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 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 A Region in Bacillus subtilisςH Required for Spo0A-Dependent Promoter Activity

1998 ◽  
Vol 180 (18) ◽  
pp. 4987-4990 ◽  
Author(s):  
Cindy M. Buckner ◽  
Charles P. Moran
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Promoter Activity ◽  
Amino Acid Substitutions ◽  
Mutant Form ◽  
Promoter Activation ◽  
Affect Expression

ABSTRACT Spo0A activates transcription in Bacillus subtilis from promoters that are used by two types of RNA polymerase, RNA polymerase containing the primary sigma factor, ςA, and RNA polymerase containing a secondary sigma factor, known as ςH. The region of ςA near positions 356 to 359 is required for Spo0A-dependent promoter activation, possibly because Spo0A interacts with this region of ςA at these promoters. To determine if the amino acids in the corresponding region of ςH are also important in Spo0A-dependent promoter activation, we examined the effects of single alanine substitutions at 10 positions in ςH (201 to 210). Two alanine substitutions in ςH, at glutamine 201 (Q201A) and at arginine 205 (R205A), significantly decreased activity from the Spo0A-dependent, ςH-dependent promoterspoIIA but did not affect expression from the ςH-dependent, Spo0A-independent promoterscitGp2 and spoVG. Therefore, promoter activation by Spo0A requires homologous regions in ςA and ςH. A mutant form of Spo0A, S231F, that suppresses the sporulation defect caused by several amino acid substitutions in ςA did not suppress the sporulation defects caused by the Q201A and R205A substitutions in ςH. This result and others indicate that different surfaces of Spo0A probably interact with ςA and ςH RNA polymerases.

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.

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

1986 ◽  
Vol 6 (10) ◽  
pp. 3470-3480 ◽  
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.

scholarly journals Epistatic interactions can moderate the antigenic effect of substitutions in hemagglutinin of influenza H3N2 virus

2018 ◽  
Author(s):  
Björn F. Koel ◽  
David F. Burke ◽  
Stefan van der Vliet ◽  
Theo M. Bestebroer ◽  
Guus F. Rimmelzwaan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Context Dependence ◽  
Single Amino Acid ◽  
H3n2 Virus ◽  
Amino Acid Substitutions ◽  
Epistatic Interactions ◽  
Context Dependent ◽  
The Impact ◽  
Influenza H3n2

AbstractWe previously showed that single amino acid substitutions at seven positions in hemagglutinin determined major antigenic change of influenza H3N2 virus. Here, the impact of two such substitutions was tested in eleven representative H3 hemagglutinins to investigate context-dependence effects. The antigenic effect of substitutions introduced at hemagglutinin position 145 was fully independent of the amino acid context of the representative hemagglutinins. Antigenic change caused by substitutions introduced at hemagglutinin position 155 was variable and context-dependent. Our results suggest that epistatic interactions with contextual amino acids in the hemagglutinin can moderate the magnitude of antigenic change.

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

1986 ◽  
Vol 6 (10) ◽  
pp. 3470-3480
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.

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 The switch between acute and persistent paramyxovirus infection caused by single amino acid substitutions in the RNA polymerase P subunit

2019 ◽  
Vol 15 (2) ◽  
pp. e1007561 ◽  
Author(s):  
Dan F. Young ◽  
Elizabeth B. Wignall-Fleming ◽  
David C. Busse ◽  
Matthew J. Pickin ◽  
Jack Hankinson ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Single Amino Acid ◽  
Amino Acid Substitutions

scholarly journals The CaaX Proteases, Afc1p and Rce1p, Have Overlapping but Distinct Substrate Specificities

2000 ◽  
Vol 20 (12) ◽  
pp. 4381-4392 ◽  
Author(s):  
Cynthia Evans Trueblood ◽  
Victor L. Boyartchuk ◽  
Elizabeth A. Picologlou ◽  
David Rozema ◽  
C. Dale Poulter ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Single Amino Acid ◽  
In Vitro Assays ◽  
Sequence Motif ◽  
Amino Acid Substitutions ◽  
In The Wild

ABSTRACT Many proteins that contain a carboxyl-terminal CaaX sequence motif, including Ras and yeast a-factor, undergo a series of sequential posttranslational processing steps. Following the initial prenylation of the cysteine, the three C-terminal amino acids are proteolytically removed, and the newly formed prenylcysteine is carboxymethylated. The specific amino acids that comprise the CaaX sequence influence whether the protein can be prenylated and proteolyzed. In this study, we evaluated processing of a-factor variants with all possible single amino acid substitutions at either the a1, the a2, or the X position of the a-factor Ca1a2X sequence, CVIA. The substrate specificity of the two known yeast CaaX proteases, Afc1p and Rce1p, was investigated in vivo. Both Afc1p and Rce1p were able to proteolyze a-factor with A, V, L, I, C, or M at the a1 position, V, L, I, C, or M at the a2 position, or any amino acid at the X position that was acceptable for prenylation of the cysteine. Eight additional a-factor variants with a1 substitutions were proteolyzed by Rce1p but not by Afc1p. In contrast, Afc1p was able to proteolyze additional a-factor variants that Rce1p may not be able to proteolyze. In vitro assays indicated that farnesylation was compromised or undetectable for 11 a-factor variants that produced no detectable halo in the wild-type AFC1 RCE1 strain. The isolation of mutations in RCE1 that improved proteolysis of a-factor-CAMQ, indicated that amino acid substitutions E139K, F189L, and Q201R in Rce1p affected its substrate specificity.

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