Published online 8 October 2010 Nucleic Acids Research, 2011, Vol. 39, No. 3 913–925 doi:10.1093/nar/gkq911 The core-independent promoter-specific interaction of primary sigma factor Hsin-Yi Yeh, Tsung-Ching Chen, Kung-Ming Liou, Hsiu-Ting Hsu, Kuei-Min Chung, Li-Ling Hsu and Ban-Yang Chang* Institute of Biochemistry, National Chung-Hsing University, Taichung 40227, Taiwan, ROC Received September 2, 2010; Revised September 22, 2010; Accepted September 23, 2010 ABSTRACT Previous studies have led to a model in which the promoter-specific recognition of prokaryotic transcription initiation factor, sigma (p), is core de- pendent. Most p functions were studied on the basis of this tenet. Here, we provide in vitro evidence demonstrating that the intact Bacillus subtilis primary sigma, pA, by itself, is able to interact spe- cifically with promoter deoxyribonucleic acid (DNA), albeit with low sequence selectivity. The core-independent promoter-specific interaction of the pA is 10 specific. However, the promoter 10 specific interaction is unable to allow the pA to discern the optimal promoter spacing. To fulfill this goal, the pA requires assistance from core RNA polymerase (RNAP). The ability of p, by itself, to interact specifically with promoter might intro- duce a critical new dimension of study in prokaryot- ic p function. INTRODUCTION The bacterial RNA polymerase holoenzyme (holo RNAP) is composed of the catalytic core RNAP, a2bb0 and the transcription initiation factor, sigma (s). The functions of s have been studied since 1969 (1). It is now clear that s confers upon holo RNAP the ability to recognize and melt promoter deoxyribonucleic acid (DNA) during transcription initiation (2–6). The bacterial s factors are classiﬁed into the s70 and s54 families (7). The s70 family members in all bacteria can be subdivided into four groups. The Group 1, or primary, ss are responsible for the transcription of house-keeping genes and are essential for viability, whereas the Groups 2–4, or alternative, ss are required for more specialized functions such as stress responses. The primary ss have four conserved regions (Regions 1–4) with each having 2–4 subregions, whereas the alter- native ss of Groups 3–4 lack Region 1 (7–9). Regions 2.4, 3.0 and 4.2 are thought to interact with the 10, extended 10 (or TG motif) and 35 promoter elements, respect- ively, according to the results of genetic and biochemical analyses of s in the context of holo RNAP (10–15). The idea for direct promoter interaction of s independent of core RNAP has been put to the test (16–21); however, it was only observed for a group 3 s, the sD of Bacillus subtilis (17). Thus, it was generally believed that the primary s has to associate with core RNAP prior to inter- action with promoter DNA. The inability of primary s, by itself, to bind promoter DNA was once ascribed to the auto-inhibition of s through direct interaction between Regions 1.1 and 4.2 of the s (22,23). However, the studies of a Thermotoga maritima s revealed that this inhibition is owing to an indirect steric and/or electrostatic mechanism (24) or to the close proximity of the negatively charged Region 1.1 to the positively charged promoter recognition domains of s, which makes s adopt a compact structure incompatible with DNA binding (25). Multiple switches are thought to be built into s to alleviate the auto-inhibition (26). They include the interactions between the ﬂap domain of the b-subunit of core RNAP and Region 4 of the s (27,28) as well as between the coiled coil domain of the b0-subunit and Region 2.2 of the s (29). Although the switch mech- anism may explain why s is unable to bind promoter DNA in a core-independent manner, direct evidence sup- porting the hypothesis that s, by itself, is unable to bind promoter DNA is still lacking. Actually, the failure to detect the core-independent promoter-speciﬁc interaction of s could be attributed to the use of a s devoid of promoter DNA-binding conformation or the use of an inappropriate condition for assaying the binding inter- action. Since how s recognizes promoter DNA would affect how we elucidate the functional mechanisms of s and core RNAP during transcription initiation, we re-investigated this issue. *To whom correspondence should be addressed. Tel: 886 4 2285 3486; Fax: 886 4 2285 3487; Email: bychang@mail.nchu.edu.tw The authors wish it to be known that, in their opinion, the ﬁrst two authors should be regarded as joint First Authors. The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.