Recently, we overcame a long-standing technical difficulty by developing general methods for expressing group II intron and related RTs with high yield and activity and applied them to group II Intron RTs from bacterial thermophiles to obtain Thermostable Group II Intron RTs (TGIRTs). We found that TGIRTs have properties beneficial for RNA-seq, including higher fidelity and processivity than retroviral RTs, and a novel template-switching activity that facilitates RNA-seq adapter addition. We also obtained a long-sought crystal structure of a full-length group II intron RT in an active conformation bound to RNA template/DNA primer and an incoming dNTP, a first for any non-LTR-retroelement RT. The structure revealed that group II intron RTs are closely related to RdRPs, as expected for an ancestral RT, and identified novel structural features that putatively contribute to their beneficial properties. It now provides a structural foundation for addressing major questions about the reverse transcription mechanisms of group II intron and other non-LTR-retroelement RTs, the evolution of RTs, and the further engineering of group II intron RTs for biotechnological applications.

Selected Publications:

Lentzsch AM, Yao J, Russell R, Lambowitz AM. Template switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq. Journal of Biological Chemistry [Internet]. 2019.

Stamos, J.L., Lentzsch, A.M., and Lambowitz, A.M. Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Significance. Mol. Cell, 68, 926-939, 2017.

Mohr, S., Ghanem, E., Smith, W., Sheeter, D., Qin, Y., King, O., Polioudakis, D., Iyer, V.R., Hunicke-Smith, S. Swamy, S., Kuersten, S., and Lambowitz, A.M. Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation sequencing. RNA 19, 958-970, 2013.

Kennell, J.C., Moran, J.V., Perlman, P.S., Butow, R.A., and Lambowitz, A.M.Reverse transcriptase activity associated with maturase-encoding group II introns in yeast mitochondria. Cell 73, 133-146, 1993.