scholarly journals Sequence and domain organization of scruin, an actin-cross-linking protein in the acrosomal process of Limulus sperm.

1995 ◽  
Vol 128 (1) ◽  
pp. 51-60 ◽  
Author(s):  
M Way ◽  
M Sanders ◽  
C Garcia ◽  
J Sakai ◽  
P Matsudaira
Keyword(s):  
Amino Acid ◽  
Actin Filaments ◽  
Limited Proteolysis ◽  
Open Reading Frames ◽  
Actin Binding ◽  
Molar Ratio ◽  
Domain Organization ◽  
Ring Canals ◽  
Drosophila Protein ◽  
Reading Frames

The acrosomal process of Limulus sperm is an 80-microns long finger of membrane supported by a crystalline bundle of actin filaments. The filaments in this bundle are crosslinked by a 102-kD protein, scruin present in a 1:1 molar ratio with actin. Recent image reconstruction of scruin decorated actin filaments at 13-A resolution shows that scruin is organized into two equally sized domains bound to separate actin subunits in the same filament. We have cloned and sequenced the gene for scruin from a Limulus testes cDNA library. The deduced amino acid sequence of scruin reflects the domain organization of scruin: it consists of a tandem pair of homologous domains joined by a linker region. The domain organization of scruin is confirmed by limited proteolysis of the purified acrosomal process. Three different proteases cleave the native protein in a 5-kD Protease-sensitive region in the middle of the molecule to generate an NH2-terminal 47-kD and a COOH-terminal 56-kD protease-resistant domains. Although the protein sequence of scruin has no homology to any known actin-binding protein, it has similarities to several proteins, including four open reading frames of unknown function in poxviruses, as well as kelch, a Drosophila protein localized to actin-rich ring canals. All proteins that show homologies to scruin are characterized by the presence of an approximately 50-amino acid residue motif that is repeated between two and seven times. Crystallographic studies reveal this motif represents a four beta-stranded fold that is characteristic of the "superbarrel" structural fold found in the sialidase family of proteins. These results suggest that the two domains of scruin seen in EM reconstructions are superbarrel folds, and they present the possibility that other members of this family may also bind actin.

scholarly journals Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein.

1983 ◽  
Vol 96 (5) ◽  
pp. 1400-1413 ◽  
Author(s):  
R Niederman ◽  
P C Amrein ◽  
J Hartwig
Keyword(s):  
Actin Filaments ◽  
Binding Protein ◽  
Three Dimensional ◽  
Actin Binding ◽  
Dimensional Structure ◽  
Molar Ratio ◽  
Computer Assisted ◽  
Three Dimensional Structure ◽  
Actin Binding Protein ◽  
Critical Point Drying

Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three-dimensional network resembling the peripheral cytoskeleton of motile cells.

scholarly journals Bundling of actin filaments by alpha-actinin depends on its molecular length.

1990 ◽  
Vol 110 (6) ◽  
pp. 2013-2024 ◽  
Author(s):  
R K Meyer ◽  
U Aebi
Keyword(s):  
Smooth Muscle ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Molar Ratio ◽  
Cross Linking ◽  
Actin Bundle ◽  
Actin Bundles ◽  
Chicken Gizzard ◽  
Molecular Length

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.

scholarly journals A Stress-Induced Bias in the Reading of the Genetic Code in Escherichia coli

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Adi Oron-Gottesman ◽  
Martina Sauert ◽  
Isabella Moll ◽  
Hanna Engelberg-Kulka
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Ribosomal Protein ◽  
Genetic Code ◽  
Open Reading Frames ◽  
Translation Machinery ◽  
E Coli ◽  
Universal Characteristic ◽  
Living Organisms ◽  
Reading Frames

