Conserved Trigger Loop Histidine of RNA Polymerase II Functions as a Positional Catalyst Primarily through Steric Effects

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.