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.

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.

In-silico structural, functional and immunogenic characterization of Taenia solium TS14 protein

2017 ◽  
Author(s):  
Chitra Joshi ◽  
Siddharth Gautam
Keyword(s):  
Amino Acid ◽  
In Silico ◽  
Mutational Analysis ◽  
Solvent Accessibility ◽  
Taenia Solium ◽  
Secretory Protein ◽  
Single Amino Acid

TS14, a Cysticercosis cellulosae derived protein, has been exploited for immunodiagnosis of cysticercosis in humans and pigs. However, the information on structure, function, stability and immunogenicity of TS14 derived from different isolates is primarily lacking. The present study deals with in-silico characterization of six TS14 isolates. High thermostability and an isoelectric point of 9.41 were recorded. Based on N-terminal amino acid residues, high resistance to intracellular proteases with extended in-vivo and in-vitro half-lives was predicted. TS14 is foreseen as a secretory protein with a signal peptide and an extracellular localization. Structural analysis of TS14 exhibited the dominance of helices in the secondary structure (92% coverage) with majority of residues showing high and medium solvent accessibility. High lysine content and presence of multiple nucleotide binding sites in TS14 suggests interaction with RNA/DNA and a role in their metabolism. Immunogenic profiling predicted presence of four distinct B-cell epitopes. Mutational analysis based on the single amino acid substitutions among six TS14 isolates demonstrated minor variations in structural stability; however, all the substitutions were well tolerated. Moreover, all the isolates revealed almost identical immunogenic profile with an equivocal potential to elicit the antibody-mediated immune response.

scholarly journals Discrimination of DNA binding sites by mutant p53 proteins.

1995 ◽  
Vol 15 (9) ◽  
pp. 5196-5202 ◽  
Author(s):  
S K Thukral ◽  
Y Lu ◽  
G C Blain ◽  
T S Harvey ◽  
V L Jacobsen
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Base Pair ◽  
Molecular Genetic ◽  
Full Length ◽  
Single Amino Acid ◽  
Wild Type ◽  
Binding Assays ◽  
Wild Type P53

Critical determinants of DNA recognition by p53 have been identified by a molecular genetic approach. The wild-type human p53 fragment containing amino acids 71 to 330 (p53(71-330)) was used for in vitro DNA binding assays, and full-length human p53 was used for transactivation assays with Saccharomyces cerevisiae. First, we defined the DNA binding specificity of the wild-type p53 fragment by using systematically altered forms of a known consensus DNA site. This refinement indicates that p53 binds with high affinity to two repeats of PuGPuCA.TGPyCPy, a further refinement of an earlier defined consensus half site PuPuPuC(A/T).(T/A) GPyPyPy. These results were further confirmed by transactivation assays of yeast by using full-length human p53 and systematically altered DNA sites. Dimers of the pentamer AGGCA oriented either head-to-head or tail-to-tail bound efficiently, but transactivation was facilitated only through head-to-head dimers. To determine the origins of specificity in DNA binding by p53, we identified mutations that lead to altered specificities of DNA binding. Single-amino-acid substitutions were made at several positions within the DNA binding domain of p53, and this set of p53 point mutants were tested with DNA site variants for DNA binding. DNA binding analyses showed that the mutants Lys-120 to Asn, Cys-277 to Gln or Arg, and Arg-283 to Gln bind to sites with noncanonical base pair changes at positions 2, 3, and 1 in the pentamer (PuGPuCA), respectively. Thus, we implicate these residues in amino acid-base pair contacts. Interestingly, mutant Cys-277 to Gln bound a consensus site as two and four monomers, as opposed to the wild-type p53 fragment, which invariably binds this site as four monomers.

scholarly journals A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model

2017 ◽  
Vol 91 (15) ◽  
Author(s):  
Sujit K. Mohanty ◽  
Bryan Donnelly ◽  
Phylicia Dupree ◽  
Inna Lobeck ◽  
Sarah Mowery ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biliary Atresia ◽  
Amino Acid Change ◽  
Reverse Genetics ◽  
Single Amino Acid ◽  
Wild Type ◽  
Neonatal Mice ◽  
Single Amino Acid Change

ABSTRACT Rotavirus infection is one of the most common causes of diarrheal illness in humans. In neonatal mice, rhesus rotavirus (RRV) can induce biliary atresia (BA), a disease resulting in inflammatory obstruction of the extrahepatic biliary tract and intrahepatic bile ducts. We previously showed that the amino acid arginine (R) within the sequence SRL (amino acids 445 to 447) in the RRV VP4 protein is required for viral binding and entry into biliary epithelial cells. To determine if this single amino acid (R) influences the pathogenicity of the virus, we generated a recombinant virus with a single amino acid mutation at this site through a reverse genetics system. We demonstrated that the RRV mutant (RRVVP4-R446G) produced less symptomatology and replicated to lower titers both in vivo and in vitro than those seen with wild-type RRV, with reduced binding in cholangiocytes. Our results demonstrate that a single amino acid change in the RRV VP4 gene influences cholangiocyte tropism and reduces pathogenicity in mice. IMPORTANCE Rotavirus is the leading cause of diarrhea in humans. Rhesus rotavirus (RRV) can also lead to biliary atresia (a neonatal human disease) in mice. We developed a reverse genetics system to create a mutant of RRV (RRVVP4-R446G) with a single amino acid change in the VP4 protein compared to that of wild-type RRV. In vitro, the mutant virus had reduced binding and infectivity in cholangiocytes. In vivo, it produced fewer symptoms and lower mortality in neonatal mice, resulting in an attenuated form of biliary atresia.

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.

