scholarly journals Interaction between FliE and FlgB, a Proximal Rod Component of the Flagellar Basal Body ofSalmonella

2000 ◽  
Vol 182 (11) ◽  
pp. 3029-3036 ◽  
Author(s):  
Tohru Minamino ◽  
Shigeru Yamaguchi ◽  
Robert M. Macnab
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Basal Body ◽  
Strong Interactions ◽  
Wild Type ◽  
Body Protein ◽  
C Terminus ◽  
Genes Encoding ◽  
And Function ◽  
Better Than

ABSTRACT FliE is a flagellar basal body protein of Salmonellawhose detailed location and function have not been established. A mutant allele of fliE, which caused extremely poor flagellation and swarming, generated extragenic suppressors, all of which mapped to flgB, one of four genes encoding the basal body rod; the fliE flgB pseudorevertants were better flagellated and swarmed better than the fliE parent, especially when the temperature was reduced from 37 to 30°C. Motility of the pseudorevertants in liquid culture was markedly better than motility on swarm plates; we interpret this to mean that reduced flagellation is less deleterious at low viscous loads. Overproduction of the mutant FliE protein improved the motility of the parentalfliE mutant and its pseudorevertants, though not to wild-type levels. Overproduction of suppressor FlgB (but not wild-type FlgB) in the fliE mutant also resulted in improved motility. The second-site FlgB mutation by itself had no phenotype; cells swarmed as well as wild-type cells. When overproduced, wild-type FliE was dominant over FliE-V99G, but the reverse was not true; that is, overproduced FliE-V99G was not negatively dominant over wild-type FliE. We conclude that the mutant protein has reduced probability of assembly but, if assembled, functions relatively well. Export of the flagellar protein FlgD, which is known to be FliE dependent, was severely impaired by the FliE-V99G mutation but was significantly improved in the suppressor strains. The FliE mutation, V99G, was close to the C terminus of the 104-amino-acid sequence; the suppressing mutations in FlgB were all either G119E or G129D, close to the C terminus of its 138-amino-acid sequence. Affinity blotting experiments between FliE as probe and various basal body proteins as targets and vice versa revealed strong interactions between FliE and FlgB; much weaker interactions between FliE and other rod proteins were observed and probably derive from the known similarities among these proteins. We suggest that FliE subunits constitute a junction zone between the MS ring and the rod and also that the proximal rod structure consists of FlgB subunits.

scholarly journals Differential Localization and Function of PB1-F2 Derived from Different Strains of Influenza A Virus

2010 ◽  
Vol 84 (19) ◽  
pp. 10051-10062 ◽  
Author(s):  
Chi-Jene Chen ◽  
Guang-Wu Chen ◽  
Ching-Ho Wang ◽  
Chih-Heng Huang ◽  
Yeau-Ching Wang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Influenza A Virus ◽  
Influenza A ◽  
Amino Acid Sequences ◽  
Wild Type ◽  
Mitochondrial Localization ◽  
Sequence Context ◽  
C Terminus ◽  
Different Strains ◽  
And Function

ABSTRACT PB1-F2 is a viral protein that is encoded by the PB1 gene of influenza A virus by alternative translation. It varies in length and sequence context among different strains. The present study examines the functions of PB1-F2 proteins derived from various human and avian viruses. While H1N1 PB1-F2 was found to target mitochondria and enhance apoptosis, H5N1 PB1-F2, surprisingly, did not localize specifically to mitochondria and displayed no ability to enhance apoptosis. Introducing Leu into positions 69 (Q69L) and 75 (H75L) in the C terminus of H5N1 PB1-F2 drove 40.7% of the protein to localize to mitochondria compared with the level of mitochondrial localization of wild-type H5N1 PB1-F2, suggesting that a Leu-rich sequence in the C terminus is important for targeting of mitochondria. However, H5N1 PB1-F2 contributes to viral RNP activity, which is responsible for viral RNA replication. Lastly, although the swine-origin influenza virus (S-OIV) contained a truncated form of PB1-F2 (12 amino acids [aa]), potential mutation in the future may enable it to contain a full-length product. Therefore, the functions of this putative S-OIV PB1-F2 (87 aa) were also investigated. Although this PB1-F2 from the mutated S-OIV shares only 54% amino acid sequence identity with that of seasonal H1N1 virus, it also increased viral RNP activity. The plaque size and growth curve of the viruses with and without S-OIV PB1-F2 differed greatly. The PB1-F2 protein has various lengths, amino acid sequences, cellular localizations, and functions in different strains, which result in strain-specific pathogenicity. Such genetic and functional diversities make it flexible and adaptable in maintaining the optimal replication efficiency and virulence for various strains of influenza A virus.

