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The sequence of a promoter determines not only the efficiency with which it forms a complex with RNA polymerase, but also the concentration of nucleoside triphosphate (NTP) required for initiating transcription. Escherichia coli ribosomal RNA (rrn P1) promoters require high initiating NTP concentrations for efficient transcription because they form unusually short-lived complexes with RNA polymerase; high initiating NTP concentrations [adenosine or guanosine triphosphate (ATP or GTP), depending on the rrn P1 promoter] are needed to bind to and stabilize the open complex. ATP and GTP concentrations, and therefore rrn P1 promoter activity, increase with growth rate. Because ribosomal RNA transcription determines the rate of ribosome synthesis, the control of ribosomal RNA transcription by NTP concentration provides a molecular explanation for the growth rate-dependent control and homeostatic regulation of ribosome synthesis.

Ribosomal RNA transcription is the rate-limiting step in ribosome synthesis in bacteria and has been investigated intensely for over half a century. Multiple mechanisms ensure that rRNA synthesis rates are appropriate for the cell's particular growth condition. Recently, important advances have been made in our understanding of rRNA transcription initiation in Escherichia coli. These include (a) a model at the atomic level of the network of protein-DNA and protein-protein interactions that recruit RNA polymerase to rRNA promoters, accounting for their extraordinary strength; (b) discovery of the nonredundant roles of two small molecule effectors, ppGpp and the initiating NTP, in regulation of rRNA transcription initiation; and (c) identification of a new component of the transcription machinery, DksA, that is absolutely required for regulation of rRNA promoter activity. Together, these advances provide clues important for our molecular understanding not only of rRNA transcription, but also of transcription in general.

The UP element, a component of bacterial promoters located upstream of the ؊35 hexamer, increases transcription by interacting with the RNA polymerase ␣-subunit. By using a modification of the SELEX procedure for identification of protein-binding sites, we selected in vitro and subsequently screened in vivo for sequences that greatly increased promoter activity when situated upstream of the Escherichia coli rrnB P1 core promoter. A set of 31 of these upstream sequences increased transcription from 136-to 326-fold in vivo, considerably more than the natural rrnB P1 UP element, and was used to derive a consensus sequence: ؊59 nnAAA(A͞T)(A͞T)T(A͞T)TTTTnnAAAAnnn ؊38. The most active selected sequence contained the derived consensus, displayed all of the properties of an UP element, and the interaction of this sequence with the ␣ C-terminal domain was similar to that of previously characterized UP elements. The identification of the UP element consensus should facilitate a detailed understanding of the ␣-DNA interaction. Based on the evolutionary conservation of the residues in ␣ responsible for interaction with UP elements, we suggest that the UP element consensus sequence should be applicable throughout eubacteria, should generally facilitate promoter prediction, and may be of use for biotechnological applications.Escherichia coli promoters recognized by the major form of RNA polymerase (RNAP E 70 , subunit composition ␣ 2 ␤␤Ј) contain up to three recognition elements. Two elements, hexamers centered approximately 10 and 35 bp upstream of the transcription start site (1, 2), interact with 70 (3). The third element, the UP element, located upstream of the Ϫ35 hexamer, binds the C-terminal domain of the RNAP ␣-subunit (␣CTD) (4, 5). The most extensively characterized UP element is an adenine (A) and thymine (T)-rich sequence located between Ϫ40 and Ϫ60 in the rrnB P1 promoter that stimulates promoter activity at least 30-fold by increasing the initial equilibrium constant (K B ) and possibly a later step(s) in the transcription initiation pathway (k f ) (4, 6). UP elements have also been described in other promoters and can function with holoenzymes containing different factors (4, 7-11).The 8-kDa ␣CTD interacts with activator proteins as well as with DNA; the 28-kDa ␣ N-terminal domain contains determinants for dimerization, assembly with the ␤-and ␤Ј-subunits, and also interacts with transcription factors (4, 5, 12-15). The two domains are connected by a flexible linker, which permits the ␣CTD to bind DNA and interact with activators at different sites upstream of the core promoter (5,12,(16)(17)(18). The ␣CTD residues involved in DNA binding are highly conserved among eubacterial ␣-subunits (19, 20); therefore, the DNA sequences recognized by ␣ are also very likely to be conserved.Consensus sequences derived previously from E. coli promoters contain highly conserved Ϫ10 and Ϫ35 hexamers, but no highly conserved upstream sequences (1, 2, 21), suggesting that UP elements are not crucial for transcription of ...

