Levansucrase of Bacillus subtilis: Effects on the Secretion Process of Single Amino Acid Substitutions in the Mature Part of the Protein

ABSTRACT At the onset of endospore formation in Bacillus subtilis, the DNA binding protein Spo0A activates transcription from two types of promoters. The first type includes the spoIIG and spoIIE promoters, which are used by σA-RNA polymerase, whereas the second type includes the spoIIA promoter, which is used by RNA polymerase containing the secondary sigma factor σH. Previous genetic analyses have identified specific amino acids in α-helix E of Spo0A that are important for activation of Spo0A-dependent, σA-dependent promoters. However, these amino acids are not required for activation of the σH-dependent spoIIA promoter. We now report the effects of additional single-amino-acid substitutions and the effects of deletions in α-helix E. The effects of alanine substitutions revealed one new position (239) in Spo0A that appears to be specifically required for activation of the σA-dependent promoters. Based on the effects of a deletion mutation, we suggest that α-helix E in Spo0A is not directly involved in interaction with σH-RNA polymerase.

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Surfaces of Spo0A and RNA Polymerase Sigma Factor A That Interact at the spoIIG Promoter in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.186.1.200-206.2004 ◽

2004 ◽

Vol 186 (1) ◽

pp. 200-206 ◽

Cited By ~ 9

Author(s):

Amrita Kumar ◽

Cindy Buckner Starke ◽

Mark DeZalia ◽

Charles P. Moran

Keyword(s):

Bacillus Subtilis ◽

Amino Acid ◽

Rna Polymerase ◽

Sigma Factor ◽

Dna Binding Protein ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

Promoter Activation ◽

Interacting Surfaces

ABSTRACT In Bacillus subtilis, the DNA binding protein Spo0A activates transcription from two classes of promoters, those used by RNA polymerase containing the primary sigma factor, σA (e.g., spoIIG), and those used by RNA polymerase containing the secondary sigma factor, σH (e.g., spoIIA). Several single amino acid substitutions in region 4 of σA define positions in σA that are specifically required for Spo0A-dependent promoter activation. Similarly, several single amino acid substitutions in Spo0A define positions in Spo0A that are required for σA-dependent promoter activation but not for other functions of Spo0A. It is unknown whether these amino acids in Spo0A interact directly with those in region 4 of σA or whether they interact with another subunit of RNA polymerase to effect promoter activation. Here we report the identification of a new amino acid in region 4 of σA, arginine at position 355 (R355), that is involved in Spo0A-dependent promoter activation. To further investigate the role of R355, we used the coordinates of Spo0A and sigma region 4, each in complex with DNA, to build a model for the interaction of σA and Spo0A at the spoIIG promoter. We tested the model by examining the effects of amino acid substitutions in the putative interacting surfaces of these molecules. As predicted by the model, we found genetic evidence for interaction of R355 of σA with glutamine at position 221 of Spo0A. These results appear to define the surfaces of Spo0A and σA that directly interact during activation of the spoIIG promoter.

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Single amino acid substitutions in the reactive site of antithrombin leading to thrombosis. Congenital substitution of arginine 393 to cysteine in antithrombin Northwick Park and to histidine in antithrombin Glasgow.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)60605-2 ◽

1988 ◽

Vol 263 (12) ◽

pp. 5589-5593 ◽

Cited By ~ 4

Author(s):

H Erdjument ◽

D A Lane ◽

M Panico ◽

V Di Marzo ◽

H R Morris

Keyword(s):

Amino Acid ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

Reactive Site

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A Single Point Mutation, Asn16→Lys, Dictates the Temperature-Sensitivity of the Reovirus tsG453 Mutant

Viruses ◽

10.3390/v13020289 ◽

2021 ◽

Vol 13 (2) ◽

pp. 289

Author(s):

Kathleen K. M. Glover ◽

Danica M. Sutherland ◽

Terence S. Dermody ◽

Kevin M. Coombs

Keyword(s):

Amino Acid ◽

Temperature Sensitivity ◽

Reverse Genetics ◽

Core Protein ◽

Single Point ◽

Single Amino Acid ◽

Single Point Mutation ◽

Temperature Sensitive ◽

Amino Acid Substitutions ◽

Gene Encoding

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

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Natural variants in SARS-CoV-2 Spike protein pinpoint structural and functional hotspots with implications for prophylaxis and therapeutic strategies

