Solution structure and stability of the anti-sigma factor AsiA: Implications for novel functions

Abstract

Anti-sigma factors regulate prokaryotic gene expression through interactions with specific sigma factors. The bacteriophage T4 anti-sigma factor AsiA is a molecular switch that both inhibits transcription from bacterial promoters and phage early promoters and promotes transcription at phage middle promoters through its interaction with the primary sigma factor of Escherichia coli, 70 . AsiA is an all-helical, symmetric dimer in solution. The solution structure of the AsiA dimer reveals a novel helical fold for the protomer. Furthermore, the AsiA protomer, surprisingly, contains a helix-turn-helix DNA binding motif, predicting a potential new role for AsiA. The AsiA dimer interface includes a substantial hydrophobic component, and results of hydrogen͞deuterium exchange studies suggest that the dimer interface is the most stable region of the AsiA dimer. In addition, the residues that form the dimer interface are those that are involved in binding to 70 . The results promote a model whereby the AsiA dimer maintains the active hydrophobic surfaces and delivers them to 70 , where an AsiA protomer is displaced from the dimer via the interaction of 70 with the same residues in AsiA that constitute the dimer interface. R egulation of prokaryotic transcription involves the interaction of sigma ( ) factors, which bind to the core RNA polymerase to impart promoter recognition specificity, with cognate anti-sigma (anti-) factors that inhibit function. Most of the numerous examples of ͞anti-pairs involve alternative factors, those activated in response to specific stimuli, as opposed to primary factors, which are essential for survival and responsible for the bulk of transcription, including the housekeeping genes (reviewed in refs. 1-4). An important exception is the interaction of 70 of Escherichia coli, namesake for a family of sequence conserved primary factors, with the anti-factor AsiA.The bacteriophage T4-encoded AsiA protein, product of the asiA gene (5), and the first anti-factor to be discovered, binds tightly to the 70 subunit of the E. coli RNA polymerase holoenzyme (6-10), altering the specificity of the complex toward both phage and host promoters. Following infection by bacteriophage T4, the E. coli RNA polymerase is recruited to sequentially transcribe genes from the T4 early, middle, and late promoters. T4 early promoters contain bacterial-like consensus DNA sequences that allow for their immediate recognition by the unmodified RNA polymerase, and the T4 early genes include the asiA gene. Shortly thereafter, transcription at early promoters is inhibited, along with transcription at bacterial promoters, by phage-induced modifications of the RNA polymerase, one of which is the tight association of AsiA with 70 . This interaction inhibits 70 -dependent transcription at early promoters by blocking recognition by 70 of the conserved sequence element centered at position Ϫ35 (11). Furthermore, AsiA not only has the ability to function as an anti-factor, it also has the ability to promote transcription. In conce...

On the Role of the Escherichia coli RNA Polymerase σ70 Region 4.2 and α-Subunit C-terminal Domains in Promoter Complex Formation on the Extended –10 galP1 Promoter

“…In addition, AsiA abolished DNase I hypersensitivity at Ϫ67 and Ϫ57, consistent with the results in Fig. 2 (25).…”

Bacteriophage T4 Genome

“…This interaction interferes with the recognition of Ϫ35 promoter sequences and at the same time stimulates T4 middle-mode transcription (180,425,1103,1104). The AsiA- 70 interaction is regarded as the pivotal event in the transition between T4 early and middle transcription: in vitro it both inhibits the recognition of most host promoters and early T4 promoters and stimulates T4 middle-mode transcription (180,425,848,849,1104). However, in vivo, defective asiA mutants do not prolong early transcription (858), suggesting that other proteins (i.e., ModA and ModB) turn off most early T4 promoters.…”

“…Approximately 8 bits of R s information are required for MotA to recognize the MotA box sequence. T4 middle promoters are all located on the minus strand ( (12,180,1104). This interaction interferes with the recognition of Ϫ35 promoter sequences and at the same time stimulates T4 middle-mode transcription (180,425,1103,1104).…”

The Bacteriophage T4 Transcription Activator MotA Interacts with the Far-C-Terminal Region of the σ70Subunit ofEscherichia coliRNA Polymerase

“…Nuclear magnetic resonance and crystallographic studies indicate that the 211 amino acids of MotA are organized into an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a small flexible linker (15,33,34 (44,55,56). Binding sites for AsiA have been mapped within C-terminal amino acids (regions 4.1 and 4.2) of 70 (8,49,50,53,59). Residues within region 4.2 normally contact the Ϫ35 element of host promoter DNA (5,9,17,29,54).…”

“…Binding sites for AsiA have been mapped within C-terminal amino acids (regions 4.1 and 4.2) of 70 (8,49,50,53,59). Residues within region 4.2 normally contact the Ϫ35 element of host promoter DNA (5,9,17,29,54).…”