ABSTRACT Escherichia coli mazEF is an extensively studied stress-induced toxin-antitoxin (TA) system. The toxin MazF is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress, the induced MazF generates a stress-induced translation machinery (STM), composed of MazF-processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Here, we further characterized the STM system, finding that MazF cleaves only ACA sites located in the open reading frames of processed mRNAs, while out-of-frame ACAs are resistant. This in-frame ACA cleavage of MazF seems to depend on MazF binding to an extracellular-death-factor (EDF)-like element in ribosomal protein bS1 (bacterial S1), apparently causing MazF to be part of STM ribosomes. Furthermore, due to the in-frame MazF cleavage of ACAs under stress, a bias occurs in the reading of the genetic code causing the amino acid threonine to be encoded only by its synonym codon ACC, ACU, or ACG, instead of by ACA. IMPORTANCE The genetic code is a universal characteristic of all living organisms. It defines the set of rules by which nucleotide triplets specify which amino acid will be incorporated into a protein. Our results represent the first existing report on a stress-induced bias in the reading of the genetic code. We found that in E. coli , under stress, the amino acid threonine is encoded only by its synonym codon ACC, ACU, or ACG, instead of by ACA. This is because under stress, MazF generates a stress-induced translation machinery (STM) in which MazF cleaves in-frame ACA sites of the processed mRNAs.

scholarly journals Isolation and Characterization of Genes Responsible for Naphthalene Degradation from Thermophilic Naphthalene Degrader, Geobacillus sp. JF8

2019 ◽  
Vol 8 (1) ◽  
pp. 44 ◽  
Author(s):  
Daisuke Miyazawa ◽  
Le Thi Ha Thanh ◽  
Akio Tani ◽  
Masaki Shintani ◽  
Nguyen Hoang Loc ◽  
...  
Keyword(s):  
Amino Acid ◽  
Thermophilic Bacterium ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Pcr Analysis ◽  
Naphthalene Degradation ◽  
Isolation And Characterization ◽  
Dihydrodiol Dehydrogenase ◽  
Reading Frames

Geobacillus sp. JF8 is a thermophilic biphenyl and naphthalene degrader. To identify the naphthalene degradation genes, cis-naphthalene dihydrodiol dehydrogenase was purified from naphthalene-grown cells, and its N-terminal amino acid sequence was determined. Using a DNA probe encoding the N-terminal region of the dehydrogenase, a 10-kb DNA fragment was isolated. Upstream of nahB, a gene for dehydrogenase, there were two open reading frames which were designated as nahAc and nahAd, respectively. The products of nahAc and nahAd were predicted to be alpha and beta subunit of ring-hydroxylating dioxygenases, respectively. Phylogenetic analysis of amino acid sequences of NahB indicated that it did not belong to the cis-dihydrodiol dehydrogenase group that includes those of classical naphthalene degradation pathways. Downstream of nahB, four open reading frames were found, and their products were predicted as meta-cleavage product hydrolase, monooxygenase, dehydrogenase, and gentisate 1,2-dioxygenase, respectively. A reverse transcriptase-PCR analysis showed that transcription of nahAcAd was induced by naphthalene. These findings indicate that we successfully identified genes involved in the upper pathway of naphthalene degradation from a thermophilic bacterium.

scholarly journals Interactions of tensin with actin and identification of its three distinct actin-binding domains.

1994 ◽  
Vol 125 (5) ◽  
pp. 1067-1075 ◽  
Author(s):  
S H Lo ◽  
P A Janmey ◽  
J H Hartwig ◽  
L B Chen
Keyword(s):  
Amino Acids ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Sh2 Domain ◽  
Actin Binding ◽  
Molar Ratio ◽  
Actin Assembly ◽  
Binding Domains ◽  
Focal Contacts ◽  
End Capping

Tensin, a 200-kD phosphoprotein of focal contacts, contains sequence homologies to Src (SH2 domain), and several actin-binding proteins. These features suggest that tensin may link the cell membrane to the cytoskeleton and respond directly to tyrosine kinase signalling pathways. Here we identify three distinct actin-binding domains within tensin. Recombinant tensin purified after overexpression by a baculovirus system binds to actin filaments with Kd = 0.1 microM, cross-links actin filaments at a molar ratio of 1:10 (tensin/actin), and retards actin assembly by barbed end capping with Kd = 20 nM. Tensin fragments were constructed and expressed as fusion proteins to map domains having these activities. Three regions from tensin interact with actin: two regions composed of amino acids 1 to 263 and 263 to 463, cosediment with F-actin but do not alter the kinetics of actin assembly; a region composed of amino acids 888-989, with sequence homology to insertin, retards actin polymerization. A claw-shaped tensin dimer would have six potential actin-binding sites and could embrace the ends of two actin filaments at focal contacts.

