scholarly journals A Region in the Bacillus subtilisTranscription Factor Spo0A That Is Important for spoIIGPromoter Activation

1998 ◽  
Vol 180 (14) ◽  
pp. 3578-3583 ◽  
Author(s):  
Cindy M. Buckner ◽  
Ghislain Schyns ◽  
Charles P. Moran
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Amino Acid Substitution ◽  
Dna Binding Protein ◽  
Single Amino Acid ◽  
Promoter Activation ◽  
Additional Amino Acid

ABSTRACT Spo0A is a DNA binding protein in Bacillus subtilisrequired for the activation of spoIIG and other promoters at the onset of endospore formation. Activation of some of these promoters may involve interaction of Spo0A and the ςAsubunit of RNA polymerase. Previous studies identified two single-amino-acid substitutions in ςA, K356E and H359R, that specifically impaired Spo0A-dependent transcription in vivo. Here we report the identification of an amino acid substitution in Spo0A (S231F) that suppressed the sporulation deficiency due to the H359R substitution in ςA. We also found that the S231F substitution partially restored use of the spoIIG promoter by the ςA H359R RNA polymerase in vitro. Alanine substitutions in the 231 region of Spo0A revealed an additional amino acid residue important for spoIIG promoter activation, I229. This amino acid substitution in Spo0A did not affect repression of abrB transcription, indicating that the alanine-substituted Spo0A was not defective in DNA binding. Moreover, the alanine-substituted Spo0A protein activated the spoIIApromoter; therefore, this region of Spo0A is probably not required for Spo0A-dependent, ςH-directed transcription. These and other results suggest that the region of Spo0A near position 229 is involved in ςA-dependent promoter activation.

Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

1992 ◽  
Vol 12 (5) ◽  
pp. 2372-2382
Author(s):  
K M Arndt ◽  
S L Ricupero ◽  
D M Eisenmann ◽  
F Winston
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Repeat ◽  
Single Amino Acid ◽  
Wild Type ◽  
Dna Binding Specificity ◽  
Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

scholarly journals Virus Susceptibility Analyses from a Phase IV Clinical Trial of Inhaled Zanamivir Treatment in Children Infected with Influenza

2013 ◽  
Vol 57 (4) ◽  
pp. 1677-1684 ◽  
Author(s):  
Phillip J. Yates ◽  
Nalini Mehta ◽  
Joseph Horton ◽  
Margaret Tisdale
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Drug Pressure ◽  
Receptor Binding Site ◽  
Virus Susceptibility ◽  
Susceptibility Data

ABSTRACTA zanamivir postapproval efficacy study was conducted in children (n= 279) in Japan during three influenza seasons. Pharyngeal swab specimens (n= 714) were obtained for detailed resistance analysis. From 371 cultured viruses, 3 viruses (A/H1N1) from two subjects showed reduced susceptibility to zanamivir at day 1 (before treatment), 1 had an N74S amino acid substitution (fold shift, 46), and 2 (day 1 and day 2) had a Q136K amino acid substitution (fold shifts, 292 and 301). Q136K was detected only in cultured virus and not in the swab. From the remaining 118 cultured viruses obtained during or after treatment with zanamivir, no shifts in virus susceptibility were detected. Neuraminidase (NA) population sequencing showed that viruses from 12 subjects had emergent amino acid substitutions, but 3 with susceptibility data were not zanamivir resistant. The remainder may be natural variants. Further analysis is planned. Hemagglutinin (HA) sequencing showed that viruses from 20 subjects had 9 HA amino acid substitutions that were previously implicated in resistance to neuraminidase inhibitors inin vitroassays or that were close to the receptor binding site. Their role inin vivoresistance appears to be less important but is not well understood. NA clonal sequence analysis was undertaken to determine if minority species of resistant viruses were present. A total of 1,682 clones from 90 subjects were analyzed. Single clones from 12 subjects contained amino acid substitutions close to the NA active site. It is unclear whether these single amino acid substitutions could have been amplified after drug pressure or are just chance mutations introduced during PCR.

scholarly journals α-Helix E of Spo0A Is Required for σA- but Not for σH-Dependent Promoter Activation in Bacillus subtilis

2004 ◽  
Vol 186 (4) ◽  
pp. 1078-1083 ◽  
Author(s):  
Amrita Kumar ◽  
James A. Brannigan ◽  
Charles P. Moran
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Dna Binding Protein ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Promoter Activation ◽  
Α Helix

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.

scholarly journals Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro.

1992 ◽  
Vol 12 (5) ◽  
pp. 2372-2382 ◽  
Author(s):  
K M Arndt ◽  
S L Ricupero ◽  
D M Eisenmann ◽  
F Winston
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Repeat ◽  
Single Amino Acid ◽  
Wild Type ◽  
Dna Binding Specificity ◽  
Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

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

2004 ◽  
Vol 186 (1) ◽  
pp. 200-206 ◽  
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.

scholarly journals In Vivo Destabilization and Functional Defects of the Xeroderma Pigmentosum C Protein Caused by a Pathogenic Missense Mutation

2007 ◽  
Vol 27 (19) ◽  
pp. 6606-6614 ◽  
Author(s):  
Gentaro Yasuda ◽  
Ryotaro Nishi ◽  
Eriko Watanabe ◽  
Toshio Mori ◽  
Shigenori Iwai ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Amino Acid Substitution ◽  
Xeroderma Pigmentosum ◽  
Excision Repair ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Dependent Manner ◽  
Damaged Dna

