Molecular flexibility and discontinuous translocation of a non-templated polymerase

Little is known regarding the translocation of non-templated nucleic acid polymerases with respect to single-stranded primers. VP55, the vaccinia virus poly(A) polymerase, translocates as it processively adds a approximately 3-7 adenylate tail to primers possessing only three ribouridylate residues (as an (rU)(2)-N(15)-rU motif), and a approximately 25-30 adenylate tail to primers that are more U-rich. Here, three models were addressed for the translocation of VP55 with respect to its primer, namely: (a) rigid protein/rigid nucleic acid; (b) flexible protein/rigid nucleic acid; (c) rigid protein/flexible nucleic acid. Analysis of free and covalently VP55-attached primers favored either (b) or a version of (c) incorporating a passive steric block, and suggested two regions of relative motion between polymerase and primer. Inclusion of a 6nt uridylate-rich patch at the primer 3' end switched the polymerase from approximately 3-7 nt to approximately 25-30 nt tail addition without affecting initial binding affinity. By synthesizing this patch as a (rU/dC) pool, discontinuous polymerase movements could be detected.     

Capacity for RNA synthesis in 70S ribosome-deficient plastids of heat-bleached rye leaves

In the leaves of rye seedlings (Secale cereale L.) grown at an elevated temperature of 32°C the formation of plastidic 70S ribosomes is specifically prevented. The resulting plastid ribosome-deficient leaves, which are chlorotic in light, represent a system for the identification of translation products of the 80S ribosomes among the chloroplastic proteins. Searching for the primary heat-sensitive event causing the 70S ribosome-deficiency, the thermostability of the chloroplastic capacity for RNA synthesis was investigated. The RNA polymerase activity of isolated normal chloroplasts from 22°-grown rye leaves was not inactivated in vitro at temperatures between 30° and 40°C. The ribosome-deficient plastids purified from bleached 32°-grown leaf parts contained significant RNA polymerase activity which was, however, lower than in functional chloroplasts. After application of [³H]uridine to intact leaf tissues [³H]uridine incorporation was found in ribosome-deficient plastids of 32°C-grown leaves. The amount of incorporation was similar to that in the control chloroplasts from 22°C-grown leaves. According to these results, it is unlikely that the non-permissive temperature (32°C) causes a general inactivation of the chloroplastic RNA synthesis in rye leaves.     

Nuclear localization signal-dependent and -independent movements of Drosophila melanogaster dUTPase isoforms during nuclear cleavage

Two dUTPase isoforms (23 kDa and 21 kDa) are present in the fruitfly with the sole difference of an N-terminal extension. In Drosophila embryo, both isoforms are detected inside the nucleus. Here, we investigated the function of the N-terminal segment using eYFP-dUTPase constructs. In Schneider 2 cells, only the 23 kDa construct showed nuclear localization arguing that it may contain a nuclear localization signal (NLS). Sequence comparisons identified a lysine-rich nonapeptide with similarity to the human c-myc NLS. In Drosophila embryos during nuclear cleavages, the 23 kDa isoform showed the expected localization shifts. Contrariwise, although the 21 kDa isoform was excluded from the nuclei during interphase, it was shifted to the nucleus during prophase and forthcoming mitotic steps. The observed dynamic localization character showed strict timing to the nuclear cleavage phases and explained how both isoforms can be present within the nuclear microenvironment, although at different stages of cell cycle.     

Hexokinase and not glycogen synthase controls the flux through the glycogen synthesis pathway in frog oocytes

Here we set out to evaluate the role of hexokinase and glycogen synthase in the control of glycogen synthesis in vivo. We used metabolic control analysis (MCA) to determine the flux control coefficient for each of the enzymes involved in the pathway. Acute microinjection experiments in frog oocytes were specifically designed to change the endogenous activities of the enzymes, either by directly injecting increasing amounts of a given enzyme (HK, PGM and UGPase) or by microinjection of a positive allosteric effector (glc-6P for GS). Values of 0.61 ± 0.07, 0.19 ± 0.03, 0.13 ± 0.03, and −0.06 ± 0.08 were obtained for the flux control coefficients of hexokinase EC 2.7.1.1 (HK), phosphoglucomutase EC 5.4.2.1 (PGM), UDPglucose pyrophosphorylase EC 2.7.7.9 (UGPase) and glycogen synthase EC 2.4.1.11 (GS), respectively. These values satisfy the summation theorem since the sum of the control coefficients for all the enzymes of the pathway is 0.87. The results show that, in frog oocytes, glycogen synthesis through the direct pathway is under the control of hexokinase. Phosphoglucomutase and UDPG-pyrophosphorylase have a modest influence, while the control exerted by glycogen synthase is null.     

