Deciphering Kinetics of RNA Polymerase I Multi-Nucleotide Transcription

Abstract

RNA polymerases carry out the first step in gene expression, transcription of DNA into RNA. Eukaryotes, unlike prokaryotes, contain at least three specialized, nuclear, multi-subunit RNA polymerases (Pol I-III). RNA Polymerase I (Pol I) synthesizes the majority of ribosomal RNA. Nearly 60% of total transcription is devoted to ribosomal RNA synthesis, making it one of the cell's most energy consuming tasks. Thus, a detailed understanding of Pol I transcription is of key importance to understanding the mechanisms that govern ribosomal RNA synthesis. Previously published single adenosine (A) incorporation experiments from our lab indicate that the rate limiting step for nucleotide addition is the formation of the phosphodiester bond between the RNA strand and the incoming nucleotide (∼180 s−1). Here we present an innovative rapid-mixing kinetic assay and parameter optimization strategy in MATLAB to determine the elementary rate constants for multi-nucleotide addition by Pol I transcription elongation at single nucleotide resolution. This approach allows us to quantify each elongated RNA intermediate in a single experiment. Our current results indicate that incorporation of guanosine (G) is faster than adenosine (A), and Pol I exhibits tighter binding for guanosine compared to adenosine. This may be a consequence of Watson-Crick base pairing where guanine base pairs with cytosine forming three hydrogen bonds while adenine forms two hydrogen bonds with thymine. The local sequence context surrounding each individual nucleotide being transcribed also appears to influence the rate constants describing Pol I transcription. An additional transitory intermediate was also observed that largely exhibited no nucleotide dependence. The identity of this intermediate is actively being investigated. These results will make advancements towards elucidating the general rules governing the rates of transcription and provide a starting point for a quantitative comparison between the three eukaryotic multi-subunit RNA polymerases.