Basic mechanism of transcription by RNA polymerase II

Abstract

RNA polymerase II-like enzymes carry out transcription of genomes in Eukaryota, Archaea, and some viruses. They also exhibit fundamental similarity to RNA polymerases from bacteria, chloroplasts, and mitochondria. In this review we take an inventory of recent studies illuminating different steps of basic transcription mechanism, likely common for most multi-subunit RNA polymerases. Through the amalgamation of structural and computational chemistry data we attempt to highlight the most feasible reaction pathway for the two-metal nucleotidyl transfer mechanism, and to evaluate the way catalysis can be linked to translocation in the mechano-chemical cycle catalyzed by RNA polymerase II. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.

Figure 1. Orthologous bacterial RNA polymerase-like core in Eubacteria (A, 3lu0), Eukarya (B, 1nik), and Archaea (C, 3hkz)

All enzymes are surface, light teal, β′-like subunits are deep teal, β-like subunits are yellow-orange, α-like subunits are red and warm pink, ω-like subunits are purple-blue.

Figure 1. Orthologous bacterial RNA polymerase-like core in Eubacteria (A, 3lu0), Eukarya (B, 1nik), and Archaea (C, 3hkz)

All enzymes are surface, light teal, β′-like subunits are deep teal, β-like subunits are yellow-orange, α-like subunits are red and warm pink, ω-like subunits are purple-blue.

Figure 1. Orthologous bacterial RNA polymerase-like core in Eubacteria (A, 3lu0), Eukarya (B, 1nik), and Archaea (C, 3hkz)

All enzymes are surface, light teal, β′-like subunits are deep teal, β-like subunits are yellow-orange, α-like subunits are red and warm pink, ω-like subunits are purple-blue.

Figure 2. Active site of yeast RNA polymerase II (2e2h)

Template DNA is cartoon grey, except for the templating nucleotide (black), RNA is cartoon light pink, except for the 3′ nucleotide (hot pink). Substrate GTP is orange sticks. Catalytic Mg²⁺ cations are solid magenta spheres, catalytic tetrad (Asp481, 483, 485, and 837) is red sticks. Trigger loop is green cartoon, residues Gln1078, Leu1081, and His1085 are green sticks. The rest of amino acids are light teal sticks.

Figure 3. Structural variants of the trigger loop in yeast RNA polymerase II

Elements of the active site (from GTP-bound 2e2h) are represented as in Fig. 2. (substrate is orange sticks, catalytic Mg²⁺ cations are solid magenta spheres, catalytic tetrad is red sticks) Superimposed are trigger loops (cartoon) from NTP-free enzyme (1sfo, purple blue), GMPCPP-bound 2e2j (yellow) and 2nvt (marine), and UTP-bound 2nvz (red).

Figure 4. Two-metal catalysis of NTP condensation by yeast RNA polymerase II

A. RNA 3′ OH is deprotonated by the OH⁻ from the bulk solvent as substrate’s O_β is protonated by His1085. B. Nucleophilic attack by RNA 3′ O⁻ on substrate’s P_α. C. Substrate in a form of NMP added to the 3′ end of RNA, pyrophosphate in a form of protonated (Mg-PP_i)⁻ leaves active site. This figure was adapted from Carvalho et al, and shows reaction mechanism as a sequence of distinct steps, for clarity and ease of analysis. Proton-inventory experiments reported by Castro et al. indicated that these reaction can take place within the same associative-like transition state.

Figure 4. Two-metal catalysis of NTP condensation by yeast RNA polymerase II

A. RNA 3′ OH is deprotonated by the OH⁻ from the bulk solvent as substrate’s O_β is protonated by His1085. B. Nucleophilic attack by RNA 3′ O⁻ on substrate’s P_α. C. Substrate in a form of NMP added to the 3′ end of RNA, pyrophosphate in a form of protonated (Mg-PP_i)⁻ leaves active site. This figure was adapted from Carvalho et al, and shows reaction mechanism as a sequence of distinct steps, for clarity and ease of analysis. Proton-inventory experiments reported by Castro et al. indicated that these reaction can take place within the same associative-like transition state.

Figure 4. Two-metal catalysis of NTP condensation by yeast RNA polymerase II

A. RNA 3′ OH is deprotonated by the OH⁻ from the bulk solvent as substrate’s O_β is protonated by His1085. B. Nucleophilic attack by RNA 3′ O⁻ on substrate’s P_α. C. Substrate in a form of NMP added to the 3′ end of RNA, pyrophosphate in a form of protonated (Mg-PP_i)⁻ leaves active site. This figure was adapted from Carvalho et al, and shows reaction mechanism as a sequence of distinct steps, for clarity and ease of analysis. Proton-inventory experiments reported by Castro et al. indicated that these reaction can take place within the same associative-like transition state.

Figure 5. Hopping pathway of pyrophosphate release from the active site of yeast RNA polymerase II

Elements of the active site (from GTP-bound 2e2h) are represented as in Fig. 2. Amino acid residues implicated in facilitating pyrophosphate release are green sticks with green semitransparent spheres.