Abstract
In all domains of life, multisubunit RNA polymerases (RNAPs) catalyze both the extension of mRNA transcripts by nucleotide addition and the hydrolysis of RNA, which enables proofreading by removal of misincorporated nucleotides. A highly conserved catalytic module within RNAPs called the trigger loop (TL) functions as the key controller of these activities. The TL is proposed to act as a positional catalyst of phosphoryl transfer and transcript cleavage via electrostatic and steric contacts with substrates in its folded helical form. The function of a near-universally conserved TL histidine that contacts NTP phosphates is of particular interest. Despite its exceptional conservation, substitutions of the TL His with Gln support efficient catalysis in bacterial and yeast RNAPs. Unlike bacterial TLs, which contain a nearby Arg, the TL His is the only acid-base catalyst candidate in the eukaryotic RNAPII TL. Nonetheless, replacement of the TL His with Leu is reported to support cell growth in yeast, suggesting that even hydrogen bonding and polarity at this position may be dispensable for efficient catalysis by RNAPII. To test how a TL His-to-Leu substitution affects the enzymatic functions of RNAPII, we compared its rates of nucleotide addition, pyrophosphorolysis, and RNA hydrolysis to those of the wild-type RNAPII enzyme. The His-to-Leu substitution slightly reduced rates of phosphoryl transfer with little if any effect on intrinsic transcript cleavage. These findings indicate that the highly conserved TL His is neither an obligate acid-base catalyst nor a polar contact for NTP phosphates but instead functions as a positional catalyst mainly through steric effects.