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

Abstract

The RNA polymerase core enzyme of Escherichia coli with the catalytic activity of RNA polymerization is assembled sequentially under the order: 2␣ 3 ␣ 2 3 ␣ 2 ␤ 3 ␣ 2 ␤␤. The core enzyme gains the activities of promoter recognition and transcription initiation after binding the subunit. The subunit-subunit contact surfaces of ␤ subunit (1407 residues) were analyzed by testing complex formation between various ␤ fragments and either the ␣ 2 ␤ complex or the 70 The RNA polymerase holoenzyme of Escherichia coli is composed of the core enzyme with the subunit composition of ␣ 2 ␤␤Ј and one of seven different species of the subunit (for review, see Ref. 1). The core enzyme carries the catalytic activities for RNA polymerization, but the subunit is required for promoter recognition and transcription initiation from the promoters. The core enzyme is assembled sequentially both in vitro and in vivo under the order: 2␣ 3 ␣ 2 3 ␣ 2 ␤ 3 ␣ 2 ␤␤Ј (premature core) 3 E (active core) (for review, see Ref. 2).Genetic and biochemical studies indicated that the subunitsubunit contact sites on ␣ including the sites for ␣ dimerization and the contact sites with the ␤ and ␤Ј subunits are all located within the amino (NH 2 )-terminal domain down to residue 235 (3-5), whereas the carboxyl (COOH)-terminal domain is involved in transcription regulation through direct interactions with class I (or ␣ contact) transcription factors and DNA UP elements (1, 5-7). 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).On the contrary, relatively little is known on the subunitsubunit contact sites on the two large subunits, ␤ and ␤Ј. The mapping of ␣ subunit contact sites on the ␤ subunit was carried out using two approaches: analysis of the proteolytic cleavage pattern of the unassembled free ␤ subunit and the intermediate subassembly ␣ 2 ␤ complex (16, 17), and analysis of complex formation between various ␤ fragments and the hexahistidine (His 6 )-tagged ␣ subunit or between various His 6 -tagged ␤ fragments and the intact ␣ subunit (17). The results altogether indicate that the primary and tight contact site of the ␣ subunit is located in the central portion of the ␤ polypeptide (16, 17); but in addition, the COOH-terminal proximal region is needed as the secondary and probable regulatory site for either efficient binding of the ␣ subunit or stabilization of ␣-␤ contact (17). All of the ␤ fragment-␣ binary complexes isolated were able to bind the ␤Ј subunit, leading to formation of pseudo-core complexes, suggesting that the ␤Ј subunit con...

Systematic search for zinc‐binding proteins in Escherichia coli

“…The forward primers used included the Nde I site sequence, while the reverse primers included the Eco RI or Bam HI site sequences. The PCR products were directly inserted between Nde I and either Eco RI or Bam HI sites of an expression vector pGET1 [13]. The reverse primer also contained the hexahistidine (His) tag sequence; expression of the target proteins was as fusions with the His tag.…”

Escherichia coli RNA polymerase subunit ω and its N‐terminal domain bind full‐length β′ to facilitate incorporation into the α2β subassembly

“…For the cloning of the NTD, the DNA corresponding to the NTD was PCR amplified and cloned between the Nde I and Xho I site of pET21b. Plasmids used for expression of α, β′, β, β′‐NTD(1–842)‐His 6 and β′‐CTD(842–1407)‐His 6 were pGEMAX185 [14], pRW308 [15], pGETB, pGETC (1–842) and pGETC (842–1407) [16], respectively.…”

Escherichia coli RNA Polymerase Is the Target of the Cyclopeptide Antibiotic Microcin J25

“…To us, however, this seems unlikely. It is worth noting that the putative target site for MccJ25 lies within a region spanning amino acids 842 to 1140 (thus containing segment G), which has been shown to be exposed on the surface of RNAP (16).…”