Structural Requirements for the Interdomain Linker of α Subunit of Escherichia coli RNA Polymerase

Abstract

The carboxy-terminal domain of the alpha subunit of Escherichia coli RNA polymerase, which is connected with the core part of RNA polymerase through a long flexible linker, plays decisive roles in transcription activation by directly interacting with a large number of transcription factors and upstream (UP) element DNA. Here we constructed a set of mutant RNA polymerases, each containing a mutant alpha subunit with an altered interdomain linker. Deletion of three amino acids from the linker exhibited 50% inhibition of cAMP receptor protein- (CRP-) dependent lac P1 transcription. Deletion of six amino acids completely knocked out the activity. Insertion of three amino acids did not affect the activity, whereas 40-60% inhibition was observed after insertion of one, two, or four amino acids. Substitution of 10 consecutive glycine residues resulted in nearly 90% reduction of the CRP-dependent activity, whereas 50% activity was retained after substitution of 10 proline residues or a sequence expected to form a strong alpha-helix. Essentially the same results were obtained with UP element-dependent rrnB P1 transcription. These observations altogether suggest that (i) sufficient length of the interdomain linker is required for transcription activation mediated by the alpha carboxy-terminal domain, (ii) the linker is not totally unstructured but has structural and torsional preferences to facilitate positioning of the carboxy-terminal domain to a proper location for the interaction with CRP and UP element, and (iii) CRP-dependent activation and UP element-dependent activation share a common intermediary state in which the positioning of the alpha carboxy-terminal domain is of primary importance.

UP element-dependent transcription at the Escherichia coli rrnB P1 promoter: positional requirements and role of the RNA polymerase alpha subunit linker

“…Thirdly, amino acid sequence alignments of α subunits from eubacteria and chloroplasts reveal a non-conserved sequence of variable length corresponding to amino acids V237-P251 of the E.coli α subunit (37). Consistent with its proposed role as a linker, the introduction of substantial deletions or insertions into this region does not affect the efficiency of assembly of α into RNAP, nor do they affect transcription from activatorindependent and UP element-independent promoters (37,38). However, deletion of three amino acids from within the linker greatly reduces transcription from a promoter where the activator protein CRP binds to a site centred at -61.5 (Class I), whereas transcription is enhanced from a promoter where CRP binds at -41.5 (Class II).…”

Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling

“…The C-domain plays decisive roles in transcription activation by directly interacting with a large number of transcription factors (Igarashi et al, 1991) and with an upstream DNA element (Ross et al, 1993). The deletion of three amino acids from the linker caused a 50% inhibition of cAMP receptor proteindependent lac P1 transcription, whereas the deletion of six amino acids completely knocked out the activity (Fujita et al, 2000). Similarly, changes in the length of the disordered linker region of the Rieske protein (which allows the catalytic domain to move between two positions, proximal to cytochrome b and cytochrome c 1 ) impaired the interaction of ubiquinol with the cytochrome bc 1 complex (Nett et al, 2000).…”

Zinc finger as distance determinant in the flexible linker of intron endonuclease I- Tev I

“…Linkers between domains of many proteins have been shown to have specific structures and͞or functions. For example, the length, and to some extent the sequence, of the 26-residue flexible interdomain linker of the ␣ subunit of E. coli RNA polymerase confers promoter classdependent activity (18)(19)(20). In some cases the flexible linkers show little defined structure or sequence requirements, as in the case of the 21-residue linker of the yeast heat shock transcription factor (21).…”