TY - JOUR
T1 - Bacillus subtilis mutations that alter the pathway of phosphorylation of the anti-anti-σF factor SpoIIAA lead to a Spo- phenotype
AU - Lee, Chung Sheng
AU - Clarkson, Joanna
AU - Shu, Jwu Ching
AU - Campbell, Iain D.
AU - Yudkin, Michael D.
PY - 2001
Y1 - 2001
N2 - Sigma-F, the first sporulation-specific transcription factor of Bacillus subtilis, is regulated by an antisigma factor SpoIIAB, which can also act as a protein kinase that phosphorylates the anti-anti-sigma factor SpoIIAA. The time course of phosphorylation reaction is biphasic, a fact that has been interpreted in terms of a mechanism for sequestering SpoIIAB away from σF and thus allowing activation of σF when needed. Site-directed mutagenesis of SpoIIAA has allowed us to isolate two mutants that cannot activate σF and which are therefore Spo-. The two mutant SpoIIAA proteins, SpoIIAAL61A and SpoIIAAL90A, are phosphorylated with linear kinetics; in addition they are less able to form the stable non-covalent complex that wild-type SpoIIAA makes with SpoIIAB in the presence of ADP. The phosphorylated form of SpoIIAAL90A was hydrolysed by the specific phosphatase SpoIIE at the same rate as wild-type SpoIIAA-P, but the rate of hydrolysis of SpoIIAAL61A-P was much slower. The secondary structure and the global fold of the mutant proteins were unchanged from the wild type. The results are interpreted in terms of a model for the wild type in which SpoIIAB, after phosphorylating SpoIIAA, is released in a form that is tightly bound to ADP and which then makes a ternary complex with an unreacted SpoIIAA. We propose that it is the inability to make this ternary complex that deprives the mutant cells of a means of keeping SpoIIAB from inhibiting σF.
AB - Sigma-F, the first sporulation-specific transcription factor of Bacillus subtilis, is regulated by an antisigma factor SpoIIAB, which can also act as a protein kinase that phosphorylates the anti-anti-sigma factor SpoIIAA. The time course of phosphorylation reaction is biphasic, a fact that has been interpreted in terms of a mechanism for sequestering SpoIIAB away from σF and thus allowing activation of σF when needed. Site-directed mutagenesis of SpoIIAA has allowed us to isolate two mutants that cannot activate σF and which are therefore Spo-. The two mutant SpoIIAA proteins, SpoIIAAL61A and SpoIIAAL90A, are phosphorylated with linear kinetics; in addition they are less able to form the stable non-covalent complex that wild-type SpoIIAA makes with SpoIIAB in the presence of ADP. The phosphorylated form of SpoIIAAL90A was hydrolysed by the specific phosphatase SpoIIE at the same rate as wild-type SpoIIAA-P, but the rate of hydrolysis of SpoIIAAL61A-P was much slower. The secondary structure and the global fold of the mutant proteins were unchanged from the wild type. The results are interpreted in terms of a model for the wild type in which SpoIIAB, after phosphorylating SpoIIAA, is released in a form that is tightly bound to ADP and which then makes a ternary complex with an unreacted SpoIIAA. We propose that it is the inability to make this ternary complex that deprives the mutant cells of a means of keeping SpoIIAB from inhibiting σF.
UR - http://www.scopus.com/inward/record.url?scp=0035048176&partnerID=8YFLogxK
U2 - 10.1046/j.1365-2958.2001.02353.x
DO - 10.1046/j.1365-2958.2001.02353.x
M3 - 文章
C2 - 11298272
AN - SCOPUS:0035048176
SN - 0950-382X
VL - 40
SP - 9
EP - 19
JO - Molecular Microbiology
JF - Molecular Microbiology
IS - 1
ER -