Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2021.100971

Figure Lengend Snippet: Structure of a group II intron RT template-switching (TS) complex. A , structure of GsI-IIC RT poised for TS from a donor RNA template–DNA primer heteroduplex to an acceptor RNA template ( right ) compared with that of GsI-IIC RT bound to a continuous RNA template–DNA primer heteroduplex ( left ; Protein Data Bank ID: 6AR1). Protein regions: fingers ( salmon ), insertions ( red ), palm ( dark blue ), thumb ( green ), D domain ( gold ), acceptor RNA template ( yellow ), donor RNA template ( purple ), DNA primer ( cyan ), and dATP ( black ). T and P denote the template and primer strands, respectively. The RT0 loop is highlighted in a dotted circle . B , schematic of protein–nucleic acid interactions in the TS structure. Nucleotide positions are denoted as template (T) or primer (P) strand numbered from the templating nucleotide at the RT active site (T-1). Interactions between nucleic acid and amino acid residues are indicated by a black line (polar interaction), dotted line (nonpolar interaction), or a double-headed arrow (RNA 2′ OH H-bond). Other color codes are as in panel A . The first nucleotide of the acceptor (the U at T-4) could not be modeled and is shown in a lighter shade of yellow . C , amino acid sequence alignments of the RT0 loop ( top ) and fingertips loop ( bottom ) regions of group II intron RTs ( red ; GsI-IIC RT [E2GM63], Roseburia intestinalis [D4L313], Eubacterium rectale [D4JMT6], Ll.LtrB [P0A3U0], TeI4h [Q8DMK2], Saccharomyces cerevisiae aI2 [P03876]) and non-LTR-retroelement RTs ( black ; human LINE-1 [Ll], orf2p [O00370], R2 Bombyx mori [V9H052], and Jockey Drosophila funebris RT [P21329]). Uniprot IDs are indicated in parentheses. Protein sequences were aligned using the MAFFT algorithm and colored using ClustalX settings. RT, reverse transcriptase.

Article Snippet: Thermostable group II intron RT 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.

Techniques: Sequencing, Reverse Transcription

Journal: The Journal of Biological Chemistry

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

doi: 10.1016/j.jbc.2021.100971

Figure Lengend Snippet: The F134A mutation decreases secondary template switches in TGIRT-Seq. A , outline of the RNA-Seq workflow. Template-switching reactions using WT or F143A GsI-IIC RTs were performed with an unlabeled starter duplex with a 1-nt 3′-DNA primer overhang that was an equimolar mixture of A, C, G, and T residues (denoted N) and a 24-nt acceptor RNA of which each of the last three nucleotides was an equimolar mixture of A, C, G, and U residues (denoted N). After a 30-min preincubation, reactions were initiated by adding dNTPs, incubated at 60 °C for 15 min, and terminated by adding NaOH and heating to 95 °C for 3 min. Complementary DNA products were cleaned up by using a MinElute column (Qiagen) and then ligated to a 5′-adenylated R1R adapter using Thermostable 5′ App DNA/RNA Ligase (New England Biolabs). After another MinElute clean up, Illumina RNA-Seq capture sites (P5 and P7) and indices were added by PCR, and the resulting libraries were cleaned up by using AMPure XP beads (Beckman Coulter) prior to sequencing on an Illumina MiSeq version 2 to obtain 150-nt paired-end reads. B , bioanalyzer (Agilent; High Sensitivity DNA chip) traces of TGIRT-Seq libraries prepared via template switching at 0.4 and 4 mM dNTPs to the 24-nt acceptor RNA. The lengths indicated on the x -axis are those of internal DNA size markers, which were omitted from the trace for clarity. C , stacked bar graphs show the percentages of reads containing a single template switch from the starter duplex (1×) or multiple template switches from the 5′ end of one RNA template to the 3′ end of another (2× to 6×) in TGIRT-Seq datasets obtained with WT GsI-IIC RT and the F143A mutant at 0.4 or 4 mM dNTPs. D , stacked bar graphs showing the proportions of different nucleotides for the first, second, and third nontemplated-nucleotide additions at the 3′ end of the final complementary DNA synthesized after the last template switch in TGIRT-Seq datasets obtained with WT GsI-IIC RT and the F143A mutant at 0.4 and 4 mM dNTPs. The table below shows the percentages of 0, 1, 2, or 3 nt NTAs for each condition. E , stacked bar graphs showing nucleotide frequencies of the 3′-terminal nucleotide of the acceptor RNA in TGIRT-Seq datasets obtained with WT GsI-IIC RT and the F143A mutant at 0.4 and 4 mM dNTPs for acceptor to acceptor (A–A) and starter duplex to acceptor (D–A) template switches. RT, reverse transcriptase; TGIRT, thermostable group II intron reverse transcriptase.

Article Snippet: Thermostable group II intron RT 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.

Techniques: Mutagenesis, RNA Sequencing, Incubation, Sequencing, Synthesized, Reverse Transcription