scholarly journals Lineage-Specific Amino Acid Substitutions in Region 2 of the RNA Polymerase σ Subunit Affect the Temperature of Promoter Opening

2008 ◽  
Vol 190 (8) ◽  
pp. 3088-3092 ◽  
Author(s):  
N. Barinova ◽  
E. Zhilina ◽  
I. Bass ◽  
V. Nikiforov ◽  
A. Kulbachinskiy
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Amino Acid Substitutions ◽  
Amino Acid Residues ◽  
Specific Amino Acid ◽  
Promoter Recognition

ABSTRACT Highly conserved amino acid residues in region 2 of the RNA polymerase σ subunit are known to participate in promoter recognition and opening. We demonstrated that nonconserved residues in this region collectively determine lineage-specific differences in the temperature of promoter opening.

scholarly journals A Region of ςK Involved in Promoter Activation by GerE in Bacillus subtilis

1999 ◽  
Vol 181 (14) ◽  
pp. 4365-4373 ◽  
Author(s):  
Kathryn H. Wade ◽  
Ghislain Schyns ◽  
Jason A. Opdyke ◽  
Charles P. Moran
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Promoter Activity ◽  
Amino Acid Substitutions ◽  
Amino Acid Residues ◽  
Transcription In Vitro ◽  
A Site ◽  
Initial Binding

ABSTRACT During endospore formation in Bacillus subtilis, the DNA binding protein GerE stimulates transcription from several promoters that are used by RNA polymerase containing ςK. GerE binds to a site on one of these promoters, cotX, that overlaps its −35 region. We tested the model that GerE interacts with ςK at the cotX promoter by seeking amino acid substitutions in ςK that interfered with GerE-dependent activation of the cotX promoter but which did not affect utilization of the ςK-dependent, GerE-independent promoter gerE. We identified two amino acid substitutions in ςK, E216K and H225Y, that decrease cotXpromoter utilization but do not affect gerE promoter activity. Alanine substitutions at these positions had similar effects. We also examined the effects of the E216A and H225Y substitutions in ςK on transcription in vitro. We found that these substitutions specifically reduced utilization of the cotXpromoter. These and other results suggest that the amino acid residues at positions 216 and 225 are required for GerE-dependentcotX promoter activity, that the histidine at position 225 of ςK may interact with GerE at the cotXpromoter, and that this interaction may facilitate the initial binding of ςK RNA polymerase to the cotX promoter. We also found that the alanine substitutions at positions 216 and 225 of ςK had no effect on utilization of the GerE-dependent promoter cotD, which contains GerE binding sites that do not overlap with its −35 region.

scholarly journals Comparing mutagenesis and simulations as tools for identifying functionally important sequence changes for protein thermal adaptation

2018 ◽  
Vol 116 (2) ◽  
pp. 679-688 ◽  
Author(s):  
Ming-ling Liao ◽  
George N. Somero ◽  
Yun-wei Dong
Keyword(s):  
Amino Acid ◽  
Thermal Adaptation ◽  
Site Directed Mutagenesis ◽  
Dynamics Simulation ◽  
Amino Acid Substitutions ◽  
Thermal Stabilities ◽  
Specific Amino Acid ◽  
Enzyme Thermal Stability ◽  
And Function

Comparative studies of orthologous proteins of species evolved at different temperatures have revealed consistent patterns of temperature-related variation in thermal stabilities of structure and function. However, the precise mechanisms by which interspecific variations in sequence foster these adaptive changes remain largely unknown. Here, we compare orthologs of cytosolic malate dehydrogenase (cMDH) from marine molluscs adapted to temperatures ranging from −1.9 °C (Antarctica) to ∼55 °C (South China coast) and show how amino acid usage in different regions of the enzyme (surface, intermediate depth, and protein core) varies with adaptation temperature. This eukaryotic enzyme follows some but not all of the rules established in comparisons of archaeal and bacterial proteins. To link the effects of specific amino acid substitutions with adaptive variations in enzyme thermal stability, we combined site-directed mutagenesis (SDM) and in vitro protein experimentation with in silico mutagenesis using molecular dynamics simulation (MDS) techniques. SDM and MDS methods generally but not invariably yielded common effects on protein stability. MDS analysis is shown to provide insights into how specific amino acid substitutions affect the conformational flexibilities of mobile regions (MRs) of the enzyme that are essential for binding and catalysis. Whereas these substitutions invariably lie outside of the MRs, they effectively transmit their flexibility-modulating effects to the MRs through linked interactions among surface residues. This discovery illustrates that regions of the protein surface lying outside of the site of catalysis can help establish an enzyme’s thermal responses and foster evolutionary adaptation of function.

scholarly journals Genetic Diversity of the cagA gene of Helicobacter pylori strains from Sudanese Patients with Different Gastroduodenal Diseases

2019 ◽  
Author(s):  
Hadeel Gassim Hassan ◽  
Abeer Babiker Idris ◽  
Mohamed A. Hassan ◽  
Hisham N. Altayb ◽  
Kyakonye Yasin ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Amino Acid ◽  
Novel Mutation ◽  
Amino Acid Residues ◽  
Specific Amino Acid ◽  
Gastrointestinal Malignancy ◽  
High Incidence ◽  
H Pylori ◽  
High Level ◽  
Chloride Method