Characterization of a cDNA encoding a novel plant poly(A) polymerase

We have isolated cDNA clones encoding a novel factor (PAP-I) that is a component of a multi-subunit poly(A) polymerase from pea seedlings. The encoded protein, when isolated from appropriately engineered Escherichia coli, was active as a poly(A) polymerase, either with an associated RNA binding cofactor (PAP-III) or with free poly(A) as an RNA substrate. The latter observation indicates that PAP-I is in fact a poly(A) polymerase. PAP-I bore a striking resemblance to an as yet uncharacterized cyanobacterial protein. This observation suggested a possible chloroplast localization for PAP-I. This hypothesis was tested and found to be substantiated; immunoblot analysis identified PAP-I in chloroplast but not nuclear extracts. Our results suggest that PAP-I is a component of the machinery that adds poly(A) to chloroplast RNAs.     

Lamin A/C speckles mediate spatial organization of splicing factor compartments and RNA polymerase II transcription

The cylindrical shape of the fission yeast cell is generated by linear polarized growth from its cell ends. Using immunofluorescence and live imaging microscopy, we have investigated the roles of the cell end marker tea1p in generating linear polarized growth. We found that tea1p is primarily transported on plus ends of microtubules from the vicinity of the nucleus to the cell ends, and that its movement near the nucleus is independent of the kinesin tea2p. Deletion analysis identified a coiled-coil domain in tea1p essential for its retention at cell ends, and demonstrated that tea1p exerts different functions dependent on its location. On the tips of microtubules, tea1p prevents the curling of microtubules around the cell ends, whereas it is required for maintaining linear cell growth and for retention of polarity factors such as the Dyrk kinase pom1p, the CLIP170-like tip1p, and tea2p at the cell ends. We propose that tea1p has roles in organizing the microtubule cytoskeleton on the tips of microtubules, and in the retention of factors at the cell ends necessary for the cell to grow in a straight line.     

Maturation of mammalian H/ACA box snoRNAs: PAPD5-dependent adenylation and PARN-dependent trimming

Small nucleolar and small Cajal body RNAs (snoRNAs and scaRNAs) of the H/ACA box and C/D box type are generated by exonucleolytic shortening of longer precursors. Removal of the last few nucleotides at the 3' end is known to be a distinct step. We report that, in human cells, knock-down of the poly(A) specific ribonuclease (PARN), previously implicated only in mRNA metabolism, causes the accumulation of oligoadenylated processing intermediates of H/ACA box but not C/D box RNAs. In agreement with a role of PARN in snoRNA and scaRNA processing, the enzyme is concentrated in nucleoli and Cajal bodies. Oligo(A) tails are attached to a short stub of intron sequence remaining beyond the mature 3' end of the snoRNAs. The noncanonical poly(A) polymerase PAPD5 is responsible for addition of the oligo(A) tails. We suggest that deadenylation is coupled to clean 3' end trimming, which might serve to enhance snoRNA stability.     

Caenorhabditis elegans: Decay of isocitrate lyase during larval development

Changes in the levels of isocitrate lyase, malate synthase, catalase, fumarase, and NADP⁺-isocitrate dehydrogenase have been investigated during larval development of the free-living soil nematode Caenorhabditis elegans in the presence and absence of Escherichia coli. The specific activities of isocitrate lyase, malate synthase, and catalase are maximal at the time of egg hatching and, thereafter, decline during larval development when larvae feed on E. coli, whereas in the absence of E. coli specific activities of the same enzymes increase for 12 hr and subsequently remain constant. There is, however, no change in specific activity of fumarase or NADP⁺-isocitrate dehydrogenase during the same developmental period, in either case. Cycloheximide at 100 μM arrests the decline of isocitrate lyase during development of feeding larvae but has no effect upon the appearance of isocitrate lyase during starvation. The latter is true also for 15 mM itaconate. There is inactivation of isocitrate lyase in crude extracts of frozen worms in comparison to that in analogous extracts prepared from freshly harvested nematodes.     

