DNA-binding determinants of the alpha subunit of RNA polymerase: novel DNA-binding domain architecture.

Abstract

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.

A nested set of C‐terminal deletions of the α subunit of Escherichia coli RNA polymerase define regions concerned with assembly, proteolysis, stabilization and transcriptional activation in vivo

“…On the two solution structures of the a C-terminal domain (Gaal et al 1996;Jeon et al 1995), this residue lies at the C-terminal end of the 'activator' helix H1(a 1 ) (see below). Clearly, the extreme C-terminal 13 residues play a key role, either indirectly by promoting the a C-terminal domain to fold in the appropriate conformation, thereby C-terminal truncated a derivatives The assembly levels of particular a deletions.…”

“…The boundaries correlate well with the limits of functional regions as defined by the assembly and transcriptional proficiencies of a variants determined in vivo and in vitro. Secondary structure is from Jeon et al 1995 andGaal et al 1996. Abbreviation: NSH, non-standard helix.…”

“…Finally, it is useful to summarize these in vivo results in the light of the two very recent NMR solution structures of the C-terminal domain (Jeon et al 1995;Gaal et al 1996). On these solution structures, the domain contains four helices (H1-H4 or a 1 -a 4 ) (Jeon et al 1995;Gaal et al 1996).…”

“…The 'stabilization' region lying beyond M316 comprises part of one of the two long loops (249-263 and 310-329) that enclose the hydrophobic core of the domain and contact each other through F249 and W321/I326 (Jeon et al 1995). According to the structure of Gaal et al 1996, the stabilization region is closer to the fourth helix. Deletion of the extreme C-terminus (residues 317-329) removes one of the loops protecting the susceptible H1-H2 (or a 1 ¹a 2 ) interhelical bridge and, through the absence of any loop¹loop interaction, may destabilize the N-terminal loop of the C-terminal domain, thus further exposing the K271 region.…”

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

“…This long unstructured linker allows the α-CTD to occupy different positions relative to the α NTD and RNAP (Blatter et al, 1994;Busby and Ebright, 1994;Ebright and Busby, 1995). The α-CTD (8 kDa) plays a regulatory role by providing the contact surfaces for trans-acting regulatory protein factors and cis-acting DNA elements Gaal et al, 1996;Murakami et al, 1996). The α CTD binds the DNA minor groove upstream of the -35 hexamer using a helix-hairpin-helix (HhH) DNA binding motif (Jeon et al, 1995;Gaal et al, 1996;Shao and Grishin, 2000;Ross et al, 2001); this motif is called the 265 determinant (Gaal et al, 1996;Murakami et al, 1996).…”