A cysteine-tethered DNA cleavage agent has been used to locate the position of region 2.5 of 70 in transcriptionally competent complexes between Escherichia coli RNA polymerase and promoters. In this study we have engineered 70 to introduce a unique cysteine residue at a number of positions in region 2.5. Mutant proteins were purified, and in each case, the single cysteine residue used as the target for covalent coupling of the DNA cleavage agent p-bromoacetamidobenzyl-EDTA⅐Fe (FeBABE). RNA polymerase core reconstituted with tagged derivatives was shown to be transcriptionally active. Hydroxyl radical-based DNA cleavage mediated by tethered FeBABE was observed for each derivative of RNA polymerase in the open complex. Our results show that region 2.5 is in close proximity to promoter DNA just upstream of the ؊10 hexamer. This positioning is independent of promoter sequence. A model for the interaction of this region of with promoter DNA is discussed.Promoter recognition requires sequence-specific contacts by the transcriptional apparatus. At most promoters these contacts are made upstream from the transcription start point. Once the transcriptional apparatus has bound the promoter to form a closed complex, an isomerization event occurs to generate the open complex, forming the single-stranded template required for transcription. The bacterium Escherichia coli provides a good model for understanding protein-DNA interactions during transcription initiation. E. coli uses a single core RNA polymerase for transcription elongation with subunit composition ␣ 2 Ј. Promoter specificity is principally afforded by a separate subunit, , which associates with the core enzyme to give holoenzyme (RNAP) 1 but dissociates once sequence-specific promoter DNA contacts are no longer required (1). The 70 subunit, encoded by rpoD, is one of several subunits utilized by E. coli and is responsible for directing the transcription of most genes during vegetative growth. RNAP is capable of sequence-specific transcription initiation in the absence of other transcription factors. Factor-independent transcription is reliant on the ability of 70 to make stable contacts with the promoter DNA (1, 2). E. coli promoters contain two very conserved motifs, the Ϫ10 and Ϫ35 hexamers (3), and several less-conserved sequences including the UP element (4) and the extended Ϫ10 motif (5). The extended Ϫ10 motif (5Ј-TGXTATAAT-3Ј) can drive factor-independent transcription at several bacterial promoters lacking homology to the consensus within the Ϫ35 region (6, 7). Therefore this TGX motif is able to compensate for a poor Ϫ35 hexamer. The TG motif has been shown to be important for promoter activity in several other bacterial species (8 -11). Work from many laboratories has defined the regions within RNA polymerase that are responsible for sequence-specific contacts within promoter DNA. Regions 2.4 and 4.2 of 70 contact the Ϫ10 and Ϫ35 hexamers, respectively (1, 2), whereas the C-terminal domain of the ␣ subunit (␣CTD) contacts the UP element (4). Recen...
No abstract
The methylated form of the Ada protein ( me Ada) binds the ada and aidB promoters between 60 and 40 base pairs upstream from the transcription start and activates transcription of the Escherichia coli ada and aidB genes. This region is also a binding site for the ␣ subunit of RNA polymerase and resembles the rrnB P1 UP element in A/T content and location relative to the core promoter. In this report, we show that deletion of the C-terminal domain of the ␣ subunit severely decreases me Ada-independent binding of RNA polymerase to ada and aidB, affecting transcription initiation at these promoters. We provide evidence that me Ada activates transcription by direct interaction with the C-terminal domain of RNA polymerase 70 subunit (amino acids 574 -613). Several negatively charged residues in the 70 C-terminal domain are important for transcription activation by me Ada; in particular, a glutamic acid to valine substitution at position 575 has a dramatic effect on me Ada-dependent transcription. Based on these observations, we propose that the role of the ␣ subunit at ada and aidB is to allow initial binding of RNA polymerase to the promoters. However, transcription initiation is dependent on me Ada-70 interaction.Transcription activation is one of the principal strategies used by bacteria to respond to external stimuli and to adapt to a changing environment. Most Escherichia coli activators stimulate transcription by establishing protein-protein interaction with RNA polymerase. Different subunits of RNA polymerase can be a target for transcription activators; however, the majority of activators interact with either the ␣ or the 70 subunits (1, 2). ␣ and 70 are also the subunits of RNA polymerase responsible for specific binding to promoters; 70 contacts the Ϫ35 and Ϫ10 promoter elements (core promoter elements), whereas ␣ interacts with UP elements. At the strong rrnB P1 promoter, an UP element stimulates transcription initiation 30-fold through direct interaction with the ␣ subunit C-terminal domain (␣CTD), 1 in the absence of any other protein factors (3).Exposure of E. coli to sublethal concentrations of methylating agents such as methyl methanesulfonate (MMS) activates expression from three promoters: the ada promoter (which also controls expression of the alkB gene), the alkA promoter, and the aidB promoter. This process is called the adaptive response (4 -7). The product of the ada gene, the Ada protein, plays a dual role in the adaptive response; Ada transfers methyl groups from DNA to two of its cysteine residues, thereby functioning as a DNA repair protein. Upon self-methylation, Ada is converted into an activator able to stimulate transcription of the adaptive response genes, including its own (8 -10). Ada is a 39-kDa protein organized in two independently structured domains, each with one methyl-acceptor cysteine (11). Methylation of Cys-69, in the N-terminal domain, triggers specific DNA binding by Ada and is required for transcription activation. In contrast, Ada protein singly methylated at Cys-321 is...
These results confirm that DHEA ST activity is diminished in liver disease and that the reduction is due to diminished enzyme presence. Further studies are required to show whether the reduction has any pathogenetic significance or is merely a consequence of disease.
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