Exploitation of a Chemical Nuclease to Investigate the Location and Orientation of the Escherichia coli RNA Polymerase α Subunit C-terminal Domains at Simple Promoters That Are Activated by Cyclic AMP Receptor Protein

Abstract

The C-terminal domain of the ␣ subunit (␣CTD) of bacterial RNA polymerase plays an important role in promoter recognition. It is known that ␣CTD binds to the DNA minor groove at different locations at different promoters via a surface-exposed determinant, the 265 determinant. Here we describe experiments that permit us to determine the location and orientation of binding of ␣CTD at any promoter. In these experiments, a DNA cleavage reagent is attached to specific locations on opposite faces of the RNA polymerase ␣ subunit. After incorporation of the tagged ␣ subunits into holo-RNA polymerase, patterns of DNA cleavage due to the reagent are determined in open complexes. The locations of DNA cleavage due to the reagent attached at different positions allow the position and orientation of ␣CTD to be deduced. Here we present data from experiments with simple Escherichia coli promoters that are activated by the cyclic AMP receptor protein.RNA synthesis in bacteria is due to holo-RNA polymerase (RNAP), 1 which is a multisubunit complex with subunit composition ␣ 2 ␤␤Ј. It is well known that the major factor ensuring "correct" gene expression in bacteria is the efficiency with which RNAP recognizes the promoters of different genes. Although the principal determinants for promoter recognition reside in the RNAP subunit, at many promoters, the ␣ subunits also play a key role (1-3). Each RNAP ␣ subunit consists of two independently folding domains connected by a flexible linker. The N-terminal domain (␣NTD; residues 8 -235 in Escherichia coli ␣) carries determinants for the interaction of the ␣ subunits with the rest of RNAP, whereas the C-terminal domain (␣CTD; residues 249 -329 in E. coli ␣) carries determinants for interaction with promoter DNA elements and with certain transcription factors (4, 5). At some promoters, the interaction of ␣CTD with specific DNA sequence elements (known as UP elements) is essential for optimal transcription initiation. At other promoters, optimal expression depends on activator proteins that function by making a direct contact with ␣CTD, thereby recruiting RNAP to the promoter (3).High resolution structures for E. coli ␣CTD, either free or bound to the UP element DNA, have been obtained (6 -8). When bound to DNA, ␣CTD contacts the minor groove (9, 10). Genetic analyses, together with the structural studies, have identified residues in two helix-loop-helix motifs that are responsible for DNA binding. Thus, Arg 265 , Asn 268 , Gly 296 , Lys 298 , and Ser 299 appear to be the crucial ␣ subunit residues that make contact with the DNA minor groove (reviewed in Ref.3 and see Ref. 8). These residues are part of a small segment of the surface of ␣CTD known as the 265 determinant. The linker joining ␣NTD and ␣CTD contains at least 13 amino acids and appears to be very flexible and unstructured (11). A consequence of this is that ␣CTD can bind at different locations at different promoters. At promoters where RNAP initiates transcription without the need for an activator protein, one ␣CTD contacts t...

Additional Determinants within Escherichia coli FNR Activating Region 1 and RNA Polymerase α Subunit Required for Transcription Activation

“…This result agrees with recent work (2) using an artificial chemical nuclease, which demonstrated that a single subunit of ␣CTD binds immediately upstream of FNR at a class II promoter in a position and orientation that would place ␣CTD region 285-288 in close proximity to FNR-AR1 region 184-192. Taken together, these data suggest that the region of ␣CTD required for activation by FNR and CRP overlap since CRP also requires region 285-288 of ␣CTD to activate transcription (27,28) and that ␣CTD binds in a similar orientation relative to both FNR and CRP at a class II promoter (18).…”

CRP Binding and Transcription Activation at CRP-S Sites

“…[29][30][31] Although UP elements are usually located between −40 and − 60, DNA bending by CRP or IHF has been shown to allow αCTD to bind UP elements at −80 and − 90. 32,33 P pilA variants in which the A + T runs were replaced or translocated were constructed to test whether these A + T runs are important for promoter activity. As shown in Fig.…”

Three-dimensional EM structure of an intact activator-dependent transcription initiation complex

“…At Class I CAP-dependent promoters, one ␣CTD protomer-interchangeably ␣CTD I or ␣CT-D II -interacts with CAP (1,2,(12)(13)(14)(15). The other ␣CTD protomer interacts nonspecifically with upstream DNA (1,2,12,13,16).…”