Transient-State Kinetic Analysis of the RNA Polymerase II Nucleotide Incorporation Mechanism

Abstract

Eukaryotic RNA polymerase II (Pol II) is an essential enzyme that lies at the core of eukaryotic biology. Due to its pivotal role in gene expression, Pol II has been subjected to a substantial number of investigations. We aim to further our understanding of Pol II nucleotide incorporation by utilizing transient-state kinetic techniques to examine Pol II single nucleotide addition on the millisecond time scale. We analyzed Saccharomyces cerevisiae Pol II incorporation of ATP or an ATP analog, Sp-ATP-α-S. Here we have measured the rate constants governing individual steps of the Pol II transcription cycle in the presence of ATP or Sp-ATP-α-S. These results suggest that Pol II catalyzes nucleotide incorporation by binding the next cognate nucleotide and immediately catalyzes bond formation and bond formation is either followed by a conformational change or pyrophosphate release. By comparing our previously published RNA polymerase I (Pol I) and Pol I lacking the A12 subunit (Pol I ΔA12) results that we collected under the same conditions with the identical technique, we show that Pol II and Pol I ΔA12 exhibit similar nucleotide addition mechanisms. This observation indicates that removal of the A12 subunit from Pol I results in a Pol II like enzyme. Taken together, these data further our collective understanding of Pol II's nucleotide incorporation mechanism and the evolutionary divergence of RNA polymerases across the three domains of life.

High-throughput Kinetics using Capillary Electrophoresis and Robotics (HiKER) platform used to Study T7, T3, and Sp6 RNA Polymerase Misincorporation

“…A recalculation of the k obs for correct nucleotide addition using the sequencing misincorporation frequency value of ∼110 misincorporations per million bases and the average misincorporation k obs value of ∼0.23 min -1 reported here gives a predicted value of ∼33 s -1 . This suggests that correct nucleotide addition by Sp6 RNAP is much slower compared to T7 and T3 RNAP, but more comparable to multi-subunit RNAP’s found in Escherichia coli and eukaryotes [38, 40, 41, 48, 50, 54].…”

Protocol for monitoring and analyzing single nucleotide incorporation by S. cerevisiae RNA polymerases

“…For RNA polymerases that do not possess intrinsic nuclease activity, no dimers will be present. 8 Note: If you observe blank gel images or ‘smeared’ bands, it is possible RNases were introduced to the experiment. See problem 5 in troubleshooting for more details.…”

“… Various model-independent and model-dependent analyses can and have been employed to calculate rate constants governing the single nucleotide addition reaction catalyzed by RNA polymerases. 1 , 5 , 4 , 8 , 9 , 10 , 11 , 12 Our research group has published a MATLAB toolbox, MENOTR (Multi-Start Evolutionary Nonlinear OpTimizeR), 7 to facilitate parameter optimization of these, often difficult to fit, time courses. Note: It is important to highlight that downstream analyses will reveal the slowest step(s) of the reaction.…”

“…The experiment and analysis described here is unable to uncover the identity of the slowest step. To investigate whether the slowest step is phosphodiester bond formation, our group has successfully perturbed the reaction by providing a slowly hydrolysable ATP analog, Sp-ATP-α-S. 4 , 8 , 10 …”

“…Various model-independent and model-dependent analyses can and have been employed to calculate rate constants governing the single nucleotide addition reaction catalyzed by RNA polymerases. 1 , 5 , 4 , 8 , 9 , 10 , 11 , 12 …”