Dissecting chemical interactions governing RNA polymerase II transcriptional fidelity

Abstract

Maintaining high transcriptional fidelity is essential to life. For all eukaryotic organisms, RNA polymerase II (Pol II) is responsible for messenger RNA synthesis from the DNA template. Three key checkpoint steps are important in controlling Pol II transcriptional fidelity: nucleotide selection and incorporation, RNA transcript extension, and proofreading. Some types of DNA damage significantly reduce transcriptional fidelity. However, the chemical interactions governing each individual checkpoint step of Pol II transcriptional fidelity and the molecular basis of how subtle DNA base damage leads to significant losses of transcriptional fidelity are not fully understood. Here we use a series of "hydrogen bond deficient" nucleoside analogues to dissect chemical interactions governing Pol II transcriptional fidelity. We find that whereas hydrogen bonds between a Watson-Crick base pair of template DNA and incoming NTP are critical for efficient incorporation, they are not required for efficient transcript extension from this matched 3'-RNA end. In sharp contrast, the fidelity of extension is strongly dependent on the discrimination of an incorrect pattern of hydrogen bonds. We show that U:T wobble base interactions are critical to prevent extension of this mismatch by Pol II. Additionally, both hydrogen bonding and base stacking play important roles in controlling Pol II proofreading activity. Strong base stacking at the 3'-RNA terminus can compensate for loss of hydrogen bonds. Finally, we show that Pol II can distinguish very subtle size differences in template bases. The current work provides the first systematic evaluation of electrostatic and steric effects in controlling Pol II transcriptional fidelity.

Fig. 1. Pol II elongation complexes containing site-specific nonpolar nucleoside analogs

(a) Detailed structure of the active site of the Pol II elongation complex bound with a matched GTP. 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. (b) The structures of nonpolar nucleoside analogs H, F, L, B, I compared with thymidine (T). (c) Cutaway view of Pol II elongation complex. Pol II is revealed as a tan surface. RNA, template DNA and non-template DNA are shown in red, cyan and green, respectively. The nonpolar nucleoside analog is highlighted in magenta. (d) Scaffold sequences of RNA, template DNA and non-template DNA in this study are depicted in red, cyan and green, respectively. X refers to H, F, L, B, I, and T.

Fig. 2. Pol II can use nonpolar nucleoside analogs as DNA templates for nucleotide incorporation and further RNA transcript elongation

(a) The single base resolution denatured PAGE-urea gel image of RNA transcripts shows nucleotide incorporation and bypass at varied times (0, 5, and 30 min respectively). Aliquots of Pol II elongation complex (40 nM) were mixed with equal volumes of elongation buffer containing 250 μM of NTP mixture or ATP at 22 °C in elongation buffer (20 mM Tris-HCl (pH = 7.5), 40 mM KCl, 5 mM MgCl₂). The initial RNA primer (10 nt) was 5’-³²P-labeled. The incorporation of ATP or the extension of RNA transcripts with NTP was monitored by the increases in RNA primer length (upper bands). Positions of full-length (FL) RNA transcripts and lengths of short RNA transcripts (products) are shown on the left. (b) Image of representative denaturing PAGE-urea gel of Pol II transcription products using template L in the presence of 25, 50, 100, 200, 500, and 1000 μM of NTP, ATP, GTP, CTP and UTP, respectively. ATP and UTP are incorporated more efficiently than CTP and GTP.

Fig. 3. Nonpolar template substitutions alter nucleotide incorporation specificity and discrimination

Specificity constants governing nucleotide incorporation for the correct nucleotide, ATP (a), and a mismatched nucleotide, UTP (b) for each of the nonpolar template analogs. (c) Nucleotide discrimination for ATP over UTP incorporation. Data for T, H, F, B, L, and I are shown in blue, red, green, yellow, cyan, and orange, respectively. All error bars (standard deviation) are derived from three experiments.

Fig. 4. Subsequent nucleotide incorporation beyond matched and mismatched 3’-RNA terminus

Specificity constants for subsequent nucleotide incorporation following either a matched (a) or mismatched (b) 3’-RNA terminus. (c) Nonpolar substitutions decrease the relative discrimination for nucleotide extension. Data for T, F, and I are shown in blue, green, and orange, respectively. All error bars (standard deviation) are derived from three experiments.

Fig. 5. Nonpolar template substitutions alter TFIIS-mediated cleavage kinetics

TFIIS cleavage scaffolds containing adenosine (a) or uracil (b) at the 3’RNA terminus paired with various nonpolar analogs. Data for T, H, F, B, L, and I are shown in blue, red, green, yellow, cyan, and orange, respectively. All error bars (standard deviation) are derived from three experiments.

Fig. 6. Nonpolar analogs affect the three key checkpoint steps of Pol II transcription fidelity

Scheme of different states of RNA/DNA hybrid within the Pol II active site during the transcription elongation along T template (a), F template (b), and I template (c). Three key checkpoint steps for Pol II transcription fidelity are depicted: (1) nucleotide selection and incorporation, (2) RNA transcript extension, and (3) proofreading. DNA and RNA strands in Pol II transcribing complex are shown in blue and red. The matched and mismatched nucleotides, and their template base are shown in green, orange, and cyan, 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 width of the solid lines corresponds to the rates. The dotted line indicates a very slow reaction. Increase and decrease of rates are shown in green and red, respectively.