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