Scientific Reports ◽

10.1038/s41598-021-92641-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Suman Pokhrel ◽

Benjamin R. Kraemer ◽

Scott Burkholz ◽

Daria Mochly-Rosen

Keyword(s):

Amino Acid ◽

Severe Disease ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

Severe Respiratory Distress ◽

S Protein ◽

Individual Sequences ◽

Natural Variants ◽

Novel Coronavirus ◽

The Individual

AbstractIn December 2019, a novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of pneumonia with severe respiratory distress and outbreaks in Wuhan, China. The rapid and global spread of SARS-CoV-2 resulted in the coronavirus 2019 (COVID-19) pandemic. Earlier during the pandemic, there were limited genetic viral variations. As millions of people became infected, multiple single amino acid substitutions emerged. Many of these substitutions have no consequences. However, some of the new variants show a greater infection rate, more severe disease, and reduced sensitivity to current prophylaxes and treatments. Of particular importance in SARS-CoV-2 transmission are mutations that occur in the Spike (S) protein, the protein on the viral outer envelope that binds to the human angiotensin-converting enzyme receptor (hACE2). Here, we conducted a comprehensive analysis of 441,168 individual virus sequences isolated from humans throughout the world. From the individual sequences, we identified 3540 unique amino acid substitutions in the S protein. Analysis of these different variants in the S protein pinpointed important functional and structural sites in the protein. This information may guide the development of effective vaccines and therapeutics to help arrest the spread of the COVID-19 pandemic.

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Influence of single amino acid substitutions on electrophoretic mobility of sodium dodecyl sulfate-protein complexes

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(78)90907-5 ◽

1978 ◽

Vol 82 (2) ◽

pp. 532-539 ◽

Cited By ~ 155

Author(s):

Wilfried W. de Jong ◽

Anneke Zweers ◽

Louis H. Cohen

Keyword(s):

Amino Acid ◽

Sodium Dodecyl Sulfate ◽

Electrophoretic Mobility ◽

Protein Complexes ◽

Sodium Dodecyl ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

Dodecyl Sulfate

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Single Amino Acid Substitutions Disrupt Tetramer Formation in the Dihydroneopterin Aldolase Enzyme ofPneumocystis carinii†

Biochemistry ◽

10.1021/bi980540x ◽

1998 ◽

Vol 37 (33) ◽

pp. 11629-11636 ◽

Cited By ~ 8

Author(s):

M. Carmen Thomas ◽

Stuart P. Ballantine ◽

Susanne S. Bethell ◽

Satty Bains ◽

Paul Kellam ◽

...

Keyword(s):

Amino Acid ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

Tetramer Formation ◽

Dihydroneopterin Aldolase

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Pathogenic alleles in microtubule, secretory granule and extracellular matrix-related genes in familial keratoconus

Human Molecular Genetics ◽

10.1093/hmg/ddab075 ◽

2021 ◽

Author(s):

Vishal Shinde ◽

Nara Sobreira ◽

Elizabeth S Wohler ◽

George Maiti ◽

Nan Hu ◽

...

Keyword(s):

Amino Acid ◽

Cell Cultures ◽

Cell Biology ◽

Stromal Cell ◽

Primary Cilia ◽

Single Amino Acid ◽

European Ancestry ◽

Base Substitution ◽

Amino Acid Substitutions ◽

Functional Link

Abstract Keratoconus is a common corneal defect with a complex genetic basis. By whole exome sequencing of affected members from 11 multiplex families of European ancestry, we identified 23 rare, heterozygous, potentially pathogenic variants in 8 genes. These include nonsynonymous single amino acid substitutions in HSPG2, EML6 and CENPF in two families each, and in NBEAL2, LRP1B, PIK3CG and MRGPRD in three families each; ITGAX had nonsynonymous single amino acid substitutions in two families and an indel with a base substitution producing a nonsense allele in the third family. Only HSPG2, EML6 and CENPF have been associated with ocular phenotypes previously. With the exception of MRGPRD and ITGAX, we detected the transcript and encoded protein of the remaining genes in the cornea and corneal cell cultures. Cultured stromal cells showed cytoplasmic punctate staining of NBEAL2, staining of the fibrillar cytoskeletal network by EML6, while CENPF localized to the basal body of primary cilia. We inhibited the expression of HSPG2, EML6, NBEAL2 and CENPF in stromal cell cultures and assayed for the expression of COL1A1 as a readout of corneal matrix production. An upregulation in COL1A1 after siRNA inhibition indicated their functional link to stromal cell biology. For ITGAX, encoding a leukocyte integrin, we assayed its level in the sera of 3 affected families compared with 10 unrelated controls to detect an increase in all affecteds. Our study identified genes that regulate the cytoskeleton, protein trafficking and secretion, barrier tissue function and response to injury and inflammation, as being relevant to keratoconus.

