation factors guide progression of the ribosome to the conformation that can enter the elongation cycle. In particular, IF2 both accelerates the rate of subunit joining and actively promotes the transition to the elongation-competent conformation. Blocking GTP hydrolysis by IF2 results in 70S complexes formed in the conformation unable to enter elongation. We observe that rapid GTP hydrolysis by IF2 drives the transition to the elongation-competent conformation, thus committing the ribosome to enter the elongation cycle.

Topics: Carbocyanines; Eukaryotic Initiation Factor-2; Fluorescence Resonance Energy Transfer; Guanosine Triphosphate; Hydrolysis; Kinetics; Models, Molecular; Peptide Chain Initiation, Translational; Peptide Initiation Factors; Prokaryotic Initiation Factor-2; Protein Binding; Protein Conformation; Ribosomes; RNA, Messenger; RNA, Transfer

Single-molecule imaging techniques were used to reveal the binding of individual cyclic adenosine 3',5'-monophosphate molecules to heterotrimeric guanine nucleotide-binding protein coupled receptors on the surface of living Dictyostelium discoideum cells. The binding sites were uniformly distributed and diffused rapidly in the plane of the membrane. The probabilities of individual association and dissociation events were greater for receptors at the anterior end of the cell. Agonist-induced receptor phosphorylation had little effect on any of the monitored properties, whereas G protein coupling influenced the binding kinetics. These observations illustrate the dynamic properties of receptors involved in gradient sensing and suggest that these may be polarized in chemotactic cells.

Topics: Animals; Carbocyanines; Cell Membrane; Chemotaxis; Cyclic AMP; Dictyostelium; Diffusion; Guanosine Diphosphate; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Kinetics; Microscopy, Fluorescence; Mutation; Phosphorylation; Pseudopodia; Receptors, Cyclic AMP; Signal Transduction