Tagetitoxin Inhibits RNA Polymerase through Trapping of the Trigger Loop

Artsimovitch, Irina; Svetlov, Vladimir; Nemetski, Sondra Maureen; Epshtein, Vitaly; Cardozo, Timothy; Nudler, Evgeny

doi:10.1074/jbc.m111.300889

Cited by 31 publications

(68 citation statements)

References 33 publications

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“…The structure of bacterial RNA polymerase with the inhibitor Tagetitoxin (Tgt) indicated that Tgt stabilizes an inactive state by changing metal ion binding in the active site [91,93]. However, a recent EC-Tgt model based on biochemical analysis and molecular dynamic simulations suggested that Tgt inhibits transcription by trapping the trigger loop in an inactive state [94]. Thus the mechanism of Tgt inhibition remains debated [95,96].…”

Section: Translocation and Inhibitionmentioning

confidence: 99%

Structural basis of transcription elongation

Martínez-Rucobo

Cramer

2013

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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For transcription elongation, all cellular RNA polymerases form a stable elongation complex (EC) with the DNA template and the RNA transcript. Since the millennium, a wealth of structural information and complementary functional studies provided a detailed three-dimensional picture of the EC and many of its functional states. Here we summarize these studies that elucidated EC structure and maintenance, nucleotide selection and addition, translocation, elongation inhibition, pausing and proofreading, backtracking, arrest and reactivation, processivity, DNA lesion-induced stalling, lesion bypass, and transcriptional mutagenesis. In the future, additional structural and functional studies of elongation factors that control the EC and their possible allosteric modes of action should result in a more complete understanding of the dynamic molecular mechanisms underlying transcription elongation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.

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Section: Translocation and Inhibitionmentioning

confidence: 99%

Structural basis of transcription elongation

Martínez-Rucobo

Cramer

2013

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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“…Interestingly, small molecules may further modify the mobile active centres. The antibiotic tagetitoxin binds in the vicinity of the catalytic residues of the TL (Artsimovitch et al, 2011;Vassylyev et al, 2005). Whilst not affecting the catalytic properties of the TL active centre (Yuzenkova et al, 2013), tagetitoxin modifies it so that, after phosphodiester bond formation, RNAP with the 'TL+tagetitoxin' active centre has a delayed translocation (Yuzenkova et al, 2013).…”

Section: Multiple Active Centres Of Rnapmentioning

confidence: 99%

Multiple personalities of the RNA polymerase active centre

Zenkin

2014

Microbiology

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Transcription in all living organisms is accomplished by highly conserved multi-subunit RNA polymerases (RNAPs). Our understanding of the functioning of the active centre of RNAPs has transformed recently with the finding that a conserved flexible domain near the active centre, the trigger loop (TL), participates directly in the catalysis of RNA synthesis and serves as a major determinant for fidelity of transcription. It also appears that the TL is involved in the unique ability of RNAPs to exchange catalytic activities of the active centre. In this phenomenon the TL is replaced by a transcription factor which changes the amino acid content and, as a result, the catalytic properties of the active centre. The existence of a number of transcription factors that act through substitution of the TL suggests that the RNAP has several different active centres to choose from in response to external or internal signals.A video of this Prize Lecture, presented at the Society for General Microbiology Annual Conference 2014, can be viewed via this link: https: //www.youtube.com/watch?v=79Z7iXVEPo4 TRIGGER LOOP: A NEW CATALYTIC DOMAIN OF THE RNA POLYMERASE ACTIVE CENTRE Multi-subunit RNAPs are the enzymes that perform transcription in all living organisms. RNAPs are highly conserved, and emerged before the divergence of the bacteria and archaea/eukaryote lineages. All RNAPs share the invariantly conserved catalytic core of five subunits: b, b9, 2a and v (bacterial nomenclature is used throughout). The two largest subunits, b and b9, form the catalytic cleft where the reaction of addition of nucleoside monophosphates (NMPs) to the growing RNA takes place. As with many other nucleic-acid-managing enzymes (Steitz & Steitz, 1993) and all nucleic-acid-polymerizing enzymes (Steitz, 1998), RNAP uses a two-metal-ion (Mg 2+ ) mechanism to catalyse the phosphotransfer reaction. One of the metal ions, Mg 2+ I, is chelated by the invariant triad of aspartates of the b9 subunit, whilst the other one, Mg 2+ II, is brought by substrates, e.g. by the incoming nucleoside triphosphate (NTP). Mg 2+ I stabilizes the negative charge on the attacking oxygen of the hydroxyl group of the 39 NMP of RNA. Mg 2+ II assists the leaving of the pyrophosphate, and both metal ions ligate the non-bridging oxygen to stabilize the pentacovalent transition state. Substitutions in the aspartate triad lead to almost full inactivation of the enzyme (Zaychikov et al., 1996).The emergence of the first crystal structure of multi-subunit RNAP (Zhang et al., 1999) ignited structure-based functional analysis of the enzyme by many researchers. We were interested in the disordered loop (referred to initially as the G-loop and, later, as the TL) of the highly conserved G domain of the b9 subunit. In the structure, this disordered region was located .20 Å from Mg 2+ I. However, we found that deletion of this loop had a dramatic effect on catalysis by slowing down NMP addition~10 4 times -an effect close to that of mutations in the aspartate triad. Three years later we foun...

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“…We identified a plausible Tgt-binding site 16 near the previously reported binding site in the T. thermophilus RNAP holoenzyme. 2 Binding of Tgt to this site is sterically incompatible with the fully-folded TL but is compatible with an alternative, "inactive" state stabilized by a contact between Tgt and the TL residue β' Arg1239 (Arg933 in the E. coli β' subunit).…”

Section: Tgt Contacts With the Tlmentioning

confidence: 92%