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Journal of Bacteriology, March 2006, p. 2222-2232, Vol. 188, No. 6
0021-9193/06/$08.00+0     doi:10.1128/JB.188.6.2222-2232.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Evidence that the Promoter Can Influence Assembly of Antitermination Complexes at Downstream RNA Sites

Ying Zhou,^1, Ting Shi,^1, Mark A. Mozola,^1, Eric R. Olson,^1,¶ Karla Henthorn,^1,|| Susan Brown,² Gary N. Gussin,² and David I. Friedman^1*

Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan 48109,¹ Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242²

Received 12 October 2005/ Accepted 4 January 2006

The N protein of phage acts with Escherichia coli Nus proteins at RNA sites, NUT, to modify RNA polymerase (RNAP) to a form that overrides transcription terminators. These interactions have been thought to be the primary determinants of the effectiveness of N-mediated antitermination. We present evidence that the associated promoter, in this case the early P_R promoter, can influence N-mediated modification of RNAP even though modification occurs at a site (NUTR) located downstream of the intervening cro gene. As predicted by genetic analysis and confirmed by in vivo transcription studies, a combination of two mutations in P_R, at positions –14 and –45 (yielding P_R-GA), reduces effectiveness of N modification, while an additional mutation at position –30 (yielding P_R-GCA) suppresses this effect. In vivo, the level of P_R-GA-directed transcription was twice as great as the wild-type level, while transcription directed by P_R-GCA was the same as that directed by the wild-type promoter. However, the rate of open complex formation at P_R-GA in vitro was roughly one-third the rate for wild-type P_R. We ascribe this apparent discrepancy to an effect of the mutations in P_R-GCA on promoter clearance. Based on the in vivo experiments, one plausible explanation for our results is that increased transcription can lead to a failure to form active antitermination complexes with NUT RNA, which, in turn, causes failure to read through downstream termination sites. By blocking antitermination and thus expression of late functions, the effect of increased transcription through nut sites could be physiologically important in maintaining proper regulation of gene expression early in phage development.

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* Corresponding author. Mailing address: Department of Microbiology and Immunology, 5641 Medical Science Bldg. II, University of Michigan, Ann Arbor, MI 48109-0620. Phone: (734) 763-3142. Fax: (734) 764-3562. E-mail: davidfri{at}umich.edu.

Present address: Department of Medicine, University of Chicago, Chicago, IL 60637.

Present address: Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195.

Present address: Neogen Corporation, 620 Lesher Place, Lansing, MI 48912.

^¶ Present address: Vertex Pharmaceuticals, 130 Waverly, Cambridge, MA 02139.

^|| Present address: Division of Math, Science, and Engineering, Northern Virginia Community College, Annandale, VA 22003.

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Journal of Bacteriology, March 2006, p. 2222-2232, Vol. 188, No. 6
0021-9193/06/$08.00+0     doi:10.1128/JB.188.6.2222-2232.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.