Sorting out theLeMANs: endo-β-mannanase genes and their encoded proteins in tomato

2007 ◽  
Vol 17 (3) ◽  
pp. 143-154 ◽  
Author(s):  
Xuemei Gong ◽  
J. Derek Bewley
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gibberellic Acid ◽  
Cell Walls ◽  
Plant Cell Walls ◽  
Tomato Seeds ◽  
C Terminus ◽  
Inactive Form ◽  
Genes Encoding ◽  
Hydrolysis Of

AbstractEndo-β-mannanase (EC 3.2.1.78) is involved in the hydrolysis of mannan-type polysaccharides that are present in plant cell walls, especially those of the seed endosperm. The genes encoding the endo-β-mannanases have been studied extensively in tomato (Solanum lycopersicum), and five genes (LeMAN1,LeMAN2,LeMAN3,LeMAN4andLeMAN5) and/or their products have been isolated and characterized.LeMAN1,LeMAN2andLeMAN3are expressed in tomato seeds,LeMAN4in the fruit andLeMAN5in the flower.LeMAN5andLeMAN2are now considered to be the same gene, and the former is re-designated asLeMAN2*. Transcripts ofLeMANs1, 2and3are detected only in the endosperm of tomato seeds, and their synthesis is promoted by gibberellic acid.LeMAN4, in the fruit, occurs asLeMAN4aandLeMAN4igenes that encode an active or inactive form of endo-β-mannanase, respectively.LeMAN1–4 enzymes encoded by these genes share 80% similarity in amino acid sequence. In tomato, the leucine amino acid present near to the C-terminus of the endo-β-mannanase is the most important for achieving full activity of the enzyme.

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

1997 ◽  
Vol 75 (6) ◽  
pp. 687-696 ◽  
Author(s):  
Tamo Fukamizo ◽  
Ryszard Brzezinski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Binding ◽  
Structure And Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Streptomyces Sp ◽  
Binding Cleft ◽  
And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

scholarly journals Terminal deoxynucleotidyltransferase. Alignment of α- and β-subunits of the core enzyme along the primary translation product

1985 ◽  
Vol 227 (3) ◽  
pp. 1003-1007 ◽  
Author(s):  
C M Beach ◽  
S K Chan ◽  
T C Vanaman ◽  
M S Coleman
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Beta Subunits ◽  
Isolation And Purification ◽  
C Terminus ◽  
Human Lymphoblast ◽  
Catalytically Active ◽  
Single Polypeptide ◽  
Dna Nucleotide Sequence

Terminal deoxynucleotidyltransferase exists in multiple Mr forms, all apparently generated from a single polypeptide of 62kDa. On isolation and purification, the smallest catalytically active protein of this enzyme consists of two subunits, alpha (12kDa) and beta (30kDa). Recently a complementary-DNA nucleotide sequence has been reported for a portion of the enzyme from human lymphoblast. We have pinpointed the locations of the alpha- and beta-subunits within the elucidated nucleotide sequence. From these data, the portions of the nucleotide sequence coding for the catalytically important area of the transferase can be estimated. Here the amino acid sequence of a number of tryptic peptides from calf alpha- and beta-subunits is presented. Because of the striking homology between the amino acid sequence of the calf enzyme and that predicted for human lymphoblast enzyme, it is possible for us to conclude that the alpha-subunit was generated from the C-terminus of the precursor protein and the beta-subunit was non-overlapping and proximal.

scholarly journals Alterations in flight muscle ultrastructure and function in Drosophila tropomyosin mutants.

