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Journal of Bacteriology, September 1999, p. 5296-5302, Vol. 181, No. 17
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

A- and T-Tract-Mediated Intrinsic Curvature in Native DNA between the Binding Site of the Upstream Activator NtrC and the nifLA Promoter of Klebsiella pneumoniae Facilitates Transcription

Amrita Kaur Cheema, Nirupam Roy Choudhury, and H. K. Das*

Genetic Engineering Unit and Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India

Received 15 March 1999/Accepted 22 June 1999

The nif promoters of Klebsiella pneumoniae must be activated by proteins bound to upstream sequences which are thought to interact with the sigma 54-RNA polymerase holoenzyme by DNA looping. NifA is the activator for most of the promoters, and integration host factor (IHF) mediates the DNA looping. While NtrC is the activator for the nifLA promoter, no IHF appears to be involved. There are two A tracts and one T tract between the upstream enhancer and the nifLA promoter. This DNA segment exhibits anomalous electrophoretic mobility, suggesting intrinsic sequence-induced curvature in the DNA. On the one hand, mutation of the A tracts or T tract individually or together, or deletion of the A tracts and the T tract reduces the anomaly; on the other hand, creation of two additional A tracts enhances the anomaly. Intrinsic curvature in the DNA has been confirmed by circular permutation analysis after cloning the DNA fragment in the vector pBend 2 and also by electron microscopy. Computer simulation with the DNA base sequence is also suggestive of intrinsic curvature. A transcriptional fusion with the Escherichia coli lacZ gene of the DNA fragment containing the nifLA promoter and the wild-type or the mutated upstream sequences was constructed, and in vivo transcription in K. pneumoniae and E. coli was monitored. There was indeed very good correlation between the extent of intrinsic curvature of the DNA and transcription from the promoter, suggesting that DNA curvature due to the A tracts and the T tract was necessary for transcription in vivo from the nifLA promoter of K. pneumoniae.

* Corresponding author. Mailing address: Genetic Engineering Unit, Jawaharlal Nehru University, New Delhi 110067, India. Phone: 91(011)610-1044.. Fax: 91(011)616-5886. E-mail: hirendas{at}hotmail.com.

Journal of Bacteriology, September 1999, p. 5296-5302, Vol. 181, No. 17
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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