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Journal of Bacteriology, September 1999, p. 5771-5782, Vol. 181, No. 18
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Multiple Mechanisms Are Used for Growth Rate and Stringent Control of leuV Transcriptional Initiation in Escherichia coli

Dmitry K. Pokholok,1,dagger Maria Redlak,1 Charles L. Turnbough Jr.,2 Sara Dylla,2 and Walter M. Holmes1,*

Institute of Structural Biology and Drug Discovery and Department of Microbiology and Immunology, Medical College of Virginia of Virginia Commonwealth University, Richmond, Virginia 23219-0133,1 and Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 352942

Received 26 February 1999/Accepted 12 July 1999

Expression of the Escherichia coli leuV operon, which contains three tRNA1Leu genes, is regulated by several mechanisms including growth-rate-dependent control (GRDC) and stringent control (SC). Structural variants of the leuV promoter which differentially affect these regulatory responses have been identified, suggesting that promoter targets for GRDC and SC may be different and that GRDC of the leuV promoter occurs in the absence of guanosine 3',5'-bisdiphosphate. To determine the mechanisms of the leuV promoter regulation, we have examined the stability of promoter open complexes and the effects of nucleotide triphosphate (NTP) concentration on the efficiency of the leuV promoter and its structural variants in vitro and in vivo. The leuV promoter open complexes were an order of magnitude more stable to heparin challenge than those of rrnBp1. The major initiating nucleotide GTP as well as other NTPs increased the stability of the leuV promoter open complexes. When the cellular level of purine triphosphates was increased at slower growth rates by pyrimidine limitation, a 10% reduction in leuV promoter activity was seen. It therefore appears that transcription initiation from the leuV promoter is less sensitive to changes in intracellular NTP concentration than that from rrnBp1. Comparative analysis of regulation of the leuV promoter with and without upstream activating sequences (UAS) demonstrated that the binding site for factor of inversion stimulation (FIS) located in UAS is essential for maximal GRDC. Moreover, the presence of UAS overcame the effects of leuV promoter mutations, which abolished GRDC of the leuV core promoter. However, although the presence of putative FIS binding site was essential for optimal GRDC, both mutant and wild-type leuV promoters containing UAS showed improved GRDC in a fis mutant background, suggesting that FIS protein is an important but not unique participant in the regulation of the leuV promoter.

* Corresponding author. Mailing address: ISBDD, Suite 212, 800 East Leigh St., Richmond, VA 23219. Phone: (804) 828-2327. Fax: (804) 828-9946. E-mail: HOLMES{at}hsc.vcu.edu.

dagger Present address: Whitehead Institute/MIT, Cambridge, MA 02142.

Journal of Bacteriology, September 1999, p. 5771-5782, Vol. 181, No. 18
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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