Identification of an UP element consensus sequence for bacterial promoters

Abstract

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 ...

The UP Element Is Necessary but Not Sufficient for Growth Rate-Dependent Control of the Escherichia coli guaB Promoter

“…Comparison of AϩT-rich sequences used in this work with the consensus sequence for UP elements. Consensus UP element sequences and the putative P guaB UP element (positions Ϫ59 to Ϫ38 relative to the guaB transcription start site) are aligned with the 22-bp general consensus sequence for UP elements (10). Nonconserved positions are indicated by a lowercase "n." Mismatches to the general consensus UP element sequence are shown in boldface.…”

“…The consensus 4541 UP element was made by combining optimal proximal and distal UP element subsites (11). The consensus 4192 UP element was selected by a SELEX procedure (10). The predicted P guaB proximal UP element subsite contains P guaB sequences from position Ϫ46 to Ϫ38, whereas the predicted P guaB distal UP element subsite contains sequences from Ϫ59 to Ϫ47.…”

“…In addition, an AϩT-rich sequence located between positions Ϫ59 and Ϫ38 at P guaB matches the consensus 22-bp UP element sequence [5Ј-nnAA A(A/T)(A/T)T(A/T)TTTTnnAAAAnnn-3Ј; n represents a nonconserved position]) at 13 out of 15 conserved positions ( Fig. 1), but its role as an UP element has not been confirmed (10,19). The term growth rate-dependent control (GRDC) is used to refer to situations where the abundance of a particular transcript or protein product represents an increasing fraction of the cell mass at higher growth rates.…”

Mutational Analysis of Transcriptional Initiation in Bacteria

“…We captured these patterns by determining a consensus sequence. Consensus sequences are usually determined by analyzing the top 5-20% of the promoters (Estrem, Gaal, Gourse, & Ross, 1998). We used this method to determine a consensus sequence from the 12 mutant promoters that had a strength of 50% or higher compared to Ptac.…”

DNA Binding Specificity of Mu Transcription Factor C and Crystallization of C : DNA Complex

“…The hexamers are only contacted by the σ subunit of the RNAP holoenzyme (Thomas et al, 1996;Gross et al, 1998). In some promoters an AT-rich region known as the UP element is present just upstream of the -35 hexamer; and this region contains two distinct subsites (proximal and distal), which are recognized by the α-CTD (Ross et al, 1993;Gaal et al, 1996;Estrem et al, 1998). The promoter strength is a function of the entire promoter, with very strong promoters like the rrnB promoter having sequences close to the consensus.…”