AbstractBackgroundThere is an increase in the prevalence of Helicobacter pylori infection in Sudan, accompanied by a high incidence of upper gastrointestinal malignancy. The cytotoxin-associated gene cagA gene is a marker of a pathogenicity island (PAI) in H. pylori and plays a crucial role in determining the clinical outcome of Helicobacter infections.ObjectiveThis study aimed to determine the frequency and heterogeneity of the cagA gene of H. pylori and correlate the presence of cagA gene with clinical outcomes.Materials and methodsFifty endoscopy biopsies were collected from Fedail and Soba hospitals in Khartoum state. DNA was extracted using the Guanidine chloride method followed by PCR to amplify 16S rRNA and cagA gene of H. pylori using specific primers. DNA amplicons of cagA gene were purified and sequenced. Bioinformatics and statistical analysis were done to characterize and to test the association between cagA gene and gastric complications.ResultsCagA gene was detected in 20/37(54%) of the samples that were found positive for H. pylori. There was no association between endoscopy finding and the presence of the cagA gene (p = 0.225). Specific amino acid variations were found at seven loci related to strains from a patient with duodenitis, gastric ulcer, and gastric atrophy (R448H, T457K, S460L, IT463-464VA, D470E, A482Q, KNV490-491-492TKT) while mutations in cancerous strain were A439P, T457P, and H500Y.ConclusionDisease-specific variations of cagA of H. pylori strains, in the region of amino acid residues 428-510, were evident among Sudanese patients with different gastroduodenal diseases. A novel mutation (K458N) was detected in a patient with duodenitis, which affects the positive electrostatic surface of cagA. Phylogenetic analysis showed a high level of diversity of cagA from Sudanese H. pylori strains.

scholarly journals RPC53 encodes a subunit of Saccharomyces cerevisiae RNA polymerase C (III) whose inactivation leads to a predominantly G1 arrest.

1992 ◽  
Vol 12 (10) ◽  
pp. 4314-4326 ◽  
Author(s):  
C Mann ◽  
J Y Micouin ◽  
N Chiannilkulchai ◽  
I Treich ◽  
J M Buhler ◽  
...  
Keyword(s):  
Cell Division ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Essential Gene ◽  
Temperature Sensitive ◽  
G1 Arrest ◽  
Restrictive Temperature ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Carboxy Terminal

RPC53 is shown to be an essential gene encoding the C53 subunit specifically associated with yeast RNA polymerase C (III). Temperature-sensitive rpc53 mutants were generated and showed a rapid inhibition of tRNA synthesis after transfer to the restrictive temperature. Unexpectedly, the rpc53 mutants preferentially arrested their cell division in the G1 phase as large, round, unbudded cells. The RPC53 DNA sequence is predicted to code for a hydrophilic M(r)-46,916 protein enriched in charged amino acid residues. The carboxy-terminal 136 amino acids of C53 are significantly similar (25% identical amino acid residues) to the same region of the human BN51 protein. The BN51 cDNA was originally isolated by its ability to complement a temperature-sensitive hamster cell mutant that undergoes a G1 cell division arrest, as is true for the rpc53 mutants.

Protein truncation is required for the activation of the c-myb proto-oncogene

1991 ◽  
Vol 11 (8) ◽  
pp. 3987-3996
Author(s):  
F A Grässer ◽  
T Graf ◽  
J S Lipsick
Keyword(s):  
Amino Acid ◽  
Bone Marrow Cells ◽  
Protein Product ◽  
Full Length ◽  
Carboxyl Terminus ◽  
Amino Acid Substitutions ◽  
Amino Acid Residues ◽  
Avian Myeloblastosis Virus ◽  
Virally Encoded ◽  
Myb Proteins

The protein product of the v-myb oncogene of avian myeloblastosis virus, v-Myb, differs from its normal cellular counterpart, c-Myb, by (i) expression under the control of a strong viral long terminal repeat, (ii) truncation of both its amino and carboxyl termini, (iii) replacement of these termini by virally encoded residues, and (iv) substitution of 11 amino acid residues. We had previously shown that neither the virally encoded termini nor the amino acid substitutions are required for transformation by v-Myb. We have now constructed avian retroviruses that express full-length or singly truncated forms of c-Myb and have tested them for the transformation of chicken bone marrow cells. We conclude that truncation of either the amino or carboxyl terminus of c-Myb is sufficient for transformation. In contrast, the overexpression of full-length c-Myb does not result in transformation. We have also shown that the amino acid substitutions of v-Myb by themselves are not sufficient for the activation of c-Myb. Rather, the presence of either the normal amino or carboxyl terminus of c-Myb can suppress transformation when fused to v-Myb. Cells transformed by c-Myb proteins truncated at either their amino or carboxyl terminus appear to be granulated promyelocytes that express the Mim-1 protein. Cells transformed by a doubly truncated c-Myb protein are not granulated but do express the Mim-1 protein, in contrast to monoblasts transformed by v-Myb that neither contain granules nor express Mim-1. These results suggest that various alterations of c-Myb itself may determine the lineage of differentiating hematopoietic cells.

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