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Structural basis for template switching by a group II intron-encoded non-LTR-retroelement reverse transcriptase

Lentzsch, Alfred M. and Stamos, Jennifer L. and Yao, Jun and Russell, Rick and Lambowitz, Alan M. (2021) Structural basis for template switching by a group II intron-encoded non-LTR-retroelement reverse transcriptase. . (Unpublished)

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Reverse transcriptases (RTs) can template switch during cDNA synthesis, enabling them to join discontinuous nucleic acid sequences. Template switching plays crucial roles in retroviral replication and recombination, is used for adapter addition in RNA-seq, and may contribute to retroelement fitness by enabling continuous cDNA synthesis on damaged templates. Here, we determined an X-ray crystal structure of a template-switching complex of a group II intron RT bound simultaneously to an acceptor RNA and donor RNA template/DNA heteroduplex with a 1-nt 3'-DNA overhang. The latter mimics a completed cDNA after non-templated addition (NTA) of a nucleotide complementary to the 3' nucleotide of the acceptor as required for efficient template switching. The structure showed that the 3' end of the acceptor RNA binds in a pocket formed by an N-terminal extension (NTE) present in non-long-terminal-repeat (LTR)-retroelement RTs and the RT fingertips loop, with the 3' nucleotide of the acceptor base paired to the 1-nt 3'-DNA overhang and its penultimate nucleotide base paired to the incoming dNTP at the RT active site. Analysis of structure-guided mutations identified amino acids that contribute to acceptor RNA binding and a phenylalanine near the RT active site that mediates NTA. Mutation of the latter residue decreased multiple sequential template switches in RNA-seq. Our results provide new insights into the mechanisms of template switching and NTA by RTs, suggest how these reactions could be improved for RNA-seq, and reveal common structural features for template switching by non-LTR-retroelement RTs and viral RNA-dependent RNA polymerases.

Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription Paper
Lentzsch, Alfred M.0000-0002-8150-0511
Stamos, Jennifer L.0000-0003-1156-3944
Yao, Jun0000-0002-1232-1587
Russell, Rick0000-0002-4879-8563
Lambowitz, Alan M.0000-0001-6036-2423
Alternate Title:Group II intron RT template-switching structure
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license. This version posted May 13, 2021. High-throughput sequencing was done by the Genomic Sequencing and Analysis Facility at the University of Texas at Austin. The Center for Biomedical Research Support (CBRS) at the University of Texas at Austin provided high-performance computing resources. Data availability: The RNA-seq data generated for this article was uploaded to BioProject ID:PRJNA723603. The structural data generated for this paper was uploaded to the Protein Data Bank with PDB ID 7K9Y. All remaining data are contained within the article. Author contributions: A.M. Lambowitz initiated the project. A.M. Lambowitz, A.M. Lentzsch and R.R. devised the experiments. A.M .Lentzsch conducted all the experiments. J.Y. analyzed the RNA-seq data. A.M. Lentzsch purified and crystallized the GsI-IIC RT protein. A.M. Lentzsch and J.S. solved the crystal structure. All authors wrote and edited the article. This work was supported by National Institutes of Health Grants R35 GM136216 (to A.M. Lambowitz) and R35 GM131777 (to R.R.). Beamline 5.0.1 of the Advanced Light Source, a U.S. DOE Office of Science User Facility under Contract No. DEAC02-05CH11231, is supported in part by the ALS-ENABLE program funded by the National Institutes of Health, National Institute of General Medical Sciences, grant P30 GM124169-01. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Competing Interest Statement: Thermostable group II intron reverse transcriptase enzymes and methods for their use are the subject of patents and patent applications that have been licensed by the University of Texas and East Tennessee State University to InGex, LLC. A. M. Lambowitz, some former and present members of the Lambowitz laboratory, and the University of Texas are minority equity holders in InGex, LLC, and receive royalty payments from the sale of TGIRT enzymes and kits employing TGIRT template-switching activity and from the sublicensing of intellectual property to other companies.
Funding AgencyGrant Number
NIHR35 GM136216
NIHR35 GM131777
Department of Energy (DOE)DEAC02-05CH11231
NIHP30 GM124169-01
Record Number:CaltechAUTHORS:20210514-115225325
Persistent URL:
Official Citation:Structural basis for template switching by a group II intron-encoded non-LTR-retroelement reverse transcriptase. Alfred M. Lentzsch, Jennifer L. Stamos, Jun Yao, Rick Russell, Alan M. Lambowitz. bioRxiv 2021.05.13.443781; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:109134
Deposited By: Tony Diaz
Deposited On:14 May 2021 20:59
Last Modified:16 Nov 2021 19:34

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