ABSTRACT Xeroderma pigmentosum group C (XPC) protein plays an essential role in DNA damage recognition in mammalian global genome nucleotide excision repair (NER). Here, we analyze the functional basis of NER inactivation caused by a single amino acid substitution (Trp to Ser at position 690) in XPC, previously identified in the XPC patient XP13PV. The Trp690Ser change dramatically affects the in vivo stability of the XPC protein, thereby causing a significant reduction of its steady-state level in XP13PV fibroblasts. Despite normal heterotrimeric complex formation and physical interactions with other NER factors, the mutant XPC protein lacks binding affinity for both undamaged and damaged DNA. Thus, this single amino acid substitution is sufficient to compromise XPC function through both quantitative and qualitative alterations of the protein. Although the mutant XPC fails to recognize damaged DNA, it is still capable of accumulating in a UV-damaged DNA-binding protein (UV-DDB)-dependent manner to UV-damaged subnuclear domains. However, the NER factors transcription factor IIH and XPA failed to colocalize stably with the mutant XPC. As well as highlighting the importance of UV-DDB in recruiting XPC to UV-damaged sites, these findings demonstrate the role of DNA binding by XPC in the assembly of subsequent NER intermediate complexes.

A novel polymorphism of human gene has an amino acid substitution (V365M) that decreases enzymatic activity in vitro and in vivo

2004 ◽  
Vol 76 (6) ◽  
pp. 519-527 ◽  
Author(s):  
T FUKAMI ◽  
M NAKAJIMA ◽  
R YOSHIDA ◽  
Y TSUCHIYA ◽  
Y FUJIKI ◽  
...  
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Amino Acid Substitution ◽  
Human Gene

scholarly journals The DNA-binding protein HTa from Thermoplasma acidophilum is an archaeal histone analog

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Antoine Hocher ◽  
Maria Rojec ◽  
Jacob B Swadling ◽  
Alexander Esin ◽  
Tobias Warnecke
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Coli Strain ◽  
Micrococcal Nuclease ◽  
Thermoplasma Acidophilum ◽  
Chromatin Architecture ◽  
Archaeal Histone

Histones are a principal constituent of chromatin in eukaryotes and fundamental to our understanding of eukaryotic gene regulation. In archaea, histones are widespread but not universal: several lineages have lost histone genes. What prompted or facilitated these losses and how archaea without histones organize their chromatin remains largely unknown. Here, we elucidate primary chromatin architecture in an archaeon without histones, Thermoplasma acidophilum, which harbors a HU family protein (HTa) that protects part of the genome from micrococcal nuclease digestion. Charting HTa-based chromatin architecture in vitro, in vivo and in an HTa-expressing E. coli strain, we present evidence that HTa is an archaeal histone analog. HTa preferentially binds to GC-rich sequences, exhibits invariant positioning throughout the growth cycle, and shows archaeal histone-like oligomerization behavior. Our results suggest that HTa, a DNA-binding protein of bacterial origin, has converged onto an architectural role filled by histones in other archaea.

scholarly journals Single amino acid mutations effect Zika virus replication in vitro and virulence in vivo

2020 ◽  
Author(s):  
Nicole M. Collette ◽  
Victoria H.I. Lao ◽  
Dina R. Weilhammer ◽  
Barbara Zingg ◽  
Shoshana D. Cohen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Zika Virus ◽  
Model Systems ◽  
Single Amino Acid ◽  
Replication Rate ◽  
Adult Mice ◽  
Naturally Occurring ◽  
Viral Virulence

AbstractThe 2014-2016 Zika virus (ZIKV) epidemic in the Americas resulted in large deposits of next-generation sequencing data from clinical samples. This resource was mined to identify emerging mutations and trends in mutations as the outbreak progressed over time. Information on transmission dynamics, prevalence and persistence of intra-host mutants, and the position of a mutation on a protein were then used to prioritize 544 reported mutations based on their ability to impact ZIKV phenotype. Using this criteria, six mutants (representing naturally occurring mutations) were generated as synthetic infectious clones using a 2015 Puerto Rican epidemic strain PRVABC59 as the parental backbone. The phenotypes of these naturally occurring variants were examined using both cell culture and murine model systems. Mutants had distinct phenotypes, including changes in replication rate, embryo death, and decreased head size. In particular, a NS2B mutant previously detected during in vivo studies in rhesus macaques was found to cause lethal infections in adult mice, abortions in pregnant females, and increased viral genome copies in both brain tissue and blood of female mice. Additionally, mutants with changes in the region of NS3 that interfaces with NS5 during replication displayed reduced replication in the blood of adult mice. This analytical pathway, integrating both bioinformatic and wet lab experiments, provides a foundation for understanding how naturally occurring single mutations affect disease outcome and can be used to predict the of severity of future ZIKV outbreaks.Author summaryTo determine if naturally occurring individual mutations in the Zika virus epidemic genotype effect viral virulence or replication rate in vitro or in vivo, we generated an infectious clone representing the epidemic genotype of stain Puerto Rico, 2015. Using this clone, six mutants were created by changing nucleotides in the genome to cause one to two amino acid substitutions in the encoded proteins. The six mutants we generated represent mutations that differentiated the early epidemic genotype from genotypes that were either ancestral or that occurred later in the epidemic. We assayed each mutant for changes in growth rate, and for virulence in adult mice and pregnant mice. Three of the mutants caused catastrophic embryo effects including increased embryonic death or significant decrease in head diameter. Three other mutants that had mutations in a genome region associated with replication resulted in changes in in vitro and in vivo replication rates. These results illustrate the potential impact of individual mutations in viral phenotype.

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