In vitro ribosomal ribonucleoprotein transport. Temperature-induced "graded unlocking" of nuclei

We examine the effect of temperature on the export of ribosomal precursor particles from nuclei isolated from Tetrahymena. A new phenomenon is observed. Temperature does affect not only the export rate, but also the maximal portion of particles exported. At 8 degrees C, for example, the export kinetics reveals a significantly lower saturation plateau which does not equilibrate with the higher plateau at 28 degrees C even after 3 h. This nonequilibration is not due to (i) a different physical quality of the exported particles, (ii) a degradation of the nuclear rRNA, (iii) a backward import of exported particles into nuclei, (iv) an irreversible inactivation of potentially transportable nuclear ribosomal ribonucleoprotein (rRNP) particles, or (v) a thermodynamic equilibrium between transportable rRNP particles associated with nuclei and those exported from nuclei. We conclude, therefore, that potentially transportable rRNP particles are somehow "locked" in nuclei at low temperature and temperature raising induces a "graded unlocking."     

Comparison of tRNA motions in the free and ribosomal bound structures

A general method is presented that allows the separation of the rigid body motions from the nonrigid body motions of structural subunits when bound in a complex. The application presented considers the motions of the tRNAs: free, bound to the ribosome and to a synthase. We observe that both the rigid body and nonrigid body motions of the structural subunits are highly controlled by the large ribosomal assembly and are important for the functional motions of the assembly. For the intact ribosome, its major parts, the 30S and the 50S subunits, are found to have counterrotational motions in the first few slowest modes, which are consistent with the experimentally observed ratchet motion. The tRNAs are found to have on average approximately 72-75% rigid body motions and principally translational motions within the first 100 slow modes of the complex. Although the three tRNAs exhibit different apparent total motions, after the rigid body motions are removed, the remaining internal motions of all three tRNAs are essentially the same. The direction of the translational motions of the tRNAs are in the same direction as the requisite translocation step, especially in the first slowest mode. Surprisingly the small intrinsically flexible mRNA has all of its internal motions completely inhibited and shows mainly a rigid-body translation in the slow modes of the ribosome complex. On the other hand, the required nonrigid body motions of the tRNA during translocation reveal that the anticodon-stem-loop, as well as the acceptor arm, of the tRNA enjoy a large mobility but act as rigid structural units. In summary, the ribosome exerts its control by enforcing rigidity in the functional parts of the tRNAs as well as in the mRNA.     

Large-scale motions within ribosomal 50S subunits as demonstrated using photolabile oligonucleotides

Photolabile oligonucleotides (PHONTs) bind to rRNA sequences to which they are complementary and, on photolysis, incorporate into neighboring ribosomal components. Here we report on photocrosslinking results obtained with PHONTs targeting 23S rRNA nucleotides 1882-1892, in the long lateral arm of the 50S subunit (PHONT 1892), and 1085-1093, in the L11 binding domain (PHONT 1093). Photolysis of the PHONT 1892.50S and PHONT 1093.50S complexes leads to formation of 'long-range' crosslinks from C1892 to U1094/A1095 and G1950, and from G1093 to U1712/1716 and U1926, that are clearly incompatible with published crystal structures of 50S subunits. These results provide strong evidence that within the 50S subunit (a) the L11 binding domain can extend in an arm-like fashion, accessing large areas of the ribosome, and (b) the lateral arm can bend about the noncanonical helix at its center. Such motions may have functional relevance in identifying regions that undergo major conformational change as the ribosome moves through its catalytic cycle.     

Plant genetics: unlocking the secrets of self-incompatibility

At last, clear evidence has been obtained, from transformation of the pollen incompatibility reaction of Brassica, showing that angiosperm self-incompatibility involves separate genes for the pollen and pistil incompatibility recognition processes.     

Sirtuins: a conserved key unlocking AceCS activity

Bacterial acetyl-coenzyme A (acetyl-CoA) synthetase (AceCS), an evolutionarily conserved enzyme that converts acetate to acetyl-CoA, is activated by sirtuin-mediated deacetylation. Two recent studies show that this mechanism of regulation is also crucial for mammalian AceCS activity, indicating that control of metabolism at the step of converting acetate to acetyl-CoA is conserved. These findings highlight a metabolic regulatory network controlled by sirtuins that has implications for the mechanisms of calorie restriction and modulation of mammalian lifespan.     