1996 ◽  
Vol 135 (3) ◽  
pp. 673-687 ◽  
Author(s):  
A J Kreuz ◽  
A Simcox ◽  
D Maughan
Keyword(s):  
Amino Acid ◽  
Power Output ◽  
Flight Muscle ◽  
Structure And Function ◽  
Wild Type ◽  
Muscle Tropomyosin ◽  
Ultrastructure And Function ◽  
And Function ◽  
Net Power Output ◽  
Current Report

Drosophila indirect flight muscle (IFM) contains two different types of tropomyosin: a standard 284-amino acid muscle tropomyosin, Ifm-TmI, encoded by the TmI gene, and two > 400 amino acid tropomyosins, TnH-33 and TnH-34, encoded by TmII. The two IFM-specific TnH isoforms are unique tropomyosins with a COOH-terminal extension of approximately 200 residues which is hydrophobic and rich in prolines. Previous analysis of a hypomorphic TmI mutant, Ifm(3)3, demonstrated that Ifm-TmI is necessary for proper myofibrillar assembly, but no null TmI mutant or TmII mutant which affects the TnH isoforms have been reported. In the current report, we show that four flightless mutants (Warmke et al., 1989) are alleles of TmI, and characterize a deficiency which deletes both TmI and TmII. We find that haploidy of TmI causes myofibrillar disruptions and flightless behavior, but that haploidy of TmII causes neither. Single fiber mechanics demonstrates that power output is much lower in the TmI haploid line (32% of wild-type) than in the TmII haploid line (73% of wild-type). In myofibers nearly depleted of Ifm-TmI, net power output is virtually abolished (< 1% of wild-type) despite the presence of an organized fibrillar core (approximately 20% of wild-type). The results suggest Ifm-TmI (the standard tropomyosin) plays a key role in fiber structure, power production, and flight, with reduced Ifm-TmI expression producing corresponding changes of IFM structure and function. In contrast, reduced expression of the TnH isoforms has an unexpectedly mild effect on IFM structure and function.

scholarly journals Directed Evolution of AraC for Improved Compatibility of Arabinose- and Lactose-Inducible Promoters

2007 ◽  
Vol 73 (18) ◽  
pp. 5711-5715 ◽  
Author(s):  
Sung Kuk Lee ◽  
Howard H. Chou ◽  
Brian F. Pfleger ◽  
Jack D. Newman ◽  
Yasuo Yoshikuni ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Cross Talk ◽  
Biological Systems ◽  
Wild Type ◽  
Dimerization Domain ◽  
Expression Levels ◽  
Inducible Promoters ◽  
C Terminus ◽  
Increased Sensitivity ◽  
Better Than

ABSTRACT Synthetic biological systems often require multiple, independently inducible promoters in order to control the expression levels of several genes; however, cross talk between the promoters limits this ability. Here, we demonstrate the directed evolution of AraC to construct an arabinose-inducible (PBAD) system that is more compatible with IPTG (isopropyl-β-d-1-thiogalactopyranoside) induction of a lactose-inducible (Plac) system. The constructed system is 10 times more sensitive to arabinose and tolerates IPTG significantly better than the wild type. Detailed studies indicate that the AraC dimerization domain and C terminus are important for the increased sensitivity of AraC to arabinose.

scholarly journals The inherent mutational tolerance and antigenic evolvability of influenza hemagglutinin

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Bargavi Thyagarajan ◽  
Jesse D Bloom
Keyword(s):  
Amino Acid ◽  
H1n1 Influenza ◽  
Influenza Hemagglutinin ◽  
Functional Variants ◽  
Viral Hemagglutinin ◽  
Before And After ◽  
Amino Acid Mutations ◽  
And Function ◽  
Phylogenetic Models ◽  
Better Than

