TY - JOUR
T1 - The presence of an RNA:DNA hybrid that is prone to slippage promotes termination by T7 RNA polymerase
AU - Molodtsov, Vadim
AU - Anikin, Michael
AU - McAllister, William T.
N1 - Funding Information:
We thank Sergei Borukhov, Dmitry Markov, Steve Emanuel, Dmitry Temiakov, and other members of our group for helpful discussions, as well as Alexander Kukarin and Maria Savkina for preliminary experiments. This work was supported by National Institutes of Health grant GM38147 to W.T.M.
PY - 2014/9/9
Y1 - 2014/9/9
N2 - Intrinsic termination signals for multisubunit bacterial RNA polymerases (RNAPs) encode a GC-rich stem-loop structure followed by a polyuridine [poly(U)] tract, and it has been proposed that steric clash of the stem-loop with the exit pore of the RNAP imposes a shearing force on the RNA in the downstream RNA:DNA hybrid, resulting in misalignment of the active site. The structurally unrelated T7 RNAP terminates at a similar type of signal (TΦ), suggesting a common mechanism for termination. In the absence of a hairpin (passive conditions), T7 RNAP slips efficiently in both homopolymeric A and U tracts, and we have found that replacement of the U tract in TΦ with a slippage-prone A tract still allows efficient termination. Under passive conditions, incorporation of a single G residue following a poly(U) tract (which is the situation during termination at TΦ) results in a "locked" complex that is unable to extend the transcript. Our results support a model in which transmission of the shearing force generated by steric clash of the hairpin with the exit pore is promoted by the presence of a slippery tracts downstream, resulting in alterations in the active site and the formation of a locked complex that represents an early step in the termination pathway.
AB - Intrinsic termination signals for multisubunit bacterial RNA polymerases (RNAPs) encode a GC-rich stem-loop structure followed by a polyuridine [poly(U)] tract, and it has been proposed that steric clash of the stem-loop with the exit pore of the RNAP imposes a shearing force on the RNA in the downstream RNA:DNA hybrid, resulting in misalignment of the active site. The structurally unrelated T7 RNAP terminates at a similar type of signal (TΦ), suggesting a common mechanism for termination. In the absence of a hairpin (passive conditions), T7 RNAP slips efficiently in both homopolymeric A and U tracts, and we have found that replacement of the U tract in TΦ with a slippage-prone A tract still allows efficient termination. Under passive conditions, incorporation of a single G residue following a poly(U) tract (which is the situation during termination at TΦ) results in a "locked" complex that is unable to extend the transcript. Our results support a model in which transmission of the shearing force generated by steric clash of the hairpin with the exit pore is promoted by the presence of a slippery tracts downstream, resulting in alterations in the active site and the formation of a locked complex that represents an early step in the termination pathway.
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U2 - 10.1016/j.jmb.2014.06.012
DO - 10.1016/j.jmb.2014.06.012
M3 - Article
C2 - 24976131
AN - SCOPUS:84906243576
SN - 0022-2836
VL - 426
SP - 3095
EP - 3107
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 18
ER -