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Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq
| Content Provider | Scilit |
|---|---|
| Author | Lentzsch, Alfred M. Yao, Jun Russell, Rick Lambowitz, Alan M. |
| Copyright Year | 2019 |
| Description | Journal: Journal of Biological Chemistry 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 thermostable group II intron 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. |
| Related Links | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926447/pdf http://www.jbc.org/content/294/51/19764.full.pdf |
| Ending Page | 19784 |
| Page Count | 21 |
| Starting Page | 19764 |
| ISSN | 00219258 |
| e-ISSN | 1083351X |
| DOI | 10.1074/jbc.ra119.011337 |
| Journal | Journal of Biological Chemistry |
| Issue Number | 51 |
| Volume Number | 294 |
| Language | English |
| Publisher | Elsevier BV |
| Publisher Date | 2019-12-01 |
| Access Restriction | Open |
| Subject Keyword | Journal: Journal of Biological Chemistry Biochemistry and Molecular Biology Rna-dependent Rna Polymerase Group Ii Intron Reverse Transcriptase Non-templated Nucleotide Addition Reverse Transcription Structure–function Thermostable Group Ii Intron Reverse Transcriptase Transposable Element (te) |
| Content Type | Text |
| Resource Type | Article |
| Subject | Cell Biology Biochemistry Molecular Biology |