Influenza is notable for its evolutionary capacity to escape immunity targeting the viral hemagglutinin. We used deep mutational scanning to examine the extent to which a high inherent mutational tolerance contributes to this antigenic evolvability. We created mutant viruses that incorporate most of the ≈104 amino-acid mutations to hemagglutinin from A/WSN/1933 (H1N1) influenza. After passaging these viruses in tissue culture to select for functional variants, we used deep sequencing to quantify mutation frequencies before and after selection. These data enable us to infer the preference for each amino acid at each site in hemagglutinin. These inferences are consistent with existing knowledge about the protein's structure and function, and can be used to create a model that describes hemagglutinin's evolution far better than existing phylogenetic models. We show that hemagglutinin has a high inherent tolerance for mutations at antigenic sites, suggesting that this is one factor contributing to influenza's antigenic evolution.

The amino acid sequence of yeast phosphoglycerate mutase

1982 ◽  
Vol 215 (1198) ◽  
pp. 19-44 ◽  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Proteolytic Enzymes ◽  
Phosphoglycerate Mutase ◽  
Crystallographic Structure ◽  
X Ray ◽  
C Terminus ◽  
Detailed Interpretation ◽  
High Degree

The complete amino acid sequence of yeast phosphoglycerate mutase comprising 241 residues has been determined. The sequence was deduced from the two cyanogen bromide fragments, and from the peptides derived from these fragments after digestion by a number of proteolytic enzymes. Determination of this sequence now allows a detailed interpretation of the existing high-resolution X-ray crystallographic structure. A comparison of the sequence reported here with the sequences of peptides from phosphoglycerate mutases from other species, and with the sequence of erythrocyte diphosphoglycerate mutase, indicates that these enzymes have a high degree of structural homology. Autolysis of phosphoglycerate mutase by yeast extracts leads to the complete loss of mutase activity, and the formation of electrophoretically distinguishable forms (R. Sasaki, E. Sugimoto & H. Chiba, Archs Biochem. Biophys. 115, 53-61 (1966)). It is apparent from the amino acid sequence that these changes are due to the loss of an 8─12 residue peptide from the C-terminus.

Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae

1986 ◽  
Vol 6 (11) ◽  
pp. 3990-3998
Author(s):  
S Harashima ◽  
A G Hinnebusch
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Lacz Gene ◽  
Gene Products ◽  
Wild Type ◽  
Double Mutation ◽  
Double Stranded Rna ◽  
Amino Acid Availability ◽  
Genes Encoding ◽  
New Alleles

GCN4 encodes a positive regulator of multiple unlinked genes encoding amino acid biosynthetic enzymes in Saccharomyces cerevisiae. Expression of GCN4 is coupled to amino acid availability by a control mechanism involving GCD1 as a negative effector and GCN1, GCN2, and GCN3 as positive effectors of GCN4 expression. We used reversion of a gcn2 gcn3 double mutation to isolate new alleles of GCD1 and mutations in four additional GCD genes which we designate GCD10, GCD11, GCD12, and GCD13. All of the mutations lead to constitutive derepression of HIS4 transcription in the absence of the GCN2+ and GCN3+ alleles. By contrast, the gcd mutations require the wild-type GCN4 allele for their derepressing effect, suggesting that each acts by influencing the level of GCN4 activity in the cell. Consistent with this interpretation, mutations in each GCD gene lead to constitutive derepression of a GCN4::lacZ gene fusion. Thus, at least five gene products are required to maintain the normal repressed level of GCN4 expression in nonstarvation conditions. Interestingly, the gcd mutations are pleiotropic and also affect growth rate in nonstarvation conditions. In addition, certain alleles lead to a loss of M double-stranded RNA required for the killer phenotype. This pleiotropy suggests that the GCD gene products contribute to an essential cellular function, in addition to, or in conjunction with, their role in GCN4 regulation.

Sign in / Sign up

Export Citation Format

Share Document