» Articles » PMID: 11410669

Structure in Nascent RNA Leads to Termination of Slippage Transcription by T7 RNA Polymerase

Overview
Specialty Biochemistry
Date 2001 Jun 19
PMID 11410669
Citations 11
Authors
Affiliations
Soon will be listed here.
Abstract

T7 RNA polymerase presents a very simple model system for the study of fundamental aspects of transcription. Some time ago it was observed that in the presence of only GTP as a substrate, on a template encoding the initial sequence GGGA., T7 RNA polymerase will synthesize a 'ladder' of poly-G RNA products. At each step, the ratio of elongation to product release is consistently approximately 0.75 until the RNA reaches a length of approximately 13-14 nt, at which point this ratio drops precipitously. One model to explain this drop in complex stability suggests that the nascent RNA may be structurally hindered by the protein; the RNA may be exiting via a pathway not taken by normally synthesized RNA and therefore becomes sterically destabilized. The fact that the length of RNA at which this occurs is close to the length at which the transition to a stably elongating complex occurs might have led to other mechanistic proposals. Here we show instead that elongation falls off due to the cooperative formation of structure in the nascent RNA, most likely an intramolecular G-quartet structure. Replacement of GTP by 7-deaza-GTP completely abolishes this transition and G-ladder synthesis continues with a constant efficiency of elongation beyond the limit of detection. The polymerase-DNA complex creates no barrier to the growth of the nascent (slippage) RNA, rather termination is similar to that which occurs in rho-independent termination.

Citing Articles

High-throughput Kinetics using capillary Electrophoresis and Robotics (HiKER) platform used to study T7, T3, and Sp6 RNA polymerase misincorporation.

Carter Z, OBrien W, Lund S, Gardner A PLoS One. 2024; 19(12):e0312743.

PMID: 39621686 PMC: 11611218. DOI: 10.1371/journal.pone.0312743.


Dielectricity of a molecularly crowded solution accelerates NTP misincorporation during RNA-dependent RNA polymerization by T7 RNA polymerase.

Takahashi S, Matsumoto S, Chilka P, Ghosh S, Okura H, Sugimoto N Sci Rep. 2022; 12(1):1149.

PMID: 35064200 PMC: 8782835. DOI: 10.1038/s41598-022-05136-8.


Molecular basis of transcriptional pausing, stalling, and transcription-coupled repair initiation.

Oh J, Xu J, Chong J, Wang D Biochim Biophys Acta Gene Regul Mech. 2020; 1864(1):194659.

PMID: 33271312 PMC: 7836393. DOI: 10.1016/j.bbagrm.2020.194659.


Control of the polyamine biosynthesis pathway by G-quadruplexes.

Lightfoot H, Hagen T, Clery A, Allain F, Hall J Elife. 2018; 7.

PMID: 30063205 PMC: 6067879. DOI: 10.7554/eLife.36362.


Mechanism of Transcription Anti-termination in Human Mitochondria.

Hillen H, Parshin A, Agaronyan K, Morozov Y, Graber J, Chernev A Cell. 2017; 171(5):1082-1093.e13.

PMID: 29033127 PMC: 5798601. DOI: 10.1016/j.cell.2017.09.035.


References
1.
Cheetham G, Steitz T . Structure of a transcribing T7 RNA polymerase initiation complex. Science. 1999; 286(5448):2305-9. DOI: 10.1126/science.286.5448.2305. View

2.
Schick C, Martin C . Identification of specific contacts in T3 RNA polymerase-promoter interactions: kinetic analysis using small synthetic promoters. Biochemistry. 1993; 32(16):4275-80. DOI: 10.1021/bi00067a016. View

3.
Temiakov D, Mentesana P, Ma K, Mustaev A, Borukhov S, McAllister W . The specificity loop of T7 RNA polymerase interacts first with the promoter and then with the elongating transcript, suggesting a mechanism for promoter clearance. Proc Natl Acad Sci U S A. 2000; 97(26):14109-14. PMC: 18879. DOI: 10.1073/pnas.250473197. View

4.
Kuzmine I, Martin C . Pre-steady-state kinetics of initiation of transcription by T7 RNA polymerase: a new kinetic model. J Mol Biol. 2001; 305(3):559-66. DOI: 10.1006/jmbi.2000.4316. View

5.
Shen H, Kang C . Two site contact of elongating transcripts to phage T7 RNA polymerase at C-terminal regions. J Biol Chem. 2000; 276(6):4080-4. DOI: 10.1074/jbc.M008616200. View