Perturbation of the tRNA tertiary core differentially affects specific steps of the elongation cycle

The tRNA tertiary core region is important for both tRNA stability and activity in the translation elongation cycle. Here we report the effects of mutating each of two highly conserved base pairs in the tertiary core of Phe-tRNA(Phe), 18-55 and 19-56, on rate and equilibrium constants for specific steps of this cycle, beginning with formation of aminoacyl-tRNA.EF-Tu.GTP ternary complexs and culminating with translocation of A-site-bound peptidyl-tRNA into the P-site. We find that codon-dependent binding of aminoacyl-tRNA to the A/T-site and proofreading of near-cognate tRNA are sensitive to perturbation of either base pair; formation of the ternary complex and accommodation from the A/T to the A-site are sensitive to 18-55 perturbation only, and translocation of peptidyl-tRNA from the A- to P-site is insensitive to perturbation of either. These results underline the importance of the core region in promoting the efficiency and accuracy of translation, and they likely reflect different requirements for structural integrity of the core during specific steps of the elongation cycle.     

何首乌中新的二苯乙烯甙

用反相层析法从何首乌(Polygonum multiflorum Thunb.)根茎的水提物中分得2个新的二苯乙烯甙(1、 2)及9个已知化合物:没食子酸(3)、 2,6-二羟基-苯甲酸(4)、吲哚-3-(L-α-氨基-α-羟基-丙酸)甲酯(5)、 1, 2-二羟基丙烷-1-(4-羟基-苯基) (6)、大黄素(7)、大黄素-8-O-β-D-葡萄糖甙(8)、(+)-lyoniresinol-3α-O-β-D-葡萄糖甙(9)、 2, 3, 4′, 5-四羟基反式二苯乙烯-2-O-β-D-吡喃葡萄糖甙(10)和 2, 3, 4′, 5-四羟基反式二苯乙烯-2, 3-二-O-β-D-吡喃葡萄糖甙(11).新化合物结构通过理化性质与波谱分析特别是 2D NMR得以确定.化合物2表现出很强的DNA裂解活性,化合物1、2与10具有很强的抗脂质过氧化活性.     

Distinct kinetic determinants for the stepwise CCA addition to tRNA

The universally conserved CCA sequence is present at the 3′ terminal 74–76 positions of all active tRNA molecules as a functional tag to participate in ribosome protein synthesis. The CCA enzyme catalyzes CCA synthesis in three sequential steps of nucleotide addition at rapid and identical rates. However, the kinetic determinant of each addition is unknown, thus limiting the insights into the kinetic basis of CCA addition. Using our recently developed single turnover kinetics of Escherichia coli CCA enzyme as a model, we show here that the identical rate of the stepwise CCA addition is determined by distinct kinetic parameters. Specifically, the kinetics of C74 and C75 addition is controlled by the chemistry of nucleotidyl transfer, whereas the kinetics of A76 addition is controlled by a prechemistry conformational transition of the active site. In multiple turnover condition, all three steps are controlled by slow product release, indicating enzyme processivity from one addition to the next. However, the processivity decreases as the enzyme progresses to complete the CCA synthesis. Together, these results suggest the existence of a network of diverse kinetic parameters that determines the overall rate of CCA addition for tRNA maturation.     

Mechanism of N-methylation by the tRNA m1G37 methyltransferase Trm5

Trm5 is a eukaryal and archaeal tRNA methyltransferase that catalyzes methyl transfer from S-adenosylmethionine (AdoMet) to the N(1) position of G37 directly 3' to the anticodon. While the biological role of m(1)G37 in enhancing translational fidelity is well established, the catalytic mechanism of Trm5 has remained obscure. To address the mechanism of Trm5 and more broadly the mechanism of N-methylation to nucleobases, we examined the pH-activity profile of an archaeal Trm5 enzyme, and performed structure-guided mutational analysis. The data reveal a marked dependence of enzyme-catalyzed methyl transfer on hydrogen ion equilibria: the single-turnover rate constant for methylation increases by one order of magnitude from pH 6.0 to reach a plateau at pH 7.0. This suggests a mechanism involving proton transfer from G37 as the key element in catalysis. Consideration of the kinetic data in light of the Trm5-tRNA-AdoMet ternary cocrystal structure, determined in a precatalytic conformation, suggests that proton transfer is associated with an induced fit rearrangement of the complex that precedes formation of the reactive configuration in the active site. Key roles for the conserved R145 side chain in stabilizing a proposed oxyanion at G37-O(6), and for E185 as a general base to accept the proton from G37-N(1), are suggested based on the mutational analysis.     