scholarly journals Biosynthesis of the β-Methylarginine Residue of Peptidyl Nucleoside Arginomycin in Streptomyces arginensis NRRL 15941

2014 ◽  
Vol 80 (16) ◽  
pp. 5021-5027 ◽  
Author(s):  
Jun Feng ◽  
Jun Wu ◽  
Jie Gao ◽  
Zhigui Xia ◽  
Zixin Deng ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biosynthetic Pathway ◽  
Biosynthetic Gene Cluster ◽  
Open Reading Frames ◽  
Enzymatic Conversion ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Adenosyl Methionine ◽  
Bacteria And Fungi ◽  
Reading Frames

ABSTRACTThe peptidyl nucleoside arginomycin is active against Gram-positive bacteria and fungi but displays much lower toxicity to mice than its analog blasticidin S. It features a rare amino acid, β-methylarginine, which is attached to the deoxyhexose moiety via a 4′-aminoacyl bond. We here report cloning of the complete biosynthetic gene cluster for arginomycin fromStreptomyces arginensisNRRL 15941. Among the 14 putative essential open reading frames,argM, encoding an aspartate aminotransferase (AAT), and adjacentargN, encoding anS-adenosyl methionine (SAM)-dependent methyltransferase, are coupled to catalyze arginine and yield β-methylarginine inEscherichia coli. Purified ArgM can transfer the α-amino group ofl-arginine to α-ketoglutaric acid to give glutamate and thereby convertsl-arginine to 5-guanidino-2-oxopentanoic acid, which is methylated at the C-3 position by ArgN to form 5-guanidino-3-methyl-2-oxopentanoic acid. Iteratively, ArgM specifically catalyzes transamination from the donorl-aspartate to the resulting 5-guanidino-3-methyl-2-oxopentanoic acid, generating β-methylarginine. The complete and concise biosynthetic pathway for the rare and bioactive amino acid revealed by this study may pave the way for the production of β-methylarginine either by enzymatic conversion or by engineered living cells.

scholarly journals Translational Control of Protein Kinase Cη by Two Upstream Open Reading Frames

2009 ◽  
Vol 29 (22) ◽  
pp. 6140-6148 ◽  
Author(s):  
Hadas Raveh-Amit ◽  
Adva Maissel ◽  
Jonathan Poller ◽  
Liraz Marom ◽  
Orna Elroy-Stein ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Translational Control ◽  
Translational Regulation ◽  
Normal Growth ◽  
Amino Acid Starvation ◽  
Open Reading Frames ◽  
Pkc Isoforms ◽  
Upstream Open Reading Frames ◽  
Reading Frames

ABSTRACT Protein kinase C (PKC) represents a family of serine/threonine kinases that play a central role in the regulation of cell growth, differentiation, and transformation. Posttranslational control of the PKC isoforms and their activation have been extensively studied; however, not much is known about their translational regulation. Here we report that the expression of one of the PKC isoforms, PKCη, is regulated at the translational level both under normal growth conditions and during stress imposed by amino acid starvation, the latter causing a marked increase in its protein levels. The 5′ untranslated region (5′ UTR) of PKCη is unusually long and GC rich, characteristic of many oncogenes and growth regulatory genes. We have identified two conserved upstream open reading frames (uORFs) in its 5′ UTR and show their effect in suppressing the expression of PKCη in MCF-7 growing cells. While the two uORFs function as repressive elements that maintain low basal levels of PKCη in growing cells, they are required for its enhanced expression upon amino acid starvation. We show that the translational regulation during stress involves leaky scanning and is dependent on eIF-2α phosphorylation by GCN2. Our work further suggests that translational regulation could provide an additional level for controlling the expression of PKC family members, being more common than currently recognized.

scholarly journals Genomic and phylogenetic characterization of ChPV2, a novel goat PV closely related to the Xi-PV1 species infecting bovines

