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.     

PALM and STORM: unlocking live-cell super-resolution

Live-cell fluorescence light microscopy has emerged as an important tool in the study of cellular biology. The development of fluorescent markers in parallel with super-resolution imaging systems has pushed light microscopy into the realm of molecular visualization at the nanometer scale. Resolutions previously only attained with electron microscopes are now within the grasp of light microscopes. However, until recently, live-cell imaging approaches have eluded super-resolution microscopy, hampering it from reaching its full potential for revealing the dynamic interactions in biology occurring at the single molecule level. Here we examine recent advances in the super-resolution imaging of living cells by reviewing recent breakthroughs in single molecule localization microscopy methods such as PALM and STORM to achieve this important goal.     

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.     

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.     

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.     

对侧皮质锁定螺钉与锁定螺钉治疗股骨远端骨折疗效比较

目的:比较对侧皮质锁定螺钉与锁定螺钉治疗股骨远端骨折的临床疗效。方法:回顾性分析自2013年5月至2016年8月诊治的52例股骨远端骨折患者,采用对侧皮质锁定螺钉+NCB接骨板内固定治疗26例(A组:对侧皮质锁定组),采用锁定螺钉+NCB接骨板内固定治疗26例(B组:锁定螺钉组)。记录两组手术出血量和手术时间、切口长度、内固定治疗后骨折愈合时间、内固定治疗后完全负重时间、内固定治疗后并发症发生率等,在每个随访节点对每位患者进行患肢的正侧位X线平片检查,末次随访时对患肢进行膝关节功能评分,采用美国特种外科医院膝关节评分标准评定患肢功能。骨折愈合的定义为活动时骨折处无痛且在骨折正侧位X线平片上可见到断端骨皮质骨痂连接。术后并发症包括:关节僵硬、内固定断裂、骨不连以及感染等。结果:本研究52例骨折均获得至少12个月的随访。两组在手术相关指标及切口愈合等方面均无明显差异(P均0.05)。在骨折愈合以及完全负重时间方面,A组均显著短于B组(P均0.05)。末次随访时52例患者患肢膝关节功能:A组:优18例,良5例,差4例,优良率88.5%;B组:优15例,良6例,中4例,差1例,优良率80.8%。两组对比A组优良率显著高于B组(P0.05)。两组并发症对比无明显差异:A组发生骨不连2例,骨折内固定断裂2例。B组发生骨不连3例,畸形愈合2例。结论:与传统锁定螺钉相比,对侧皮质锁定螺钉在骨折愈合时间、完全负重时间、术后患肢功能优良率方面具有优势,但在并发症发生率方面没有明显差异。对侧皮质锁定螺钉的治疗指征及自身强度还有待大样本、多中心的临床研究进一步明确。