2000
DOI: 10.1074/jbc.m003884200
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Inter- and Intrasubunit Interactions during the Formation of RNA Polymerase Assembly Intermediate

Abstract: We used yeast two-hybrid and in vitro co-immobilization assays to study the interaction between the Escherichia coli RNA polymerase (RNAP) ␣ and ␤ subunits during the formation of ␣ 2 ␤, a physiological RNAP assembly intermediate. We show that a 430-amino acidlong fragment containing ␤ conserved segments F, G, H, and a short part of segment I forms a minimal domain capable of specific interaction with ␣. The ␣-interacting domain is held together by protein-protein interactions between ␤ segments F and I. Resid… Show more

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Cited by 9 publications
(6 citation statements)
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“…The beads were pelleted by centrifugation and washed twice with 500 μl of reconstitution buffer containing 20 mM imidazole so that all the unbound proteins were removed. The protein samples were eluted from the beads with 50 μl reconstitution buffer containing 250 mM imidazole and analyzed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) [24]. Molar ratio was calculated by densitometric scanning using standard proteins in Gel Doc 2000 (Bio Rad‐Quality one software).…”
Section: Methodsmentioning
confidence: 99%
“…The beads were pelleted by centrifugation and washed twice with 500 μl of reconstitution buffer containing 20 mM imidazole so that all the unbound proteins were removed. The protein samples were eluted from the beads with 50 μl reconstitution buffer containing 250 mM imidazole and analyzed by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) [24]. Molar ratio was calculated by densitometric scanning using standard proteins in Gel Doc 2000 (Bio Rad‐Quality one software).…”
Section: Methodsmentioning
confidence: 99%
“…4)) come from clearly different "domains" of the structure. 5 The i-site lies on the face of the ␤ sheet domain made up from the ␤ subunit conserved segments F, G, and H and a portion of segment I. Proteolytic studies, in vitro reconstitution, and twohybrid analysis indicate that in E. coli, this domain is capable of independent folding and specific binding to RNAP ␣ subunit (24,25) and that evolutionary conserved residue Asp 1084 in segment H is particularly important for ␣ 2 ␤ formation (25). Our results show that this domain is also capable of interacting with the initiating nucleotide ␣-phosphate through ␤Lys 1073 .…”
Section: Figmentioning
confidence: 99%
“…The large α-like subunit (RPB3 in eukaryal RNAP II, AC40 in RNAP I and RNAP III, Rpo3 (also known as RpoD) in archaea) heterodimerizes with its much smaller counterpart (RPB11 in eukaryal RNAP II, AC19 in RNAP I and RNAP III, Rpo11 (also known as RpoL) in archaea) [33-36]. Crystallographic and functional analyses indicates that large α homolog makes interaction with the second-largest (β-like) subunit through a surface that contains residue homologous to E. coli α Arg 45 [35,37], and is thus formally similar to Francisella α1. The smaller α homolog of eukaryal and archaeal RNAP thus corresponds to Francisella α2.…”
Section: Discussionmentioning
confidence: 99%