Understanding the Molecular Basis of RNA Polymerase II Transcription

Abstract

Synthetic nucleic acid analogues have profoundly advanced our knowledge of DNA and RNA, as well as the complex biological processes that involve nucleic acids. As a pivotal enzyme, eukaryotic RNA polymerase II (Pol II) is responsible for transcribing DNA into messenger RNA, which serves as a template to direct protein synthesis. Chemically modified nucleic acid analogues have greatly facilitated the structural elucidation of RNA Pol II elongation complex and understanding the key chemical interactions governing RNA Pol II transcriptional fidelity. This review addresses major progress in RNA polymerase II mechanistic studies using modified nucleic acid analogues in recent years.

Keywords: RNA polymerase II; chemical biology; nucleic acids; structural biology; transcription.

Figure 1

A) Hydrogen bonding in canonical Watson-Crick base pairs G:C and A:T. B) Cutaway view of RNA Pol II elongation complex (pdb:1R9T). RNA Pol II is revealed as a tan surface. The nascent RNA, template DNA, and non-template DNA are shown in red, cyan, and green, respectively. C) Detailed structure active site of RNA Pol II transcribing complex bound with a matched GTP (PDB: 2E2H). The bridge helix and trigger loop are shown in magenta and cyan, respectively. RNA and DNA are shown in yellow and GTP is green. Side chains are shown as sticks. Nitrogen, oxygen, and phosphate atoms are highlighted in blue, red, and orange, respectively. Mg²⁺ ions are shown in gold.

Figure 2

Structures of chemically modified nucleotides, 3′-deoxyadenosine 5′-triphosphate (1, 3′-dATP) and guanosine-5′-[(α,β)-methyleno]-triphosphate (2, GMPCPP). These modified nucleotides were used for structural studies and mechanism elucidation of the RNA transcribing complexes.

Figure 3

A three-dimensional scheme of A) forward translocation and B) proofreading, respectively, across different RNA Pol II registers (n−1, n, and n+1). Different states of RNA Pol II transcription elongation are shown in colored squares: post-translocation state (orange); pre-translocation state (yellow); nucleotide bound entry site (blue); nucleotide bound addition site (green); backtracked state (grey). Red arrows indicate the direction of transcription elongation. Straight blue arrows indicate the reverse translocation and curved blue arrows indicate the RNA cleavage. C) A two-dimensional scheme of different states of RNA/DNA hybrid within RNA Pol II active site during transcription elongation. Three key checkpoint steps for RNA Pol II transcription fidelity are depicted: (1) nucleotide selection and incorporation, (2) RNA transcript extension, and (3) proofreading. DNA and RNA strands in RNA Pol II transcribing complex are shown in green and red. The matched and mismatched nucleotides, and their template base are shown in purple, grey, and yellow, respectively. Correct incorporation and misincorporation are depicted with n and m, respectively. The positions of 3′-end of matched (n) or mismatched (m) RNA are depicted as registers of n−1, n, n+1, n+2, m+1, and m+2, respectively. The position of next nucleotide addition is shown in a dotted box. The dotted line indicates a very slow reaction.

Figure 4

A) The structures of nonpolar nucleoside analogues dH (4), dF (5), dL (6), dB (7), dI (8) compared with thymidine (3, T). B) Five forms of cytosine bases. Cytosine (9) is converted into 5mC (10) by DNA methyltransferase (DNMT). Oxidation of 5mC by ten-eleven-translocation (TET) enzymes produces 5hmC (11), 5fC (12), and 5caC (13).