Strong transcription blockage mediated by R-loop formation within a G-rich homopurine-homopyrimidine sequence localized in the vicinity of the promoter

Nucleic Acids Research
Boris P BelotserkovskiiPhilip C Hanawalt

Abstract

Guanine-rich (G-rich) homopurine-homopyrimidine nucleotide sequences can block transcription with an efficiency that depends upon their orientation, composition and length, as well as the presence of negative supercoiling or breaks in the non-template DNA strand. We report that a G-rich sequence in the non-template strand reduces the yield of T7 RNA polymerase transcription by more than an order of magnitude when positioned close (9 bp) to the promoter, in comparison to that for a distal (∼250 bp) location of the same sequence. This transcription blockage is much less pronounced for a C-rich sequence, and is not significant for an A-rich sequence. Remarkably, the blockage is not pronounced if transcription is performed in the presence of RNase H, which specifically digests the RNA strands within RNA-DNA hybrids. The blockage also becomes less pronounced upon reduced RNA polymerase concentration. Based upon these observations and those from control experiments, we conclude that the blockage is primarily due to the formation of stable RNA-DNA hybrids (R-loops), which inhibit successive rounds of transcription. Our results could be relevant to transcription dynamics in vivo (e.g. transcription 'bursting') and may also have practic...Continue Reading

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Citations

Feb 24, 2018·Nucleic Acids Research·Nalini RaghunathanJayaraman Gowrishankar
Jan 23, 2019·Nucleic Acids Research·Kaspar BurgerMonika Gullerova
Jul 12, 2019·The EMBO Journal·Julieta RivosecchiVincent Vanoosthuyse
Sep 19, 2019·Genes & Development·Deirdre C TatomerJeremy E Wilusz
Jun 5, 2020·Cold Spring Harbor Symposia on Quantitative Biology·Rosa LunaAndrés Aguilera
Feb 27, 2020·International Journal of Molecular Sciences·Antonio MaffiaSimone Sabbioneda
Apr 22, 2020·Nature Reviews. Molecular Cell Biology·Dhaval VarshneyShankar Balasubramanian
Feb 29, 2020·The Journal of Biological Chemistry·Frederic Chedin, Craig J Benham
May 10, 2018·Nature Reviews. Molecular Cell Biology·Fei Xavier ChenAli Shilatifard
Dec 30, 2017·Current Medicinal Chemistry·Nayun Kim
Dec 3, 2020·Nucleic Acids Research·Prakash KharelPavel Ivanov
Mar 31, 2021·Nucleic Acids Research·Ernest WangJiou Wang
Jun 2, 2020·Biochemistry·Maroof Khan ZafarAlicia K Byrd
Jul 25, 2021·DNA Repair·Daisy Castillo-Guzman, Frédéric Chédin
Aug 28, 2021·Biomolecules·Commodore St GermainJacqueline H Barlow
Aug 31, 2021·Cellular and Molecular Life Sciences : CMLS·Fang-Yuan TengYong Xu

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Methods Mentioned

BETA
in vitro transcription

Software Mentioned

Bio
Rad Image Lab

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