Redundancy, insult-specific sensors and thresholds: unlocking the S-phase checkpoint response

DNA damage that is not properly repaired during genomic replication is a major source of gross chromosomal rearrangements and sequence loss during cell proliferation. In higher eukaryotes such mutations increase the risk of cancer. Eukaryotic cells have multiple checkpoint responses activated by DNA damage and stalled replication forks. We focus here on fork-associated events that activate and respond to S-phase checkpoint kinases.     

Phosphorylation of histone variant regions in chromatin: unlocking the linker?

Histone variants illuminate the behavior of chromatin through their unique structures and patterns of postsynthetic modification. This review examines the literature on heteromorphous histone structures in chromatin, structures that are primary targets for histone kinases and phosphatases in vivo. Special attention is paid to certain well-studied experimental systems: mammalian culture cells, chicken erythrocytes, sea urchin sperm, wheat sprouts, Tetrahymena, and budding yeast. A common theme emerges from these studies. Specialized, highly basic structures in histone variants promote chromatin condensation in a variety of developmental situations. Before, and sometimes after condensed chromatin is formed, the chromatin is rendered soluble by phosphorylation of the heteromorphous regions, preventing their interaction with linker DNA. A simple structural model accounting for histone variation and phosphorylation is presented.     

Local motions of fluorophores

Summary We further describe the general formulation of fluorescence depolarization in which the depolarization results from exchanges between a number of oscillator orientations in thermal equilibrium. Temperature and pressure affect the polarization by changing the relative populations of the allowed orientations as well as the rate of exchanges during the fluorescence lifetime. This treatment satisfactorily describes the limited motions that fluorophores undergo when they are either attached to a macromolecule such as the local tryptophan rotations in proteins, or embedded in a biological membrane.     

Profiling hydroxycinnamoyl-coenzyme A thioesters: unlocking the back door of phenylpropanoid metabolism

In plants, 20 to 30% of photosynthetically fixed carbon is directed toward lignin and other phenylpropanoid compounds for which hydroxycinnamoyl-coenzyme A (CoA) esters are key intermediates. CoA thioesters, ubiquitous metabolites found in all living cells (often at trace levels), have traditionally been challenging to measure. Here we report a hydrophilic interaction liquid chromatography (HILIC) method, coupled with tandem mass spectrometry (MS/MS), that allows simultaneous sensitive quantification of previously undetectable hydroxycinnamoyl-CoA esters and an extended range of acyl-CoAs from plant tissues. This method provides rapid liquid chromatography (LC) analysis (10 min/sample) and the ability for qualitative assessment of acyl-CoAs by MS/MS precursor ion scanning.     

Mass spectrometric analysis of formalin-fixed paraffin-embedded tissue: unlocking the proteome within

The predominance of tissues stored worldwide in hospitals and clinical laboratories exist in formalin-fixed paraffin-embedded (FFPE) blocks that are generated by simple and well-established protocols. Although generation of FFPE tissues has facilitated their characterization by such techniques as histopathology, they have proven refractory to biomarker discovery investigations using state-of-the-art MS-based proteomic methodologies. Very recently new methods have been developed that enable proteins extracted from FFPE tissues to be analyzed by MS. This review will highlight and discuss those efforts that have led to this exciting recent progress. Although these developments are quite new, the ability to conduct MS-based proteomic analyses of FFPE tissues opens heretofore intractable clinical samples for discovery-based biomarker research.     

Passive deformations and active motions of leukocytes

The purpose of this paper is to review the development of continuum mechanics models of single leukocytes in both passive deformations and active motions and to indicate some future directions. Models of passive deformations describe the overall rheological behavior of single leukocytes under externally applied forces and predict the average mechanical properties from experimental data. Various "apparent" viscoelastic coefficients are obtained depending on the models assumed and the types of test used. Models of spontaneous motions postulate active driving mechanisms which must be derived internally from the cell itself and probably have different bases for different kind of motions. For pseudopod protrusion on leukocytes, energy transduction from chemical potential to mechanical work associated with actin polymerization at the tip of the projection is assumed to supply the motive power. For pseudopod retraction, active contraction due to actin-myosin interaction is assumed to be the driving force. The feasibility of the hypotheses are tested via numerical examples and comparison of the theoretical results with experimental measurements.