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Anouk Willemsen ◽  
Alexander van den Boom ◽  
Julienne Dietz ◽  
Seval Bilge Dagalp ◽  
Firat Dogan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Phylogenetic Analyses ◽  
Regulatory Elements ◽  
Open Reading Frames ◽  
Capra Hircus ◽  
Phylogenetic Position ◽  
Reading Frame ◽  
Phylogenetic Characterization ◽  
Ruminant Species ◽  
Reading Frames

Abstract Background Papillomaviruses (PVs) infecting artiodactyls are very diverse, and only second in number to PVs infecting primates. PVs associated to lesions in economically important ruminant species have been isolated from cattle and sheep. Methods Potential PV DNA from teat lesions of a Damascus goat was isolated, cloned and sequenced. The PV genome was analyzed using bioinformatics approaches to detect open reading frames and to predict potential features of encoded proteins as well as putative regulatory elements. Sequence comparison and phylogenetic analyses using the concatenated E1E2L2L1 nucleotide and amino acid alignments was used to reveal the relationship of the new PV to the known PV diversity and its closest relevants. Results We isolated and characterized the full-genome of novel Capra hircus papillomavirus. We identified the E6, E7, E1, E2, L2, L1 open reading frames with protein coding potential and putative active elements in the ChPV2 proteins and putative regulatory genome elements. Sequence similarities of L1 and phylogenetic analyses using concatenated E1E2L2L1 nucleotide and amino acid alignments suggest the classification as a new PV type designated ChPV2 with a phylogenetic position within the XiPV genus, basal to the XiPV1 species. ChPV2 is not closely related to ChPV1, the other known goat PV isolated from healthy skin, although both of them belong confidently into a clade composed of PVs infecting cervids and bovids. Interestingly, ChPV2 contains an E6 open reading frame whereas all closely related PVs do not Conclusion ChPV2 is a novel goat PV closely related to the Xi-PV1 species infecting bovines. Phylogenetic relationships and genome architecture of ChPV2 and closely related PV types suggest at least two independent E6 losses within the XiPV clade.

scholarly journals Formation of actin filament bundles in the ring canals of developing Drosophila follicles.

1996 ◽  
Vol 133 (1) ◽  
pp. 61-74 ◽  
Author(s):  
L G Tilney ◽  
M S Tilney ◽  
G M Guild
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Actin Binding ◽  
Nurse Cells ◽  
Ring Canal ◽  
Contractile Ring ◽  
Thin Sections ◽  
Development Stages ◽  
Ring Canals ◽  
Filament Bundles

Growing the intracellular bridges that connect nurse cells with each o ther and to the developing oocyte is vital for egg development. These ring canals increase from 0.5 microns in diameter at stage 2 to 10 microns in diameter at stage 11. Thin sections cut horizontally as you would cut a bagel, show that there is a layer of circumferentially oriented actin filaments attached to the plasma membrane at the periphery of each canal. By decoration with subfragment 1 of myosin we find actin filaments of mixed polarities in the ring such as found in the "contractile ring" formed during cytokinesis. In vertical sections through the canal the actin filaments appear as dense dots. At stage 2 there are 82 actin filaments in the ring, by stage 6 there are 717 and by stage 10 there are 726. Taking into account the diameter, this indicates that there is 170 microns of actin filaments/canal at stage 2 (pi x 0.5 microns x 82), 14,000 microns at stage 9 and approximately 23,000 microns at stage 11 or one inch of actin filament! The density of actin filaments remains unchanged throughout development. What is particularly striking is that by stages 4-5, the ring of actin filaments has achieved its maximum thickness, even though the diameter has not yet increased significantly. Thereafter, the diameter increases. Throughout development, stages 2-11, the canal length also increases. Although the density (number of actin filaments/micron2) through a canal remains constant from stage 5 on, the actin filaments appear as a net of interconnected bundles. Further information on this net of bundles comes from studying mutant animals that lack kelch, a protein located in the ring canal that has homology to the actin binding protein, scruin. In this mutant, the actin filaments form normally but individual bundles that comprise the fibers of the net are not bound tightly together. Some bundles enter into the ring canal lumen but do not completely occlude the lumen. all these observations lay the groundwork for our understanding of how a noncontractile ring increases in thickness, diameter, and length during development.

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