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Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2019 Dec 20; Vol. 294 (51), pp. 19764-19784. Date of Electronic Publication: 2019 Nov 11. - Publication Year :
- 2019
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Abstract
- The reverse transcriptases (RTs) encoded by mobile group II introns and other non-LTR retroelements differ from retroviral RTs in being able to template-switch efficiently from the 5' end of one template to the 3' end of another with little or no complementarity between the donor and acceptor templates. Here, to establish a complete kinetic framework for the reaction and to identify conditions that more efficiently capture acceptor RNAs or DNAs, we used a t hermostable g roup II i ntron RT (TGIRT; GsI-IIC RT) that can template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3'-DNA overhang end. We found that the rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3'-DNA overhang complementary to the 3' nucleotide of the acceptor RNA, suggesting a role for nontemplated nucleotide addition of a complementary nucleotide to the 3' end of cDNAs synthesized from natural templates. Longer 3'-DNA overhangs progressively decreased the template-switching rate, even when complementary to the 3' end of the acceptor template. The reliance on only a single bp with the 3' nucleotide of the acceptor together with discrimination against mismatches and the high processivity of group II intron RTs enable synthesis of full-length DNA copies of nucleic acids beginning directly at their 3' end. We discuss the possible biological functions of the template-switching activity of group II intron- and other non-LTR retroelement-encoded RTs, as well as the optimization of this activity for adapter addition in RNA- and DNA-Seq protocols.<br /> (© 2019 Lentzsch et al.)
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 294
- Issue :
- 51
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 31712313
- Full Text :
- https://doi.org/10.1074/jbc.RA119.011337