M-CSA Mechanism and Catalytic Site Atlas

DNA-directed RNA polymerase

DNA-dependent RNA polymerases bind to promoter DNA in transcription initiation, opens the duplex strands, and can convert from initiation to elongation phase conformation. More specifically, RNA polymerase II transcribes mRNA in the nucleus of eukaryotes, with closely related enzymes in bacteria and some viruses. RNA synthesis occurs through cycles of repeated ribonucleotide incorporation. While RNAPII is apart of a family containing multiple subunits, there are also single subunit RNAPs such as in the T7 bacteriophage which display high similarity to DNA polymerases.

Reference Protein and Structure

Sequence: P04050 (2.7.7.6) (Sequence Homologues) (PDB Homologues)
Biological species: Saccharomyces cerevisiae S288c (Baker's yeast)
PDB: 2e2h - RNA polymerase II elongation complex at 5 mM Mg2+ with GTP (3.95 Å)
Catalytic CATH Domains: 2.40.40.20 2.40.270.10 (see all for 2e2h)
Cofactors: Magnesium(2+) (2)

Click To Show Structure

Enzyme Reaction (EC:2.7.7.6)

nucleoside triphosphate(4-)

CHEBI:61557

RNA (poly(A))

CHEBI:8756

→

diphosphate(3-)

CHEBI:33019

RNA polyanion

CHEBI:83400

Enzyme reaction links: IntEnz ENZYME ExplorEnz

Alternative enzyme names: C RNA formation factors, C ribonucleic acid formation factors, DNA-dependent RNA nucleotidyltransferase, DNA-dependent RNA polymerase, DNA-dependent ribonucleate nucleotidyltransferase, RNA nucleotidyltransferase, RNA nucleotidyltransferase (DNA-directed), RNA polymerase, RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA transcriptase, Deoxyribonucleic acid-dependent ribonucleic acid polymerase, Ribonucleate nucleotidyltransferase, Ribonucleate polymerase, Ribonucleic acid nucleotidyltransferase, Ribonucleic acid polymerase, Ribonucleic acid transcriptase, Ribonucleic polymerase, Ribonucleic transcriptase, Transcriptase,

Enzyme Mechanism

Mechanism Proposal 1 ☆☆

Summary
Step 1
Step 2
Products
All Steps

Introduction

RNA polymerases catalyse the nucleophilic attack of a bound nucleoside 5'-triphosphate by the 3'-hydroxyl of an RNA primer, resulting in the incorporation of a nucleoside monophosphate into RNA and the release of pyrophosphate. This is thought to occur using two-metal catalysis. In RNA polymerase II, two magnesium ions are coordinated by four aspartates (3'OH of RNA also proposed to weakly coordinate to Mg2+A). Mg2+A is proposed to lower the pKa around the attacking hydroxyl while Mg2+B is there to stabilise the negative charges during transition state. More recently, computational evidence suggests the reaction proceeds by nucleophilic attack by a recently deprotonated 3' OH group (by a solvent hydroxide ion), on the alpha NTP phosphate. The leaving group, pyrophosphate is also protonated by His1085. There has however been evidence to rule out His1085 acting as a general acid (and a nearby Arginine), where another residue that remains unknown carries out this function.

Catalytic Residues Roles

UniProt	PDB* (2e2h)
His1085	His1085A(D)	Proposed to act as a general acid to protonate the leaving group, pyrophosphate. However, there has been a recent paper disproving it's role as an acid-base catalyst and instead involved in a positional catalyst.	proton acceptor, proton donor
Asp485, Asp483, Asp481, Asp837	Asp485A(D), Asp483A(D), Asp481A(D), Asp837B(E)	Coordinates to the two magnesium metal ions.	metal ligand

*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

bimolecular nucleophilic substitution, proton transfer, overall product formed, overall reactant used, inferred reaction step, native state of enzyme regenerated

References

Carvalho AT et al. (2011), J Chem Theory Comput, 7, 1177-1188. The Catalytic Mechanism of RNA Polymerase II. DOI:10.1021/ct100579w. PMID:26606364.
Mishanina TV et al. (2017), Proc Natl Acad Sci U S A, 114, E5103-E5112. Trigger loop of RNA polymerase is a positional, not acid-base, catalyst for both transcription and proofreading. DOI:10.1073/pnas.1702383114. PMID:28607053.
Svetlov V et al. (2013), Biochim Biophys Acta, 1829, 20-28. Basic mechanism of transcription by RNA polymerase II. DOI:10.1016/j.bbagrm.2012.08.009. PMID:22982365.
Wang D et al. (2006), Cell, 127, 941-954. Structural basis of transcription: role of the trigger loop in substrate specificity and catalysis. DOI:10.1016/j.cell.2006.11.023. PMID:17129781.

Step 1. Free hydroxyl from bulk sulvent acts as a general base and deprotonates the ribose sugar's 3' OH. This increases the 3'OH nucleophilicity to attack the alpha phosphate on the NTP. The pyrophosphate leaving group is protonated by His1085.

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