Mapping the molecular interface between the sigma(70) subunit of E. coli RNA polymerase and T4 AsiA.
Bacteriophage T4 antisigma protein AsiA (10 kDa) orchestrates a switch from the host and early viral transcription to middle viral transcription by binding to the sigma(70) subunit of E. coli RNA polymerase. The molecular determinants of sigma(70)-AsiA complex formation are not known. Here, we used combinatorial peptide chemistry, protein-protein crosslinking, and mutational analysis to study the interaction between AsiA and its target, the 33 amino acid residues-long sigma(70) peptide containing conserved region 4.2. Many region 4.2 amino acid residues contact AsiA, which likely completely occludes the DNA-binding surface of region 4.2. Though none of region 4.2 amino acid residues is singularly responsible for the very tight interaction with AsiA, sigma(70) Lys593 and Arg596 which lie outside the putative DNA recognition element of region 4.2, contribute the most. In AsiA, the first 20 amino acid residues are both necessary and sufficient for interactions with sigma(70). Our results clarify details of sigma(70)-AsiA interaction and open the way for engineering AsiA derivatives with altered specificities.
Minakhin
L
L
Waksman Institute of Microbiology, Department of Genetics, Rutgers, The State University of New Jersey, USA.
Camarero
J A
JA
Holford
M
M
Parker
C
C
Muir
T W
TW
Severinov
K
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D017122:Bacteriophage T4 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002846:Chromatography, Affinity; D020650:Combinatorial Chemistry Techniques; D003432:Cross-Linking Reagents; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D019151:Peptide Library; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits; D016415:Sequence Alignment; D012808:Sigma Factor / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
C078876:AsiA protein, Enterobacteria phage T4; D003432:Cross-Linking Reagents; D020035:Holoenzymes; D010446:Peptide Fragments; D019151:Peptide Library; D021122:Protein Subunits; D012808:Sigma Factor; D014764:Viral Proteins; D001120:Arginine; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D008239:Lysine