草鱼呼肠孤病毒RNA聚合酶基因功能区在原核细胞中的表达

草鱼呼肠孤病毒(grass carp reovirus)为我国分离、鉴定的第一株水生动物病毒.1983年,我国首次报道引起爆发性草鱼出血病的病原为草鱼出血病病毒[1,2],其后相继进行了系统的病毒形态学、生物学、生物化学及分子生物学特性等研究[3-8].自1979年Meyers T R等报道从水生动物中分离出第一株呼肠孤样病毒,迄今国际上已分离鉴定40余种水生呼肠孤病毒(aquareovirus).在这些分离株中,大多数毒株不能引起寄主的病理反应或仅表现出较弱的致病性.然而研究认为,GCRV为水生呼肠孤病毒中致病力最强的毒株[9].可见,以GCRV为模型,研究水生呼肠孤病毒的复制与致病机理具有一定的理论及实际意义.我们在对GCRV反应核心及体外转录研究中,已证实GCRV RNA聚合酶在病毒粒子中的存在及其位置[5];GCRV序列测定及定位结果显示,GCRV-VP2多肽为该病毒RNA聚合酶(RNA dependent RNA polymerase RdRp)[6,7].为了探讨草鱼呼肠孤病毒的侵染与宿主的相关性及复制机制,我们首次进行了该病毒RNA聚合酶基因(GCRV-RdRp)功能区序列在原核细胞中的表达研究,并得到高效表达融合蛋白.这一结果将为该酶的活性及特性分析提供实验依据.下面报道本研究结果.     

Entamoeba histolytica: ultrastructural localization of Ca2+-dependent nucleotidases

Localization of nucleotidases dependent on Ca²⁺ was investigated cytochemically in axenically cultivated trophozoites of Entamoeba histolytica, strain HM-1:IMSS, with an electron microscope. Ca²⁺-dependent ATPase (EC 3.6.1.3) activity was found on the plasma membrane and on the inner surface of the limiting membrane of a few cytoplasmic vacuoles. Ca²⁺-dependent ADPase, Ca²⁺-dependent thiamine pyrophosphatase, and acid phosphatase (EC 3.1.3.2) activities were detected on the inner surface of the limiting membrane of most of the cytoplasmic vacuoles but not on the plasma membrane. Cytoplasmic vacuoles with these enzymatic activities seemed similar in morphological characteristics. Moreover, the reaction product formed by Ca²⁺-dependent ADPase, Ca²⁺-dependent thiamine pyrophosphatase and acid phosphatase was demonstrable on the inner surface of the limiting membrane of vacuoles containing ingested red blood cells. The reaction product formed by these enzymes was also observed on the periphery of ingested red blood cells. The findings suggest that cytoplasmic vacuoles with these enzymatic activities are lysosomal in nature, probably phagolysosomes; therefore, the enzymes appear to be at least partially associated with primary lysosomes of E. histolytica.     

Cofactor requirements for nuclear export of Rev response element (RRE)- and constitutive transport element (CTE)-containing retroviral RNAs. An unexpected role for actin

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev-NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).     

A new regulatory principle for in vivo biochemistry: Pleiotropic low affinity regulation by the adenine nucleotides – Illustrated for the glycolytic enzymes of Saccharomyces cerevisiae

Enzymology tends to focus on highly specific effects of substrates, allosteric modifiers, and products occurring at low concentrations, because these are most informative about the enzyme’s catalytic mechanism. We hypothesized that at relatively high in vivo concentrations, important molecular monitors of the state of living cells, such as ATP, affect multiple enzymes of the former and that these interactions have gone unnoticed in enzymology.