Subunit assembly in vivo of Escherichia coli RNA polymerase: role of the amino‐terminal assembly domain of alpha subunit

Abstract

Background: The RNA polymerase core enzyme of Escherichia coli is assembled in the sequence

Rpb3, Stoichiometry and Sequence Determinants of the Assembly into Yeast RNA Polymerase II in Vivo

“…The NH 2 -terminal ␣-fragment (8) of Rpb3 (amino acids 29 -55) corresponds to the region in the ␣-subunit where a number of mutations were isolated that affected ␣-␣ dimerization (17)(18)(19)(20). In order to evaluate the importance of this fragment for the assembly of the RNA polymerase II in vivo, we constructed a deletion mutant of RPB3, lacking the coding sequence for amino acids 29 -55 (Rpb3 ␣N ), and cloned it into the p416GAL1 expression vector.…”

Positioning of two alpha subunit carboxy-terminal domains of RNA polymerase at promoters by two transcription factors

“…The ␣ subunit, consisting of 329 amino acid residues, is composed of two structural domains, each responsible for distinct functions (1)(2)(3) and each forming independent structural domains connected by a protease-sensitive flexible linker (4)(5)(6). The amino (N)-terminal domain from residues 20 to 235 plays a key role in RNA polymerase assembly by providing the contact surface for ␣ dimerization and binding of ␤ and ␤Ј subunits (7)(8)(9)(10), whereas the CTD from residues 235 to 329 plays a regulatory role by providing the contact surfaces for trans-acting protein factors and cis-acting DNA elements (11)(12)(13)(14).…”

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

“…For construction of plasmid pGET21-␣C-His 6 for high level expression of the ␣ subunit with His 6 tag at the COOH terminus, a XbaIBamHI fragment of plasmid pLAW2-H6, which was constructed for moderate expression of the His 6 -tagged ␣ subunit (14), was substituted for the corresponding segment of pET21a (Novagen).…”

“…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 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).…”