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

Abstract

The RNA polymerase core enzyme of Escherichia coli is composed of 2alpha, 1beta, and 1beta' subunits. Previously we mapped the alpha-alpha, alpha-beta, and alpha-beta' contact sites on the alpha subunit. Here we analyzed the alpha subunit contact sites on the beta subunit by using various experimental approaches: (i) comparison of the proteolytic cleavage map between the unassembled free beta subunit and the alpha2 beta complex; (ii) analysis of the binary complex formation between His6-tagged intact alpha subunit and various truncated beta fragments; and (iii) analysis of the complex formation between the alpha subunit and various His6-tagged beta fragments. The results altogether indicate that two regions of the beta subunit are involved in the full activity of alpha binding, that is, the primary contact site between residues 737 and 904 and the secondary region with assembly control activity downstream from residue 1138. All of the alpha subunit-beta fragment binary complexes identified in this study were found to bind beta' subunit and form pseudo-core complexes, indicating that the regions of beta involved in alpha subunit contact also participate in interaction with the beta' subunit.

Inter- and Intrasubunit Interactions during the Formation of RNA Polymerase Assembly Intermediate

“…18). However, similar analysis performed by the Ishihama group (19) suggested that the primary determinants of ␣ binding include conserved segments F and G, corresponding to E. coli ␤ amino acids 800 -900.…”

“…Moreover, ␤ 950 -1342 interacted with His 6 -␣ WT and His 6 -␣ R45A in vitro with the same (low) efficiency (18). On the other hand, Nomura et al (19) mapped the primary ␣-binding site to ␤ amino acids 737-904. We constructed pPC86-based two-hybrid plasmids with ␤ 800 -911 (conserved segments F and G), and ␤ 907-1231 , (conserved segments H and a small portion of I) and tested them for their ability to interact with ␣.…”

“…The impetus for this study came from the apparently contradictory results of Nomura et al (19) and Wang et al (18), who localized the ␣-binding determinants into non-overlapping fragments of E. coli ␤. Nomura et al claimed that ␤ conserved segments F and G, harbor the primary ␣-binding site; Wang et al localized the binding site to ␤ segments H and I; however, four conserved regions, F, G, H, and I, were required for strong and specific ␣ binding.…”

Intragenic suppression of trans‐dominant lethal substitutions in the conserved GEME motif of the β subunit of RNA polymerase: evidence for functional cooperativity within the C‐terminus

“…[Note that G1228D alone and in combination with six other primary mutations, but not with M1273V, with which it was initially isolated, also prevents assembly (Malik et al, manuscript in preparation).] Indeed, a recent study defines an assembly determinant for the ␣ subunit in the conserved ␤ region encompassing residues 907→1246 (and the extreme C-terminus of ␤ has been implicated in ␤ 0 association; Wang et al 1997; but see also Nomura et al 1999). Second, two of the three assembled enzymes are functional in vitro and do not blockade (cluster #1, G1228D (M1273V) and L1238P (G1271V)) whilst the two primary mutants, M1273V and G1271V, are both transcriptionally proficient and inhibitory in vitro.…”

Interaction of Recombinant Tata-Binding Protein With Mammals Genes Promoter Tata

“…В работах [22,8] показано, что за-мены в ТАТА-боксе промотора SV40 (ТАТАААА→ТАТТТАТ и ТАТАААА→ ТАТАСА) вызывают снижение сродства дрожжевого ТВР к ДНК примерно в 4 раза, нуклеотидная замена в ТАТА-боксе промотора гена hsp70 (ТАТА→ TGTA) приводит к снижению уровня транскрипции примерно в 10 раз, а аналогичные замены в промоторе гена his3 (TATA→TGTA и TATA→TATG) снижают уровень транскрипции почти в 100 раз. Wobbe и Struhl [34] пока-зали, что мутации в консенсусе Т 1 А 2 Т 3 А 4 А 5 А 6 А 7 , функционирование которого анализировали в контексте двадцати пяти промоторов, поразному снижали активность промотора.…”