Minimal requirements for reverse polymerization and tRNA repair by tRNAHis guanylyltransferase.

tRNAHis guanylyltransferase (Thg1) has unique reverse (3'-5') polymerase activity occurring in all three domains of life. Most eukaryotic Thg1 homologs are essential genes involved in tRNAHis maturation. These enzymes normally catalyze a single 5' guanylation of tRNAHis lacking the essential G-1 identity element required for aminoacylation. Recent studies suggest that archaeal type Thg1, which includes most archaeal and bacterial Thg1 enzymes is phylogenetically distant from eukaryotic Thg1. Thg1 is evolutionarily related to canonical 5'-3' forward polymerases but catalyzes reverse 3'-5'polymerization. Similar to its forward polymerase counterparts, Thg1 encodes the conserved catalytic palm domain and fingers domain. Here we investigate the minimal requirements for reverse polymerization. We show that the naturally occurring minimal Thg1 enzyme from Ignicoccus hospitalis (IhThg1), which lacks parts of the conserved fingers domain, is catalytically active. And adds all four natural nucleotides to RNA substrates, we further show that the entire fingers domain of Methanosarcina acetivorans Thg1 and Pyrobaculum aerophilum Thg1 (PaThg1) is dispensable for enzymatic activity. In addition, we identified residues in yeast Thg1 that play a part in preventing extended polymerization. Mutation of these residues with alanine resulted in extended reverse polymerization. PaThg1 was found to catalyze extended, template dependent tRNA repair, adding up to 13 nucleotides to a truncated tRNAHis substrate. Sequencing results suggest that PaThg1 fully restored the near correct sequence of the D- and acceptor stem, but also produced incompletely and incorrectly repaired tRNA products. This research forms the basis for future engineering efforts towards a high fidelity, template dependent reverse polymerase.

Pilot and Feasibility Studies in Rehabilitation Research: A Review and Educational Primer for the Physiatrist Researcher.

ABSTRACT: Pilot and feasibility studies are conducted early in the clinical research pathway to evaluate whether a future, definitive study can or should be done and, if so, how. Poor planning and reporting of pilot and feasibility studies can compromise subsequent research efforts. Inappropriate labeling of studies as pilots also compromises education. In this review, first, a systematic survey of the current state of pilot and feasibility studies in rehabilitation research was performed, and second, recommendations were made for improvements to their design and reporting. In a random sample of 100 studies, half (49.5%) were randomized trials. Thirty (30.0%) and three (3.0%) studies used "pilot" and "feasibility" in the study title, respectively. Only one third (34.0%) of studies provided a primary objective related to feasibility. Most studies (92.0%) stated an intent for hypothesis testing. Although many studies (70.0%) mentioned outcomes related to feasibility in the methods, a third (30.0%) reported additional outcomes in the results and discussion only or commented on feasibility anecdotally. The reporting of progression plans to a main study (21.0%) and progression criteria (4.0%) was infrequent. Based on these findings, it is recommended that researchers correctly label studies as a pilot or feasibility design based on accepted definitions, explicitly state feasibility objectives, outcomes, and criteria for determining success of feasibility, justify the sample size, and appropriately interpret and report the implications of feasibility findings for the main future study.