Faculty Opinions recommendation of Swing-gate model of nucleotide entry into the RNA polymerase active center.

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.

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The TFIIF-Like Rpc37/53 Dimer Lies at the Center of a Protein Network To Connect TFIIIC, Bdp1, and the RNA Polymerase III Active Center

Molecular and Cellular Biology ◽

10.1128/mcb.05151-11 ◽

2011 ◽

Vol 31 (13) ◽

pp. 2715-2728 ◽

Cited By ~ 55

Author(s):

C.-C. Wu ◽

Y.-C. Lin ◽

H.-T. Chen

Keyword(s):

Rna Polymerase ◽

Active Center ◽

Rna Polymerase Iii ◽

Protein Network ◽

Polymerase Iii

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The involvement of the aspartate triad of the active center in all catalytic activities of multisubunit RNA polymerase

Nucleic Acids Research ◽

10.1093/nar/gki688 ◽

2005 ◽

Vol 33 (13) ◽

pp. 4202-4211 ◽

Cited By ~ 44

Author(s):

V. Sosunov

Keyword(s):

Rna Polymerase ◽

Active Center ◽

Catalytic Activities

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Closing and opening of the RNA polymerase trigger loop

10.1101/817080 ◽

2019 ◽

Cited By ~ 1

Author(s):

Abhishek Mazumder ◽

Miaoxin Lin ◽

Achillefs N. Kapanidis ◽

Richard H. Ebright

Keyword(s):

Rna Polymerase ◽

Fluorescent Probe ◽

Active Center ◽

Single Molecule ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Single Molecule Fluorescence ◽

Structural Element ◽

Trigger Loop ◽

Nucleotide Addition

The RNA polymerase (RNAP) trigger loop (TL) is a mobile structural element of the RNAP active center that, based on crystal structures, has been proposed to cycle between an “unfolded”/“open” state that allows an NTP substrate to enter the active center and a “folded”/“closed” state that holds the NTP substrate in the active center. Here, by quantifying single-molecule fluorescence resonance energy transfer between a first fluorescent probe in the TL and a second fluorescent probe elsewhere in RNAP or in DNA, we detect and characterize TL closing and opening in solution. We show that the TL closes and opens on the millisecond timescale; we show that TL closing and opening provides a checkpoint for NTP complementarity, NTP ribo/deoxyribo identity, and NTP tri/di/monophosphate identity, and serves as a target for inhibitors; and we show that one cycle of TL closing and opening typically occurs in each nucleotide addition cycle in transcription elongation.

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Identification of a Specific Inhibitor of Human Scp1 Phosphatase Using the Phosphorylation Mimic Phage Display Method

Catalysts ◽

10.3390/catal9100842 ◽

2019 ◽

Vol 9 (10) ◽

pp. 842 ◽

Cited By ~ 2

Author(s):

Takuya Yoshida ◽

Kazuki Yamazaki ◽

Shunta Imai ◽

Akinori Banno ◽

Atsushi Kaneko ◽

...

Keyword(s):

Rna Polymerase ◽

Phage Display ◽

Rna Polymerase Ii ◽

Active Center ◽

Protein Phosphatases ◽

Specific Inhibitor ◽

Competitive Inhibitor ◽

Dependent Manner ◽

Simple Method ◽

Ctd Phosphatase

Protein phosphatases are divided into tyrosine (Tyr) phosphatases and serine/threonine (Ser/Thr) phosphatases. While substrate trapping mutants are frequently used to identify substrates of Tyr phosphatases, a rapid and simple method to identify Ser/Thr phosphatase substrates is yet to be developed. The TFIIF-associating component of RNA polymerase II C-terminal domain (CTD) phosphatase/small CTD phosphatase (FCP/SCP) phosphatase family is one of the three types of Ser/Thr protein phosphatases. Defects in these phosphatases are correlated with the occurrence of various diseases such as cancer and neuropathy. Recently, we developed phosphorylation mimic phage display (PMPD) method with AlF4−, a methodology to identify substrates for FCP/SCP type Ser/Thr phosphatase Scp1. Here, we report a PMPD method using BeF3− to identify novel substrate peptides bound to Scp1. After screening peptide phages, we identified peptides that bound to Scp1 in a BeF3−-dependent manner. Synthetic phosphopeptide BeM12-1, the sequence of which was isolated at the highest frequency, directly bound to Scp1. The binding was inhibited by adding BeF3−, indicating that the peptide binds to the active center of catalytic site in Scp1. The phosphorylated BeM12-1 worked as a competitive inhibitor of Scp1. Thus, PMPD method may be applicable for the identification of novel substrates and inhibitors of the FCP/SCP phosphatase family.

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