Acquisition of NFKB1-selective DNA binding by substitution of four amino acid residues from NFKB1 into RelA

1993 ◽  
Vol 13 (7) ◽  
pp. 3850-3859
Author(s):  
T A Coleman ◽  
C Kunsch ◽  
M Maher ◽  
S M Ruben ◽  
C A Rosen
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Fusion Protein ◽  
Binding Specificity ◽  
Single Amino Acid ◽  
Nf Kappa B ◽  
Dna Binding Specificity ◽  
Dna Motif ◽  
Single Amino Acid Change

The subunits of NF-kappa B, NFKB1 (formerly p50) and RelA (formerly p65), belong to a growing family of transcription factors that share extensive similarity to the c-rel proto-oncogene product. The homology extends over a highly conserved stretch of approximately 300 amino acids termed the Rel homology domain (RHD). This region has been shown to be involved in both multimerization (homo- and heterodimerization) and DNA binding. It is now generally accepted that homodimers of either subunit are capable of binding DNA that contains a kappa B site originally identified in the immunoglobulin enhancer. Recent studies have demonstrated that the individual subunits of the NF-kappa B transcription factor complex can be distinguished by their ability to bind distinct DNA sequence motifs. By using NFKB1 and RelA subunit fusion proteins, different regions within the RHD were found to confer DNA-binding and multimerization functions. A fusion protein that contains 34 N-terminal amino acids of NFKB1 and 264 amino acids of RelA displayed preferential binding to an NFKB1-selective DNA motif while dimerizing with the characteristics of RelA. Within the NFKB1 portion of this fusion protein, a single amino acid change of His to Arg altered the DNA-binding specificity to favor interaction with the RelA-selective DNA motif. Furthermore, substitution of four amino acids from NFKB1 into RelA was able to alter the DNA-binding specificity of the RelA protein to favor interaction with the NFKB1-selective site. Taken together, these findings demonstrate the presence of a distinct subdomain within the RHD involved in conferring the DNA-binding specificity of the Rel family of proteins.

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.

scholarly journals Single amino acid exchanges in separate domains of the Drosophila serendipity delta zinc finger protein cause embryonic and sex biased lethality.

Genetics ◽  
1992 ◽  
Vol 131 (4) ◽  
pp. 905-916 ◽  
Author(s):  
M Crozatier ◽  
K Kongsuwan ◽  
P Ferrer ◽  
J R Merriam ◽  
J A Lengyel ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Essential Gene ◽  
Larval Instar ◽  
Single Amino Acid ◽  
Isolation And Characterization ◽  
Finger Protein

Abstract The Drosophila serendipity (sry) delta (delta) zinc finger protein is a sequence-specific DNA binding protein, maternally inherited by the embryo and present in nuclei of transcriptionally active cells throughout fly development. We report here the isolation and characterization of four ethyl methanesulfate-induced zygotic lethal mutations of different strengths in the sry delta gene. For the stronger allele, all of the lethality occurs during late embryogenesis or the first larval instar. In the cases of the three weaker alleles, most of the lethality occurs during pupation; moreover, those adult escapers that emerge are sterile males lacking partially or completely in spermatozoa bundles. Genetic analysis of sry delta thus indicates that it is an essential gene, whose continued expression throughout the life cycle, notably during embryogenesis and pupal stage, is required for viability. Phenotypic analysis of sry delta hemizygote escaper males further suggests that sry delta may be involved in regulation of two different sets of genes: genes required for viability and genes involved in gonadal development. All four sry delta alleles are fully rescued by a wild-type copy of sry delta, but not by an additional copy of the sry beta gene, reinforcing the view that, although structurally related, these two genes exert distinct functions. Molecular characterization of the four sry delta mutations revealed that these mutations correspond to single amino acid replacements in the sry delta protein. Three of these replacements map to the same (third out of seven) zinc finger in the carboxy-terminal DNA binding domain; interestingly, none affects the zinc finger consensus residues. The fourth mutation is located in the NH2-proximal part of the protein, in a domain proposed to be involved in specific protein-protein interactions.

scholarly journals A single amino acid change in CUP2 alters its mode of DNA binding.

1990 ◽  
Vol 10 (9) ◽  
pp. 4778-4787 ◽  
Author(s):  
C Buchman ◽  
P Skroch ◽  
W Dixon ◽  
T D Tullius ◽  
M Karin
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Binding Activity ◽  
Binding Domain ◽  
Single Amino Acid ◽  
Dna Binding Domain ◽  
Sequence Motifs ◽  
Wild Type ◽  
Mobility Shift ◽  
Single Amino Acid Change

CUP2 is a copper-dependent transcriptional activator of the yeast CUP1 metallothionein gene. In the presence of Cu+ and Ag+) ions its DNA-binding domain is thought to fold as a cysteine-coordinated Cu cluster which recognizes the palindromic CUP1 upstream activation sequence (UASc). Using mobility shift, methylation interference, and DNase I and hydroxyl radical footprinting assays, we examined the interaction of wild-type and variant CUP2 proteins produced in Escherichia coli with the UASc. Our results suggest that CUP2 has a complex Cu-coordinated DNA-binding domain containing different parts that function as DNA-binding elements recognizing distinct sequence motifs embedded within the UASc. A single-amino-acid substitution of cysteine 11 with a tyrosine results in decreased Cu binding, apparent inactivation of one of the DNA-binding elements and a dramatic change in the recognition properties of CUP2. This variant protein interacts with only one part of the wild-type site and prefers to bind to a different half-site from the wild-type protein. Although the variant has about 10% of wild-type DNA-binding activity, it appears to be completely incapable of activating transcription.

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