Direct Interaction between Escherichia coli RNA Polymerase and the Zinc Ribbon Domains of DNA Topoisomerase I

Abstract

Escherichia coli DNA topoisomerase I (encoded by the topA gene) is important for maintaining steady-state DNA supercoiling and has been shown to influence vital cellular processes including transcription. Topoisomerase I activity is also needed to remove hypernegative supercoiling generated on the DNA template by the progressing RNA polymerase complex during transcription elongation. The accumulation of hypernegative supercoiling in the absence of topoisomerase I can lead to R-loop formation by the nascent transcript and template strand, leading to suppression of transcription elongation. Here we show by affinity chromatography and overlay blotting that E. coli DNA topoisomerase I interacts directly with the RNA polymerase complex. The protein-protein interaction involves the ␤ subunit of RNA polymerase and the C-terminal domains of E. coli DNA topoisomerase I, which are homologous to the zinc ribbon domains in a number of transcription factors. This direct interaction can bring the topoisomerase I relaxing activity to the site of transcription where its activity is needed. The zinc ribbon C-terminal domains of other type IA topoisomerases, including mammalian topoisomerase III, may also help link the enzyme activities to their physiological functions, potentially including replication, transcription, recombination, and repair.DNA topoisomerases are ubiquitous enzymes that have functional roles in many vital cellular processes (1, 2). Among different classes of topoisomerases, type IA topoisomerases found in archea, prokaryotes, and eukaryotes share the mechanistic feature of cutting and rejoining a single strand of DNA via a 5Ј-phosphotyrosine linkage and homologous amino acid sequences (3). Escherichia coli DNA topoisomerase I (encoded by the topA gene) is the most extensively studied example of this class of enzyme. Its most apparent physiological role is the maintenance of steady-state DNA supercoiling (4, 5). During transcription, the movement of the RNA polymerase complex on the DNA template creates local transcription-driven supercoiling with negative supercoiling generated behind the RNA polymerase and positive supercoiling generated ahead of the RNA polymerase (6, 7). DNA gyrase is needed for removing the positive supercoils, and topoisomerase I is responsible for removing the excess negative supercoils. In the absence of topoisomerase I function due to mutation in the topA gene, the accumulation of hypernegative supercoiling can lead to R-loop formation by nascent transcription and template stranding with the consequent suppression of transcription elongation (8,9).In previous studies, Tn5 transposase was found to copurify with E. coli DNA topoisomerase I and inhibit the topoisomerase I activity (10). RNA polymerase was also found to copurify with Tn5 transposase, but the copurification was reduced in extracts from a topA mutant strain, suggesting that the interaction between RNA polymerase and DNA topoisomerase I was responsible for the copurification of RNA polymerase with Tn5 transposase (10). The p...

Antimicrobial Susceptibility and SOS-Dependent Increase in Mutation Frequency Are Impacted by Escherichia coli Topoisomerase I C-Terminal Point Mutation

“…Biochemical activity assays have shown that the C-terminal region beyond Asp-760 is not required for the in vitro relaxation activity of the enzyme (26). The topA CTD mutations may, however, affect the previously identified interaction between RNA polymerase and topoisomerase I (19). This interaction could be more critical when a rapid cellular response to a stress challenge is required for survival.…”

“…The last 122 residues of the enzyme do not bind Zn(II) but also fold into a zinc ribbon structure (18). The C-terminal domains (CTD) between residues 598 and 865 contribute to the protein-protein interaction with RNA polymerase for relief of transcription-driven supercoiling during transcription elongation (19). This direct interaction with RNA polymerase may be critical for topoisomerase I function dur-ing a stress response when high rates of transcription of certain genes are needed for adaptation and survival.…”

Convergence of Molecular, Modeling, and Systems Approaches for an Understanding of the Escherichia coli Heat Shock Response

“…Enzymes for modifying rRNA (23S methylation) and tRNA (⌬ 3 -isopentyl diphosphate added adjacent to the anticodon) are encoded by the regulon; these modifications are believed to be important for growth at high temperature (4,59). Several RNA polymerase binding proteins (HepA, TopA, and NusB) believed to be important for alleviating the effects of supercoiling on transcription and two proteins implicated in ribosome protection and recycling (YfrH [Hsp15] and YfiA) are regulon members (6,29,30,50). Taken together, this brief survey indicates that 32 protect DNA and RNA in addition to proteins from the deleterious effects of excess heat and also modify the transcription, translation, and repair machinery to enable stress adaptation.…”

Regulon and promoter analysis of theE. coliheat-shock factor, σ32, reveals a multifaceted cellular response to heat stress

“…Two newly discovered regulon members, HepA and NusB, join TopA (Qi et al 1996) as general effectors of transcription, and all three may respond to altered supercoiling at high temperature. Previous work indicated that thermal induction of TopA, an RNA polymerase-associated topoisomerase (Cheng et al 2003) is necessary for normal transcription patterns at high temperature and for thermal resistance (Qi et al 1996). HepA (RapA) is also an RNA polymerase-associated protein (Sukhodolets et al 2001) specifically required for RNA polymerase recycling of tightly compacted negatively supercoiled DNA.…”