We demonstrate here that the previously described bacterial promoter upstream element (UP element) consists of two distinct subsites, each of which, by itself, can bind the RNA polymerase holoenzyme ␣ subunit carboxy-terminal domain (RNAP ␣CTD) and stimulate transcription. Using binding-site-selection experiments, we identify the consensus sequence for each subsite. The selected proximal subsites (positions −46 to −38; consensus 5-AAAAAARNR-3) stimulate transcription up to 170-fold, and the selected distal subsites (positions −57 to −47; consensus 5-AWWWWWTTTTT-3) stimulate transcription up to 16-fold. RNAP has subunit composition ␣ 2 ␤␤ and thus contains two copies of ␣CTD. Experiments with RNAP derivatives containing only one copy of ␣CTD indicate, in contrast to a previous report, that the two ␣CTDs function interchangeably with respect to UP element recognition. Furthermore, function of the consensus proximal subsite requires only one copy of ␣CTD, whereas function of the consensus distal subsite requires both copies of ␣CTD. We propose that each subsite constitutes a binding site for a copy of ␣CTD, and that binding of an ␣CTD to the proximal subsite region (through specific interactions with a consensus proximal subsite or through nonspecific interactions with a nonconsensus proximal subsite) is a prerequisite for binding of the other ␣CTD to the distal subsite.[Key Words: Promoter; RNA polymerase; ␣ subunit; UP element; transcription initiation] Received May 18, 1999; revised version accepted July 6, 1999.Bacterial promoters consist of at least three RNA polymerase (RNAP) recognition sequences: The −10 element, the −35 element, and the UP element (Hawley and McClure 1983;Ross et al. 1993). The −10 and −35 elements are recognized by the RNAP subunit (Dombroski et al. 1992), and the UP element, located upstream of the −35 element, is recognized by the RNAP ␣ subunit (Ross et al. 1993;Blatter et al. 1994). The best-characterized UP element is in the rrnB P1 promoter, in which the sequence determinants are located between positions −40 and −60 with respect to the transcription start site (Rao et al. 1994), and UP element-␣ interactions facilitate initial binding of RNAP and subsequent step(s) in transcription initiation (Rao et al. 1994;Strainic et al. 1998). A consensus UP element sequence (referred to here as the consensus full UP element), derived from binding-siteselection experiments, consists almost exclusively of A and T residues and increases promoter activity >300-fold . UP elements have been identified upstream of many bacterial and phage promoters and can function with RNAPs containing different factors (e.g., Newlands et al. 1993;Ross et al. 1993Ross et al. , 1998Fredrick et al. 1995).Each RNAP ␣ subunit consists of two domains connected by a long unstructured and/or flexible linker (Blatter et al. 1994;Jeon et al. 1997). The 28-kD aminoterminal domain (␣NTD) is responsible for dimerization of ␣ and for interaction with the remainder of RNAP (Igarashi and Ishihama 1991;Busby and Ebright 1994). The...

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SUMMARY The global regulatory nucleotide ppGpp (“magic spot”) regulates transcription from a large subset of Escherichia coli promoters, illustrating how small molecules can control gene expression promoter-specifically by interacting with RNA polymerase (RNAP) without binding to DNA. However, ppGpp’s target site on RNAP, and therefore its mechanism of action, have remained unclear. We report here a binding site for ppGpp on E. coli RNAP, identified by crosslinking, protease mapping, and analysis of mutant RNAPs that fail to respond to ppGpp. A strain with a mutant ppGpp binding site displays properties characteristic of cells defective for ppGpp synthesis. The binding site is at an interface of two RNAP subunits, ω and β′, and its position suggests an allosteric mechanism of action involving restriction of motion between two mobile RNAP modules. Identification of the binding site allows prediction of bacterial species in which ppGpp exerts its effects by targeting RNAP.

The Escherichia coil RNA polymerase oL-subunit binds through its carboxy-terminal domain (o~CTD) to a recognition element, the upstream (UP) element, in certain promoters. We used genetic and biochemical techniques to identify the residues in aCTD important for UP-element-dependent transcription and DNA binding. These residues occur in two regions of oLCTD, close to but distinct from, residues important for interactions with certain transcription activators. We used NMR spectroscopy to determine the secondary structure of ,vCTD. aCTD contains a nonstandard helix followed by four c~-helices. The two regions of ~CTD important for DNA binding correspond to the first a-helix and the loop between the third and fourth oL-helices. The o~CTD DNA-binding domain architecture is unlike any DNA-binding architecture identified to date, and we propose that aCTD has a novel mode of interaction with DNA. Our results suggest models for c~CTD-DNA and c~CTD-DNA-activator interactions during transcription initiation.