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Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes.

Molecular and Cellular Biology ◽

10.1128/mcb.6.10.3470 ◽

1986 ◽

Vol 6 (10) ◽

pp. 3470-3480 ◽

Cited By ~ 98

Author(s):

E Moran ◽

B Zerler ◽

T M Harrison ◽

M B Mathews

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Point Mutations ◽

Apparent Molecular Weight ◽

Amino Acid Position ◽

Cysteine Residue ◽

Transformation Function ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

E1a Gene

The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135. Interestingly, conversion of a cysteine residue at position 124 to glycine severely impaired the transformation function without affecting the early adenovirus gene activating functions. Single amino acid substitutions in a different region of the E1A gene had the converse effect. All the mutants produced polypeptides of sufficient stability to be detected by Western immunoblot analysis. The single amino acid substitutions at positions 124 and 135, although impairing the transformation functions, did not detectably alter the formation of the higher-apparent-molecular-weight forms of the E1A products.

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Functional effects of single amino acid substitutions in the region of Phe113 to Asp138 in the plasminogen activator inhibitor 1 molecule

Biochemical Journal ◽

10.1042/bj3310409 ◽

1998 ◽

Vol 331 (2) ◽

pp. 409-415 ◽

Cited By ~ 15

Author(s):

Guang-Chao SUI ◽

Björn WIMAN

Keyword(s):

Amino Acid ◽

Plasminogen Activator ◽

Plasminogen Activator Inhibitor ◽

Site Directed Mutagenesis ◽

Single Amino Acid ◽

Amino Acid Substitutions ◽

Plasminogen Activator Inhibitor 1 ◽

Low Activity ◽

Activator Inhibitor ◽

Pai 1

Thirteen amino acid substitutions have been introduced within the stretch Phe113 to Asp138 in the plasminogen activator inhibitor 1 (PAI-1) molecule by site-directed mutagenesis. The different proteins and wild-type (wt) PAI-1 have been overexpressed in Escherichia coliand purified by chromatography on heparin–Sepharose and on anhydrotrypsin–agarose. The PAI-1 variants have been characterized by their reactivity with tissue plasminogen activator (tPA), interactions with vitronectin or heparin, and stability. Most PAI-1 variants, except for Asp125 → Lys, Phe126 → Ser and Arg133 → Asp, displayed a high spontaneous inhibitory activity towards tPA, which did not change greatly on reactivation with 4 M guanidinium chloride, followed by dialysis at pH 5.5. The variants Asp125 → Lys and Arg133 → Asp became much more active after reactivation and they were also more rapidly transformed to inactive forms (t½ 22–31 min) at physiological pH and temperature than the other variants. However, in the presence of vitronectin they were both almost equally stable (t½ 2.3 h) as wtPAI-1 (t½ 3.0 h). The mutant Glu130 → Lys showed an increased stability, both in the absence and in the presence of vitronectin compared with wtPAI-1. Nevertheless a similar affinity between all the active PAI-1 variants and vitronectin was observed. Further, all mutants, including the three mutants with low activity, were to a large extent adsorbed on anhydrotrypsin–agarose and were eluted in a similar fashion. In accordance with these data, the three variants with a low activity were all to a large extent cleaved as a result of their reaction with tPA, suggesting that they occurred predominantly in the substrate conformation. Our results do not support the presence of a binding site for vitronectin in this part of the molecule, but rather that it might be involved in controlling the active PAI-1 to substrate transition. Partly, this region of the PAI-1 molecule (Arg115 to Arg118) seems also to be involved in the binding of heparin to PAI-1.

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