Heterogeneity of the Ca2+ sensitivity of secretion in a pituitary gonadotrope cell line and its modulation by protein kinase C and Ca2+

Modulation of the Ca2+ sensitivity and cooperativity of secretion is an important means of regulating neurotransmission and hormone secretion. Employing high-time resolution measurement of membrane capacitance (Cm) stimulated by step-like or ramp [Ca2+]i elevation, we have identified the co-existence of both a high and low Ca2+-sensitive exocytosis in an immortal pituitary gonadotrope cell line, LbetaT2. Ramp [Ca2+]i generated by slow uncaging elicited a biphasic C(m) response. The first phase of response, which represents a highly Ca2+-sensitive pool (HCSP) of vesicles, began to secrete at low [Ca2+]i concentration (<1 microM) with low Ca2+ cooperativity. In contrast, the second phase, which represents a lowly Ca2+-sensitive pool (LCSP) of vesicles, only exocytozed at higher [Ca2+]i (>5 microM) and displayed a steep Ca2+ cooperativity. The co-existence of vesicle populations with different Ca2+ sensitivities was further confirmed by flash photolysis stimuli. The size of the HCSP was approximately 30 fF under resting conditions, but was dramatically increased (approximately threefold) by application of phorbol-12-myristate-13-acetate (PMA, an activator of protein kinase C). Forskolin (an activator of protein kinase A), however, exerted no significant effect on the size of both HCSP and LCSP. GnRH (gonadotropin releasing hormone) augmented the size of both pools to a larger extent (5- and 1.7-fold increase for HCSP and LCSP, respectively). The heterogeneity of Ca2+ sensitivity from different pools of vesicles and its differential modulation by intracellular signals suggests that LbetaT2 cells are an ideal model to further unravel the mechanism underlying the modulation of Ca2+-sensing machineries for exocytosis.     

Rapid ribosomal translocation depends on the conserved 18-55 base pair in P-site transfer RNA

The L shape of tRNA is stabilized by the 'tertiary core' region, which contains base-pairing interactions between the D and T loops. Distortions of the L shape accompany tRNA movement across the ribosomal surface. Here, using single-turnover rapid kinetics assays, we determine the effects of mutations within the tertiary core of P site-bound tRNA(fMet) on three measures of the rate of translocation, the part of the elongation cycle involving the most extensive tRNA movement. Mutations in the strictly conserved G18.U55 base pair result in as much as an 80-fold decrease in the rate of translocation, demonstrating the importance of the 18-55 interaction for rapid translocation. This implicates the core region as a locus for functionally important dynamic interactions with the ribosome and leads to the proposal that translocation of ribosome-bound tRNAs may be sequential rather than concerted.     

Biological Comparison of Two Genotypes of Helicoverpa armigera Single-Nucleocapsid Nucleopolyhedrovirus

Baculovirus isolates from the same host species often show a considerable degree of variation on phenotypes. The completely sequenced genotypes C1 and G4 of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) were compared. Bioassay studies suggested that nearly double of HaSNPV G4 virus was required compared with HaSNPV C1 to achieve a similar LD₅₀, and at the LD₉₀ level the insect-killing speed for HaSNPV C1 was quicker than that of HaSNPV G4. The budded virus (BV) production of HaSNPV C1 was nearly two- to threefold higher at 24 and 48 h post-infection (p.i.) than that of HaSNPV G4. However, the kinetics of polyhedral inclusion body (PIB) formation in HzAM1 cells was similar in both the genotypes, which implied that the insect-killing speed was not influenced by PIB formation, but by the kinetics of BV production. The results suggested that the HaSNPV C1 isolate was a better choice than HaSNPV G4 virus for controlling H. armigera.     

Generation and characterization of 11 novel est derived microsatellites from Vicia faba (Fabaceae)

? Premise of the study: Simple sequence repeat (SSR) markers were developed for faba bean using expressed sequence tags (ESTs) from the NCBI database to study for genetic diversity. ? Methods and Results: A total of 11 novel EST-SSR loci were generated and characterized when tested on four populations of 29 faba bean individuals from China and Europe. The number of alleles (A) ranged from 1 to 3 in each population, and observed heterozygosity (H(O)) and expected heterozygosity (H(E)) ranged from 0 to 0.5000 and 0.6400, respectively. Furthermore, transferable analysis revealed that eight of these loci (72.73%) amplified in Pisum sativum L., six of which (75.00%) detected polymorphism. ? Conclusions: The developed markers in this study will provide valuable tools for genetic diversity, resource conservation, genetic mapping, and marker-assisted breeding of faba bean in the future.