GE23077 binds to the RNA polymerase ‘i’ and ‘i+1’ sites and prevents the binding of initiating nucleotides

Abstract

Using a combination of genetic, biochemical, and structural approaches, we show that the cyclic-peptide antibiotic GE23077 (GE) binds directly to the bacterial RNA polymerase (RNAP) active-center ‘i’ and ‘i+1’ nucleotide binding sites, preventing the binding of initiating nucleotides, and thereby preventing transcription initiation. The target-based resistance spectrum for GE is unusually small, reflecting the fact that the GE binding site on RNAP includes residues of the RNAP active center that cannot be substituted without loss of RNAP activity. The GE binding site on RNAP is different from the rifamycin binding site. Accordingly, GE and rifamycins do not exhibit cross-resistance, and GE and a rifamycin can bind simultaneously to RNAP. The GE binding site on RNAP is immediately adjacent to the rifamycin binding site. Accordingly, covalent linkage of GE to a rifamycin provides a bipartite inhibitor having very high potency and very low susceptibility to target-based resistance.DOI: http://dx.doi.org/10.7554/eLife.02450.001

Structural Basis of Transcription Initiation by Bacterial RNA Polymerase Holoenzyme

“…Previously published crystal structure of the Thermus thermophilus RNAP transcription initiation complex contains a GpA dinucleotide primer complementary to the template DNA positions Ϫ1 and ϩ1 but lacking the 5Ј-triphosphate group (8). Recently, a structure of the T. thermophilus RNAP initiating complex containing two iNTPs has been reported (9). However, the roles of the observed RNAP-iNTP contacts in transcription initiation were not tested experimentally; in addition, the structure contained a suboptimal template strand sequence around the transcription start site (see below), suggesting that it might miss some important contacts with the iNTPs.…”

Bacterial Transcription as a Target for Antibacterial Drug Development

“…Chemical derivation does not result in a great improvement of antibiotic activity (88). However, the target-dependent resistance spectrum for GE23077 was much smaller than those for RIF and other RNAP inhibitors, with resistant substitutions being obtained at only four residues (E565, G566, M681, and N684) in the ␤ subunit (86), suggesting that the i and iϩ1 sites at the active center might be a good target for drug discovery.…”

“…3D) (86). This compound demonstrated excellent activity against GE23077 or RIF-resistant RNAP, showing that the RIF binding pocket and GE23077 binding i and iϩ1 sites could be considered for use as a combined target for drug design.…”

“…3D) (86). More precisely, it was demonstrated using saturation mutagenesis that E. coli ␤ subunit residues P564, E565, G566, N568, R678, M681, N684, M685, Q688, K1065, K1073, and H1237 and ␤= residues D462, T786, and A787 were involved in binding with GE23077.…”

Real-Time Observation of Polymerase- Promoter Contact Remodeling during Transcription Initiation

“…Previous structural, biochemical, and biophysical studies [13][14][15][16][17] have provided snapshots of holoenzyme-promoter contacts, and a variety of single-molecule approaches have proved useful in dissecting additional mechanistic and kinetic details of initiation in prokaryotes 3,4,[18][19][20] and eukaryotes 21 , but key questions remain. In particular, how does RNAP remodel its contacts with the promoter DNA during the initiation phase, ultimately leading to the formation of the EC?…”