Dissecting the Extended “–10” Escherichia coli rpsB Promoter Activity and Regulation in vivo

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L. V. Aseev, L. S. Koledinskaya, and I. V. Boni^*

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia; fax: (495) 330-6538; E-mail: irina_boni@ibch.ru

^* To whom correspondence should be addressed.

Received April 21, 2014; Revision received May 15, 2014

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As we have shown previously, transcription of the rpsB-tsf operon encoding essential components of the translation machinery, a ribosomal protein S2 and an elongation factor Ts, is driven by a single promoter P_rpsB, which is highly conserved among γ-proteobacteria. P_rpsB belongs to the extended “–10” promoter class; it comprises a TGTG-extension upstream of the “–10” hexamer TATAAA, a suboptimal “–35” region TTGGTG, and a GC-rich discriminator GCGCGC that separates the “–10” element from the transcription start site. In this work, we examined an impact of site-directed mutations in the rpsB promoter region on expression of the reporter gene P_rpsB-lacZ within the E. coli chromosome as well as promoter regulation by transcription factors ppGpp and DksA upon amino acid starvation. The results show that the transcription level largely depends on both the TGTG-extension and the TTG-element in the “–35” region, as mutations in these sequences dramatically decrease the activity of the promoter. Upon induction of amino acid starvation, the rpsB promoter is negatively regulated by ppGpp due to the presence of the GC-rich discriminator, whose substitution for the AT-rich element abolished stringent control. These and other data obtained demonstrate the necessity of a natural combination of all the conserved promoter elements for efficient and regulated transcription of the essential rpsB-tsf operon.

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KEY WORDS: rpsB-tsf operon, extended “–10” promoter, site-directed mutagenesis, ppGpp/DksA, GC-rich discriminator, stringent control

DOI: 10.1134/S0006297914080057

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