TY - JOUR

T1 - Role of R-Loop Structure in Efficacy of RNA Elongation Synthesis by RNA Polymerase from Escherichia coli

AU - Timofeyeva, Nadezhda A.

AU - Tsoi, Ekaterina I.

AU - Novopashina, Darya S.

AU - Kuznetsova, Aleksandra A.

AU - Kuznetsov, Nikita A.

N1 - This work was supported by the Russian Science Foundation, grant No. 23-44-00064. Partial support by Russian-State-funded budget project No. 121031300041-4 for the routine maintenance of the equipment used is also acknowledged.

PY - 2024/11

Y1 - 2024/11

N2 - The mechanism of transcription proceeds through the formation of R-loop structures containing a DNA–RNA heteroduplex and a single-stranded DNA segment that should be placed inside the elongation complex; therefore, these nucleic acid segments are limited in length. The attachment of each nucleotide to the 3′ end of an RNA strand requires a repeating cycle of incoming nucleoside triphosphate binding, catalysis, and enzyme translocation. Within these steps of transcription elongation, RNA polymerase sequentially goes through several states and is post-translocated, catalytic, and pre-translocated. Moreover, the backward movement of the polymerase, which is essential for transcription pausing and proofreading activity, gives rise to a backtracked state. In the present study, to analyze both the efficacy of transcription elongation complex (TEC) formation and the rate of RNA synthesis, we used a set of model R-loops that mimic the pre-translocated state, post-translocated state, backtracked state, and a misincorporation event. It was shown that TEC assembly proceeds as an equilibrium process, including the simultaneous formation of a catalytically competent TEC as well as a catalytically inactive conformation. Our data suggest that the inactive complex of RNA polymerase with an R-loop undergoes slow conformational changes, resulting in a catalytically competent TEC. It was revealed that the structural features of R-loops affect the ratio between active and inactive states of the TEC, the rate of conformational rearrangements required for the induced-fit transition from the inactive state to the catalytically competent TEC, and the rates of accumulation of both the total RNA products and long RNA products.

AB - The mechanism of transcription proceeds through the formation of R-loop structures containing a DNA–RNA heteroduplex and a single-stranded DNA segment that should be placed inside the elongation complex; therefore, these nucleic acid segments are limited in length. The attachment of each nucleotide to the 3′ end of an RNA strand requires a repeating cycle of incoming nucleoside triphosphate binding, catalysis, and enzyme translocation. Within these steps of transcription elongation, RNA polymerase sequentially goes through several states and is post-translocated, catalytic, and pre-translocated. Moreover, the backward movement of the polymerase, which is essential for transcription pausing and proofreading activity, gives rise to a backtracked state. In the present study, to analyze both the efficacy of transcription elongation complex (TEC) formation and the rate of RNA synthesis, we used a set of model R-loops that mimic the pre-translocated state, post-translocated state, backtracked state, and a misincorporation event. It was shown that TEC assembly proceeds as an equilibrium process, including the simultaneous formation of a catalytically competent TEC as well as a catalytically inactive conformation. Our data suggest that the inactive complex of RNA polymerase with an R-loop undergoes slow conformational changes, resulting in a catalytically competent TEC. It was revealed that the structural features of R-loops affect the ratio between active and inactive states of the TEC, the rate of conformational rearrangements required for the induced-fit transition from the inactive state to the catalytically competent TEC, and the rates of accumulation of both the total RNA products and long RNA products.

KW - R-loop

KW - RNA polymerase

KW - enzymatic activity

KW - enzyme kinetics

KW - processivity

KW - transcriptional pausing

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85210243250&origin=inward&txGid=6d1680969a099a254a3c2fa107ec0b77

UR - https://www.mendeley.com/catalogue/8a25a90a-8294-3270-b055-5f5805b4b8bd/

U2 - 10.3390/ijms252212190

DO - 10.3390/ijms252212190

M3 - Article

C2 - 39596270

VL - 25

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 22

M1 - 12190

ER -