Structure-function analysis of a Group II Intron reverse transcriptase

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2022-05-09

Authors

Alvarado Torres, Jose Mario

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Abstract

Mobile group II introns are ribozymes and mobile genetic elements found in all branches of life. They consist of a highly structured intron RNA that encodes a reverse transcriptase (RT). This RT is utilized during retrohoming to synthesize a full-length DNA copy (cDNA) of the intron RNA which is then integrated into new genome locations. One theory suggests that RTs evolved from an RNA Dependent RNA Polymerase (RDRP) that originated in the RNA World. These ancestral RDRPs served to propagate genetic material, and during the transition to DNA-based life, RDRPs evolved into RTs. Recent structural data for Geobacillus stearothermophilus Group II Intron RT (GsI-IIC RT) suggests a close evolutionary relationship between RDRPs and group II intron RTs, including a strikingly similar RT active site and regions that function in binding the template and primer. To further probe this evolutionary relationship rational mutations were introduced into GsI-IIC RT that affected rNTP incorporation ability. Group II introns have also potentially evolved characteristics like higher fidelity and processivity compared to other RTs due to stringent accuracy requirements when reverse transcribing the catalytically active and highly structured intron RNA. Host-encoded group II intron-like RTs that have acquired other functions often have substitutions for the conserved A residue in the YADD motif at the RT active site. This suggests that the active site can be modified by evolutionary pressures to suit diverse activities. This tolerance was exploited to mutate the highly conserved YADD motif to study structure-function relationships between conserved features of previously uncharacterized classes of RTs. Current laboratory data suggests that the YXDD motif can be modified to modulate different activities, although other crucial complementary or compensatory mutations are likely necessary. Through this work I obtained data that suggests group II intron RTs evolved from ancestral RdRPs, that their constricted active site might serve to increase fidelity, and that their conserved active site motif can be modified to modulate biochemical activities.

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