tRNA修飾ランドスケープが新規タンパク質機能の進化に与える影響

2020 Fiscal Year Annual Research Report

• Project/Area Number: 20F20705
• Research Institution: Okinawa Institute of Science and Technology Graduate University
• Principal Investigator: LAURINO Paola 沖縄科学技術大学院大学, タンパク質工学・進化ユニット, 准教授 (90812256)
• Co-Investigator(Kenkyū-buntansha): CLIFTON BENJAMIN 沖縄科学技術大学院大学, タンパク質工学・進化ユニット, 外国人特別研究員
• Project Period (FY): 2020-07-29 – 2022-03-31
• Keywords: tRNA / mistranslation / RNA modification / evolution

Outline of Annual Research Achievements

Out of the three aims of the proposed research, I have nearly completed the first aim successfully.

It has been reported that TrmD, which introduces the m1G37 modification in Pro, Arg, and Leu tRNAs, is essential for growth in bacteria because it prevents +1 frameshift errors in translation of Pro codons, which otherwise induce cell death. However, this observation is puzzling given that bacteria are highly tolerant to translational frameshift errors. My first aim was to perform experimental evolution of TrmD-deficient E. coli to determine whether there are mechanisms to compensate for the loss of m1G37-modifed tRNA. I engineered eight E. coli strains with mutations in TrmD, including a knockout mutation. The mutant strains showed slow growth but were viable, in contrast to previous reports. The mutant strains showed rapid recovery of growth, mainly via tandem duplication or coding mutations in the proline-tRNA ligase gene ProS. In vitro aminoacylation assays showed that ProS variants, but not wild-type ProS, efficiently aminoacylate Pro tRNA that does not contain the m1G37 modification.

These results suggest that inefficient aminoacylation of unmodified tRNA, not translational frameshift errors, is responsible for lack of growth in the absence of the m1G37 modification and that ProS may function as a gatekeeper, preventing use of error-prone unmodified Pro-tRNA in translation. This work also shows the utility of experimental evolution for uncovering the biological functions of proteins, including essential enzymes.

Current Status of Research Progress

3: Progress in research has been slightly delayed.

Reason

The project is running slightly behind schedule due to some delays caused by COVID-19. OIST was closed between May 1 and May 18 2020, and closure of the Coli Genetic Stock Centre at Yale University from March 2020 led to delays in obtaining the E. coli strain needed to start work on Aim 1 of the project. Characterizing the effects of the mutations observed during experimental evolution has also taken slightly longer than expected due to the technical difficulty of the relevant RNA purifications and enzyme assays, which could not have been predicted at the start of the project. However, other aspects of the project, such as the next-generation sequencing analysis, progressed more smoothly than expected, so the delay to the project is minimal.

Strategy for Future Research Activity

To complete Aim 1, aminoacylation assays will be completed to confirm that　mutations in ProS increase enzyme activity with unmethylated tRNA.
In Aim 2, I will explore how tRNA modifications might affect the evolution of new protein functions via fixation of phenotypic mutations. We will perform bioinformatic analysis to identify relevant phenotypic mutations with potential functional significance, for example, conserved CC[C/U]-[C/U] motifs where unmethylated G37-induced frameshift errors would expose cryptic C-terminal signal sequences or regulatory sequences. We will then identify putative cases where phenotypic mutations were genetically fixed by frameshift mutations; these mutations could potentially represent evolutionary adaptations, for example, to facilitate new subcellular localization. Finally, we will recreate the phenotypic mutation in a suitable model system.
In Aim 3, I will explore how changes in tRNA modifications affect the course of protein evolution through general effects on mistranslation. Using a panel of E. coli strains deficient in non-essential tRNA modifying enzymes, I will identify strains that show higher fitness in the presence of an antibiotic relative to the wild-type strain. I will then perform directed evolution of an antibiotic resistance gene in the wild-type and mutant strains to determine the effect of mistranslation on the evolutionary trajectory.