Study of the interaction between bacteriophage T4 asiA and Escherichia coli sigma(70), using the yeast two-hybrid system: neutralization of asiA toxicity to E. coli cells by coexpression of a truncated sigma(70) fragment.
The interaction of T4 phage-encoded anti-sigma factor, asiA, and Escherichia coli sigma(70) was studied by using the yeast two-hybrid system. Truncation of sigma(70) to identify the minimum region involved in the interaction showed that the fragment containing amino acid residues proximal to the C terminus (residues 547 to 603) was sufficient for complexing to asiA. Studies also indicated that some of the truncated C-terminal fragments (residues 493 to 613) had higher affinity for asiA as judged by the increased beta-galactosidase activity. It is proposed that the observed higher affinity may be due to the unmasking of the binding region of asiA on the sigma protein. Advantage was taken of the increased affinity of truncated sigma(70) fragments to asiA in designing a coexpression system wherein the toxicity of asiA expression in E. coli could be neutralized and the complex of truncated sigma(70) and asiA could be expressed in large quantities and purified.
Sharma
U K
UK
AstraZeneca Pvt. Ltd., Bangalore-560 003, India.
Ravishankar
S
S
Shandil
R K
RK
Praveen
P V
PV
Balganesh
T S
TS
D001426:Bacterial Proteins / Q000378:metabolism; D017122:Bacteriophage T4 / Q000378:metabolism*; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism* / Q000821:virology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae; D017384:Sequence Deletion; D012808:Sigma Factor / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
C078876:AsiA protein, Enterobacteria phage T4; D001426:Bacterial Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012808:Sigma Factor; D014764:Viral Proteins; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases