Transcript assisted phosphodiester bond hydrolysis by eukaryotic RNA polymerase II

Hydrolysis of the phosphodiester bonds of the transcript by bacterial RNA polymerase is assisted by 3′NMP of the RNA. Here we provide evidence that this mechanism is also involved in RNA cleavage by eukaryotic RNA polymerase II, suggesting that transcript assisted hydrolysis has emerged before divergence of bacteria and archaea/eukaryotes. Multisubunit RNA polymerase (RNAP), the enzyme accomplishing transcription in all living organisms, has emerged before the divergence of bacteria and archaea/eukaryotes. Accordingly, the molecular mechanisms involved in RNA synthesis are highly conserved in evolution. Besides the synthesis of phosphodiester bonds (and pyrophosphorolysis, which is a direct reversal of this reaction) during transcript elongation, RNAP active center can catalyze the hydrolysis of the phosphodiester bonds of the nascent RNA. As well as the synthesis, the hydrolysis is catalyzed by the two metal (Mg) ion mechanism. In addition, in bacterial RNAP, a flexible domain of the active center, the Trigger Loop, plays a critical role in hydrolysis by participating in the reaction as a general base. This appears to be different from eukaryotic RNA polymerase II (RNAP II), whose Trigger Loop fails to adapt a catalytically active conformation during hydrolysis, explaining a much slower intrinsic hydrolysis by RNAP II as compared with bacterial RNAPs. Earlier, we have discovered that an NMP at the 3′ end of the RNA transcript in the bacterial elongation complex also participates in catalysis of phosphodiester bond hydrolysis. For this to occur, RNAP has to backtrack by 1 base pair, thus positioning the penultimate (second from the 3′ end of the RNA) phosphodiester bond in the active center, making it ready for cleavage. The 3′NMP disengages from the template strand and flips backward to approach the site of the reaction, and helps to chelate the second Mg ion, MgII (which otherwise is bound weakly), to position the attacking water molecule and, possibly, to participate in the catalysis as a general acid/base. Such transcript-assisted hydrolysis of the second phosphodiester bond becomes even more prominent when RNAP is stabilized in the 1 base pair backtracked state via misincorporation of NMP that is not complementary to the base in the template strand. Chemical groups of 3′AMP, CMP, GMP and UMP contribute differently during the hydrolysis. Furthermore, the nature of the misincorporation event, i.e., the base in the template strand, may also influence the involvement of the chemical groups of the 3′NMP in the reaction. The erroneously incorporated NMP can be imagined to help to excise itself from the transcript, thus contributing to the proofreading of misincorporated events, i.e., overall fidelity of transcription. The “self-correcting” function of the transcript led to the proposition that transcript assisted second phosphodiester bond hydrolysis could be an ancient feature of transcription by multisubunit RNAPs, which may have emerged before the divergence of bacterial and archaeal/ eukaryotic lineages. A possible involvement of the 3′NMP in the RNA hydrolysis by eukaryotic RNAP became evident from the crystal structure of the backtracked elongation complex of RNAP II