Published online 3 February 2004
Nucleic Acids Research, 2004, Vol. 32, No. 2 834-841
© 2004 Oxford University Press
Role of the RNA polymerase 
subunits in CII-dependent activation of the bacteriophage 
pE promoter: identification of important residues and positioning of the C-terminal domains
grzyn1,3
Division of Genomic Medicine, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield S10 2RX, UK,
1 Department of Molecular Biology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland,
2 School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK and
3 Institute of Oceanology, Polish Academy of Sciences, 
w. Wojciecha 5, 81-347 Gdynia, Poland
*To whom correspondence should be addressed. Tel: +44 114 271 2834; Fax: +44 114 271 3892; Email: m.s.thomas{at}shef.ac.uk
The bacteriophage 
CII protein stimulates the activity of three phage promoters, pE, pI and paQ, upon binding to a site overlapping the –35 element at each promoter. Here we used preparations of RNA polymerase carrying a DNA cleavage reagent attached to specific residues in the C-terminal domain of the RNA polymerase 
subunit (CTD) to demonstrate that one CTD binds near position –41 at pE, whilst the other CTD binds further upstream. The CTD bound near position –41 is oriented such that its 261 determinant is in close proximity to 
70. The location of CTD in CII-dependent complexes at the pE promoter is very similar to that found at many activator-independent promoters, and represents an alternative configuration for CTD at promoters where activators bind sites overlapping the –35 region. We also used an in vivo alanine scan analysis to show that the DNA-binding determinant of CTD is involved in stimulation of the pE promoter by CII, and this was confirmed by in vitro transcription assays. We also show that whereas the K271E substitution in CTD results in a drastic decrease in CII-dependent activation of pE, the pI and paQ promoters are less sensitive to this substitution, suggesting that the role of CTD at the three lysogenic promoters may be different.
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