Analysis of regions within the bacteriophage T4 AsiA protein involved in its binding to the sigma70 subunit of E. coli RNA polymerase and its role as a transcriptional inhibitor and co-activator.
Bacteriophage T4 AsiA, a protein of 90 amino acid residues, binds to the sigma(70) subunit of Escherichia coli RNA polymerase and inhibits host or T4 early transcription or, together with the T4 MotA protein, activates T4 middle transcription. To investigate which regions within AsiA are involved in forming a complex with sigma(70) and in providing transcriptional functions we generated random mutations throughout AsiA and targeted mutations within the C-terminal region. We tested mutant proteins for their ability to complement the growth of T4 asiA am phage under non-suppressing conditions, to inhibit E. coli growth, to interact with sigma(70) region 4 in a two-hybrid assay, to bind to sigma(70) in a native protein gel, and to inhibit or activate transcription in vitro using a T4 middle promoter that is active with RNA polymerase alone, is inhibited by AsiA, and is activated by MotA/AsiA. We find that substitutions within the N-terminal half of AsiA, at amino acid residues V14, L18, and I40, rendered the protein defective for binding to sigma(70). These residues reside at the monomer-monomer interface in recent NMR structures of the AsiA dimer. In contrast, AsiA missing the C-terminal 44 amino acid residues interacted well with sigma(70) region 4 in the two-hybrid assay, and AsiA missing the C-terminal 17 amino acid residues (Delta74-90) bound to sigma(70) and was fully competent in standard in vitro transcription assays. However, the presence of the C-terminal region delayed formation of transcriptionally competent species when the AsiA/polymerase complex was pre-incubated with the promoter in the absence of MotA. Our results suggest that amino acid residues within the N-terminal half of AsiA are involved in forming or maintaining the AsiA/sigma(70) complex. The C-terminal region of AsiA, while not absolutely required for inhibition or co-activation, aids inhibition by slowing the formation of transcription complexes between a promoter and the AsiA/polymerase complex.
Pal
Debashis
D
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, USA.
Vuthoori
Madhavi
M
Pande
Suchira
S
Wheeler
David
D
Hinton
Deborah M
DM
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D012808:Sigma Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism
C078876:AsiA protein, Enterobacteria phage T4; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C067996:MotA protein, Enterobacteria phage T4; D010446:Peptide Fragments; D012808:Sigma Factor; D014157:Transcription Factors; D014764:Viral Proteins; D004247:DNA; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase