The bacteriophage T4 transcription activator MotA interacts with the far-C-terminal region of the sigma70 subunit of Escherichia coli RNA polymerase.
Transcription from bacteriophage T4 middle promoters uses Escherichia coli RNA polymerase together with the T4 transcriptional activator MotA and the T4 coactivator AsiA. AsiA binds tightly within the C-terminal portion of the sigma70 subunit of RNA polymerase, while MotA binds to the 9-bp MotA box motif, which is centered at -30, and also interacts with sigma70. We show here that the N-terminal half of MotA (MotA(NTD)), which is thought to include the activation domain, interacts with the C-terminal region of sigma70 in an E. coli two-hybrid assay. Replacement of the C-terminal 17 residues of sigma70 with comparable sigma38 residues abolishes the interaction with MotA(NTD) in this assay, as does the introduction of the amino acid substitution R608C. Furthermore, in vitro transcription experiments indicate that a polymerase reconstituted with a sigma70 that lacks C-terminal amino acids 604 to 613 or 608 to 613 is defective for MotA-dependent activation. We also show that a proteolyzed fragment of MotA that contains the C-terminal half (MotA(CTD)) binds DNA with a K(D(app)) that is similar to that of full-length MotA. Our results support a model for MotA-dependent activation in which protein-protein contact between DNA-bound MotA and the far-C-terminal region of sigma70 helps to substitute functionally for an interaction between sigma70 and a promoter -35 element.
Pande
Suchira
S
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0830, USA.
Makela
Anna
A
Dove
Simon L
SL
Nickels
Bryce E
BE
Hochschild
Ann
A
Hinton
Deborah M
DM
D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology*; D012808:Sigma Factor / Q000737:chemistry* / Q000502:physiology; D014157:Transcription Factors / Q000737:chemistry / Q000502:physiology*; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000737:chemistry / Q000502:physiology*
D004268:DNA-Binding Proteins; C067996:MotA protein, Enterobacteria phage T4; D012808:Sigma Factor; D014157:Transcription Factors; D014764:Viral Proteins; D004247:DNA; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases