Dimeric Association of Escherichia coli RNA Polymerase α Subunits, Studied by Cleavage of Single-Cysteine α Subunits Conjugated to Iron−(S)-1-[p-(Bromoacetamido)benzyl]ethylenediaminetetraacetate

Abstract

Proximity relationships between the two associated monomers of the Escherichia coli RNA polymerase alpha subunit were studied using a set of four mutant alpha subunits, each with a single Cys residue at one of the naturally occurring positions (54, 131, 176, and 269). These mutant alpha subunits were conjugated with the cutting reagent iron-(S)-1-[p-(bromoacetamido)benzyl]ethylenediaminetetraacetate (Fe-BABE), and the peptide backbone was cleaved at locations near the modified Cys. Analysis of the cleavage sites identified segments within approximately 12 A of the conjugation site. These results show that, for intermolecular cutting, segments of the subunit assembly domain (N-terminal domain) of one subunit and the linker region between N- and C-terminal domains of the other subunit are near each other, and the N-terminal domains of both subunits are in close proximity to one another. Intramolecular cutting however, was observed only within an individual N- or C-terminal domain.

Site-Specific Incorporation of Probes into RNA Polymerase by Unnatural-Amino-Acid Mutagenesis and Staudinger–Bertozzi Ligation

“…Important biochemical and biophysical approaches for analysis of RNA polymerase (RNAP) structure and function--including affinity cleaving (1,2), photocrosslinking (3,4), and ensemble and single-molecule fluorescence resonance energy transfer (5–16)--require the ability to incorporate probes at specific sites within RNAP.…”

“…One approach to introduce a probe at a specific site within a protein is to perform site-directed mutagenesis to introduce a unique Cys residue at the site of interest followed by Cys-specific chemical modification to introduce the probe at the site of interest (1–17). This approach can be used to incorporate probes at any non-essential, solvent-accessible residue of Escherichia coli RNAP ω subunit (11), E. coli RNAP α subunit (after use of site-directed mutagenesis to substitute the pre-existing Cys residues at positions 54, 131, 176, and 269; 1), and E. coli transcription initiation factor σ 70 (after use of site-directed mutagenesis to substitute the pre-existing Cys residues at positions 132, 291, and 295; 2, 5–16).…”

“…This approach can be used to incorporate probes at any non-essential, solvent-accessible residue of Escherichia coli RNAP ω subunit (11), E. coli RNAP α subunit (after use of site-directed mutagenesis to substitute the pre-existing Cys residues at positions 54, 131, 176, and 269; 1), and E. coli transcription initiation factor σ 70 (after use of site-directed mutagenesis to substitute the pre-existing Cys residues at positions 132, 291, and 295; 2, 5–16). The resulting probe-containing ω and α derivatives enable preparation of probe-containing RNAP derivatives by in vitro reconstitution of RNAP (1,11; see 19–23), and the resulting probe-containing σ 70 derivatives enable preparation of probe-containing RNAP holoenzyme derivatives by addition to RNAP core (2, 5–16). However, this approach cannot be used to incorporate probes into the second-largest and largest RNAP subunits, β and β', since these very large polypeptides (1342 residues for E. coli β; 1407 residues for E. coli β') contain multiple pre-existing Cys residues that are essential and that therefore cannot be substituted without loss of function.…”

Functional complementation between mutations at two distant positions in Escherichia coli RNA polymerase as revealed by second-site suppression

“…It is quite conceivable that long-range secondary interaction, in addition to primary contacts, in¯uences subunit assembly signi®cantly. In order to probe the primary contact sites between interacting proteins, many approaches, including crosslinking (Hillel and Wu 1977;Chen et al 1994;McMahan and Burgess 1994;Lee and Hoover 1995), protein footprinting (Heyduk and Heyduk 1994;Nagai and Shimamoto 1997), complex formation between subunits or subunit fragments Ishihama 1995a, 1995b;Nomura et al 1998) and chemical proteolysis (Greiner et al 1996;Owens et al 1998;Miyake et al 1998) have been used. On the other hand, protein-protein contact domains can also be studied by means of genetic suppression (reviewed by Hartman and Roth 1973;Perlich 1999).The rationale for this approach is that a mutant derivative which has lost its original function can regain the wildtype phenotype as the result of a second mutation.…”

Mapping of Subunit-Subunit Contact Surfaces on the β′ Subunit of Escherichia coli RNA Polymerase

“…The NMR structure has been determined for the ␣ COOH-terminal domain (8); the structure of the ␣ NH 2 -terminal domain was determined by x-ray crystallography (9), and the two domains are connected by a long flexible linker (10). Detailed mapping of the ␣-␣, ␣-␤, and ␣-␤Ј contact sites on the ␣ subunit have been carried out by making a number of contact-defective ␣ mutants with deletion, insertion, and Ala substitution mutations (11)(12)(13)(14) or by mapping the cleavage sites in ␣ by a chemical protease conjugated at various positions of the ␣ subunit (15).…”