Mutational analysis of bacteriophage T4 AsiA: involvement of N- and C-terminal regions in binding to sigma(70) of Escherichia coli in vivo.
The T4 AsiA is an anti-sigma factor encoded by an early gene of bacteriophage T4. AsiA has been shown to inhibit T4 early promoters in vitro and expression of this protein from a plasmid causes transcriptional shut off in the host cells leading to cell death. By reasoning that mutant AsiA expression in Escherichia coli will not inhibit the host transcription and hence lead to healthy colony formation, a strategy was developed wherein inactive or partially active mutants of AsiA could be isolated. These mutants were tested for their ability to bind to sigma(70) in vivo in E. coli, monitored as a relative toxicity assay, co-purification of sigma(70), inhibition of [3H-uridine] incorporation and also in the yeast two hybrid system. A good correlation was found between the loss of toxicity of AsiA to E. coli cells and the inability of mutant AsiAs to bind to sigma(70) It was observed that deletion of C-terminal 17 amino acid residues of AsiA did not affect the activity whereas a mutant asiA lacking C-terminal 28 amino acid residues had the toxicity reduced to a large extent, suggesting that amino acid residues between 64 and 73 played a role in binding to AsiA. A mutant with a deletion of 34 amino acids in the C-terminus did not show any toxicity to E. coli cells. In the N-terminal region, deletion of five amino acid residues was tolerated but extending the deletion to ten amino acids abolished the AsiA activity completely. The conversion of glutamic acid (E10) to either leucine, serine, glutamine, tyrosine or alanine did not affect the toxicity to a great extent suggesting that a negative charge at E10 is not critical for interaction with sigma(70). The results of our in vivo studies suggest that the primary sigma(70) binding site of AsiA is in N-terminus, but, it requires the presence of C-terminal 64-73 amino acid residues for effective binding in vivo.
Sharma
Umender K
UK
AstraZeneca India Pvt. Ltd., 277 T. Chowdaiah Road, Malleswaram, Bangalore 560 003, India. umender.sharma@astrazeneca.com
Praveen
P V K
PV
Balganesh
T S
TS
D017122:Bacteriophage T4 / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D015870:Gene Expression; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017384:Sequence Deletion; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C078876:AsiA protein, Enterobacteria phage T4; D012808:Sigma Factor; D014764:Viral Proteins; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases