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

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yu Zhang ◽  
David Degen ◽  
Mary X Ho ◽  
Elena Sineva ◽  
Katherine Y Ebright ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Binding Site ◽  
Active Center ◽  
Transcription Initiation ◽  
Cyclic Peptide ◽  
Cross Resistance ◽  
Peptide Antibiotic ◽  
Nucleotide Binding Sites ◽  
Bacterial Rna ◽  
Polymerase I

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.

scholarly journals Antibacterial nucleoside-analog inhibitor of bacterial RNA polymerase: pseudouridimycin

2017 ◽  
Author(s):  
Sonia I. Maffioli ◽  
Yu Zhang ◽  
David Degen ◽  
Thomas Carzaniga ◽  
Giancarlo Del Gatto ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Drug Development ◽  
Rna Polymerase ◽  
Binding Site ◽  
Bacterial Pathogens ◽  
Nucleoside Analog ◽  
Cross Resistance ◽  
Nucleoside Triphosphate ◽  
Antibacterial Drug ◽  
Bacterial Rna

There is an urgent need for new antibacterial drugs effective against bacterial pathogens resistant to current drugs1–2. Nucleoside-analog inhibitors (NAIs) of viral nucleotide polymerases have had transformative impact in treatment of HIV3and HCV4. NAIs of bacterial RNA polymerase (RNAP) potentially could have major impact on treatment of bacterial infection, particularly because functional constraints on substitution of RNAP nucleoside triphosphate (NTP) binding sites4-5could limit resistance emergence4-5. Here we report the discovery, from microbial extract screening, of an NAI that inhibits bacterial RNAP and exhibits antibacterial activity against a broad spectrum of drug-sensitive and drug-resistant bacterial pathogens: pseudouridimycin (PUM). PUM is a novel microbial natural product consisting of a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 6'-amino-pseudouridine. PUM potently and selectively inhibits bacterial RNAP in vitro, potently and selectively inhibits bacterial growth in culture, and potently clears infection in a mouse model ofStreptococcus pyogenesperitonitis. PUM inhibits RNAP through a binding site on RNAP (the "i+1" NTP binding site) and mechanism (competition with UTP for occupancy of the "i+1" NTP binding site) that differ from those of the RNAP inhibitor and current antibacterial drug rifampin (Rif). PUM exhibits additive antibacterial activity when co-administered with Rif, exhibits no cross-resistance with Rif, and exhibits a spontaneous resistance rate an order-of-magnitude lower than that of Rif. The results provide the first example of a selective NAI of bacterial RNAP, provide an advanced lead compound for antibacterial drug development, and provide structural information and synthetic routes that enable lead optimization for antibacterial drug development.

scholarly journals Mechanism of SARS-CoV-2 polymerase stalling by remdesivir

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Goran Kokic ◽  
Hauke S. Hillen ◽  
Dimitry Tegunov ◽  
Christian Dienemann ◽  
Florian Seitz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Binding Site ◽  
Molecular Mechanism ◽  
Active Center ◽  
Rna Synthesis ◽  
Active Form ◽  
Nucleoside Triphosphate ◽  
Rna Dependent Rna Polymerase ◽  
Cryo Electron Microscopy

AbstractRemdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3ʹ-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3ʹ-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3ʹ-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication.

scholarly journals Universal functions of the σ finger in alternative σ factors during transcription initiation by bacterial RNA polymerase

RNA Biology ◽  
2021 ◽  
pp. 1-10
Author(s):  
Anastasiya Oguienko ◽  
Ivan Petushkov ◽  
Danil Pupov ◽  
Daria Esyunina ◽  
Andrey Kulbachinskiy
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Universal Functions ◽  
Bacterial Rna

The REB1 site is an essential component of a terminator for RNA polymerase I in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (1) ◽  
pp. 649-658
Author(s):  
W H Lang ◽  
R H Reeder
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Protein Binding ◽  
Binding Site ◽  
Rna Polymerase I ◽  
Recognition Sequence ◽  
Sequence Context ◽  
Yeast Saccharomyces Cerevisiae ◽  
Essential Component ◽  
Polymerase I

We have identified a terminator for transcription by RNA polymerase I in the genes coding for rRNA of the yeast Saccharomyces cerevisiae. The terminator is located 108 bp downstream of the 3' end of the mature 25S rRNA and shares several characteristics with previously studied polymerase I terminators in the vertebrates. For example, the yeast terminator is orientation dependent, is inhibited by its own sequence, and forms RNA 3' ends 17 +/- 2 bp upstream of an essential protein binding site. The recognition sequence for binding of the previously cloned REB1 protein (Q. Ju, B. E. Morrow, and J. R. Warner, Mol. Cell. Biol. 10:5226-5234, 1990) is an essential component of the terminator. In addition, the efficiency of termination depends upon sequence context extending at least 12 bp upstream of the REB1 site.

scholarly journals Structural basis of transcription inhibition by fidaxomicin (lipiarmycin A3)

2017 ◽  
Author(s):  
Wei Lin ◽  
Kalyan Das ◽  
David Degen ◽  
Abhishek Mazumder ◽  
Diego Duchi ◽  
...  
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Transcription Initiation ◽  
Resonance Energy ◽  
Active Pharmaceutical Ingredient ◽  
Conformational State ◽  
Initiation Factor ◽  
Structural Basis ◽  
Cross Resistance ◽  
Antibacterial Drug

Fidaxomicin is an antibacterial drug in clinical use in treatment ofClostridium difficilediarrhea1–2. The active pharmaceutical ingredient of fidaxomicin, lipiarmycin A3 (Lpm)1–4, is a macrocyclic antibiotic with bactericidal activity against Gram-positive bacteria and efflux-deficient strains of Gram-negative bacteria1–2, 5. Lpm functions by inhibiting bacterial RNA polymerase (RNAP)6–8. Lpm exhibits no cross-resistance with the classic RNAP inhibitor rifampin (Rif)7, 9and inhibits transcription initiation at an earlier step than Rif8–11, suggesting that the binding site and mechanism of Lpm differ from those of Rif. Efforts spanning a decade to obtain a crystal structure of RNAP in complex with Lpm have been unsuccessful. Here, we report a cryo-EM12–13structure ofMycobacterium tuberculosisRNAP holoenzyme in complex with Lpm at 3.5 Å resolution. The structure shows that Lpm binds at the base of the RNAP “clamp,” interacting with the RNAP switch region and the RNAP RNA exit channel. The binding site on RNAP for Lpm does not overlap the binding sites for other RNAP inhibitors, accounting for the absence of cross-resistance of Lpm with other RNAP inhibitors. The structure exhibits an open conformation of the RNAP clamp, with the RNAP clamp swung outward by ~17° relative to its position in catalytically competent RNAP-promoter transcription initiation complexes, suggesting that Lpm traps an open-clamp conformational state. Single-molecule fluorescence resonance energy transfer14experiments confirm that Lpm traps an open-clamp conformational state and define effects of Lpm on clamp opening and closing dynamics. We propose that Lpm inhibits transcription initiation by trapping an open-clamp conformational state, thereby preventing simultaneous engagement of transcription initiation factor σ regions 2 and 4 with promoter -10 and -35 elements. The results provide information essential to understanding the mode of action of Lpm, account for structure-activity relationships of known Lpm analogs, and suggest modifications to Lpm that could yield new, improved Lpm analogs.

scholarly journals Structural basis of RNA polymerase III transcription repression by Maf1

2019 ◽  
Author(s):  
Matthias K. Vorländer ◽  
Florence Baudin ◽  
Robyn D. Moir ◽  
René Wetzel ◽  
Wim J. H. Hagen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Binding Site ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
Structural Basis ◽  
Metabolic Efficiency ◽  
Transcription Repression ◽  
Polymerase Iii ◽  
Wide Range ◽  
Pol Iii

ABSTRACTMaf1 is a highly conserved central regulator of transcription by RNA polymerase III (Pol III), and Maf1 activity influences a wide range of phenotypes from metabolic efficiency to lifespan. Here, we present a 3.3 Å cryo-EM structure of yeast Maf1 bound to Pol III, which establishes how Maf1 achieves transcription repression. In the Maf1-bound state, Pol III elements that are involved in transcription initiation are sequestered, and the active site is sealed off due to ordering of the mobile C34 winged helix 2 domain. Specifically, the Maf1 binding site overlaps with the binding site of the Pol III transcription factor TFIIIB and DNA in the pre-initiation complex, rationalizing that binding of Maf1 and TFIIIB to Pol III are mutually exclusive. We validate our structure using variants of Maf1 with impaired transcription-inhibition activity. These results reveal the exact mechanism of Pol III inhibition by Maf1, and rationalize previous biochemical data.

Events during eucaryotic rRNA transcription initiation and elongation: conversion from the closed to the open promoter complex requires nucleotide substrates

1988 ◽  
Vol 8 (5) ◽  
pp. 1940-1946
Author(s):  
E Bateman ◽  
M R Paule
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Topological Analysis ◽  
Acanthamoeba Castellanii ◽  
Rna Polymerase I ◽  
Initiation Factor ◽  
Rrna Transcription ◽  
Promoter Complex ◽  
Polymerase I

Chemical footprinting and topological analysis were carried out on the Acanthamoeba castellanii rRNA transcription initiation factor (TIF) and RNA polymerase I complexes with DNA during transcription initiation and elongation. The results show that the binding of TIF and polymerase to the promoter does not alter the supercoiling of the DNA template and the template does not become sensitive to modification by diethylpyrocarbonate, which can identify melted DNA regions. Thus, in contrast to bacterial RNA polymerase, the eucaryotic RNA polymerase I-promoter complex is in a closed configuration preceding addition of nucleotides in vitro. Initiation and 3'-O-methyl CTP-limited translocation by RNA polymerase I results in separation of the polymerase-TIF footprints, leaving the TIF footprint unaltered. In contrast, initiation and translocation result in a significant change in the conformation of the polymerase-DNA complex, culminating in an unwound DNA region of at least 10 base pairs.

Transcription initiation from the dihydrofolate reductase promoter is positioned by HIP1 binding at the initiation site

1990 ◽  
Vol 10 (2) ◽  
pp. 653-661
Author(s):  
A L Means ◽  
P J Farnham
Keyword(s):  
Rna Polymerase ◽  
Binding Site ◽  
Rna Polymerase Ii ◽  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Simian Virus 40 ◽  
Initiation Site ◽  
Simian Virus ◽  
Sequence Element ◽  
Initiator Element

We have identified a sequence element that specifies the position of transcription initiation for the dihydrofolate reductase gene. Unlike the functionally analogous TATA box that directs RNA polymerase II to initiate transcription 30 nucleotides downstream, the positioning element of the dihydrofolate reductase promoter is located directly at the site of transcription initiation. By using DNase I footprint analysis, we have shown that a protein binds to this initiator element. Transcription initiated at the dihydrofolate reductase initiator element when 28 nucleotides were inserted between it and all other upstream sequences, or when it was placed on either side of the DNA helix, suggesting that there is no strict spatial requirement between the initiator and an upstream element. Although neither a single Sp1-binding site nor a single initiator element was sufficient for transcriptional activity, the combination of one Sp1-binding site and the dihydrofolate reductase initiator element cloned into a plasmid vector resulted in transcription starting at the initiator element. We have also shown that the simian virus 40 late major initiation site has striking sequence homology to the dihydrofolate reductase initiation site and that the same, or a similar, protein binds to both sites. Examination of the sequences at other RNA polymerase II initiation sites suggests that we have identified an element that is important in the transcription of other housekeeping genes. We have thus named the protein that binds to the initiator element HIP1 (Housekeeping Initiator Protein 1).

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