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The researchers found that a portion of the enzyme known as the
trigger loop acts like a “trap door,” swinging beneath a matching
nucleoside triphosphate (NTP) building block, to close off the active
center and form an extensive network of interactions with the NTP and
other parts of the enzyme. Those interactions leave another side chain
in the trigger loop precisely positioned, such that it may literally
“trigger” the formation of the chemical bonds that link components of
the growing RNA chain together. If the NTP is even slightly misaligned,
Kornberg said, those critical interactions fail.
The trigger loop mechanism therefore couples NTP recognition and
catalysis, ensuring the fidelity of transcription, they reported.
“Of all revelations from the structure [of the transcription machinery]
since it was first solved, this is perhaps the most fundamental since
it gets at the underlying mechanisms,” Kornberg said. “It’s long known
that the enzyme operates with high fidelity - selecting the correct
base and sugar - but it’s been a mystery how that is accomplished.”
These findings offer “an unexpected and elegant explanation that’s
both beautiful and simple, as nature invariably proves to be.”
The fundamental mechanism of transcription is conserved among cellular
RNA polymerases, the researchers explained. Common features include an
unwound region of about 15 base pairs of the DNA with some eight
residues of the RNA transcript hybridized with the DNA in the center
of the “transcription bubble.” The enzyme polymerases involved are
capable of moving both forward and backward on the DNA. Forward
movement is favored by the binding of NTPs, while backtracking occurs
especially when the enzyme encounters an impediment, such as damaged
DNA.
Kornberg’s group captured the first picture of the polymerase II
transcribing complex by X-ray crystallography in 2001. Those
structures revealed the complex with a nucleotide still in the
enzyme’s addition site, just after it had been added to the RNA
transcript.
Later X-ray structures revealed the transcribing complex with the
addition site available for entry of a matched NTP. Those crystals
uncovered a second NTP-binding site on the transcribing enzyme, dubbed
the entry site. While all NTPs can bind the entry site, only an NTP
matched for base-pairing with the DNA template binds the addition site
for attachment to the growing RNA strand, Kornberg said.
Yet the question of how the enzyme achieves such a high degree of
discrimination between matched and mismatched NTPs remained unanswered.
The chemical attraction alone between RNA bases - adenine, cytosine,
guanine, and uracil - and their complementary bases on the DNA
template strand is far from sufficient to account for the incredible
selectivity of polymerase II, Kornberg said. And the scientists didn’t
know either how the polymerase avoids substituting the NTPs that
constitute DNA for the correct RNA building blocks, molecules that
differ by only one oxygen atom.
In search of an explanation in the current study, the researchers
screened hundreds of crystals to achieve higher data quality and
resolution than ever before.
“In the course of the work, we saw something that had never been
noticed before - additional protein density beneath the matching
nucleotide,” Kornberg said.
The team traced that protein density back to a portion of the
polymerase II enzyme: the trigger loop.
“Of the 14 crystal structures now reported in which the trigger loop
was observed, only in two is it seen in that location, directly
beneath the NTP,” Kornberg said. Those were the only two crystals in
which the NTP was correctly matched to the DNA template, evidence of
the trigger loop’s “clear relationship to NTP selection.”
Further study revealed that, when a matching NTP reaches the addition
site, the trigger loop swings from its usual position some distance
away until it rests parallel to the NTP. It then forms a network of
interactions - some 20 to 30 in all - with components of the NTP, a
process that serves to “recognize all features of the NTP in the
addition site and detect its precise location,” the researchers
reported.
“The specificity is a result of the alignment with the NTP that is
critically dependent upon the base, sugar, phosphate and location when
the trigger loop swings into position,” Kornberg said. “If it is
misaligned even slightly, that set of contacts cannot occur.”
As a consequence of that alignment, to angstrom (a unit of length
equal to one hundred millionth of a centimeter) precision, a histidine
side chain of the trigger loop rests on the ß phosphate, the chemical
constituent that must have its bond broken in order for the NTP to
join the RNA chain through the formation of a phosphodiester bond,
Kornberg said. The finding suggested the side chain acts as a trigger
for bond formation.
The whole decision-making process occurs extremely rapidly, he added,
on the order of picoseconds. A picosecond is one trillionth of a
second.
“The basis for the extraordinary specificity with which RNA
polymerases transcribe DNA lies in a structural element termed the
trigger loop, which makes both direct and indirect contact with all
features of the nucleotide in the polymerase active center,” the
researchers concluded.
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