Regulatory function of the tRNA 3'-end in E. coli
Final Report Abstract
In this project, we investigated the involvement of the tRNA 3’ end in tRNA quality control and integrity surveillance in E. coli. tRNA nucleotidyltransferase (the CCA-adding enzyme), usually involved in tRNA maturation, is able to discriminate between functional and stable tRNA molecules and damaged/hypomodified transcripts that cannot participate in translation. On such non-functional tRNAs, the enzyme adds a second CCA triplet, leading to CCACCA tails that are recognized as a degradation tag. As several tRNA precursors carry 3‘-trailers of similar sequence, we set out to investigate how tRNA degrading enzymes discriminate between damaged and not yet processed tRNA molecules. We identified that RNaseT, an enzyme that frequently chops off the terminal A residue of intact tRNAs, subjects these tRNAs to a repeated quality control by the CCA-adding enzyme. While this 3’end turnover was originally described as a futile cycle, our data show that it in fact represents an essential part of the tRNA integrity control. Furthermore, based on our findings on RNase T activity, we established a very useful in vivo system consisting of an E. coli strain (with a knocked-out gene for the CCA-adding enzyme) to investigate tRNA nucleotidyltransferase activity in a quantitative way. The basal expression of RNase T removes the terminal A residue of tRNAs, converting them into inactive transcripts. A functional CCA-adding enzyme is now required to re-activate the tRNA pool. With this assay, we were able to investigate the enzymatic activity (in terms of A addition) of different tRNA nucleotidyltransferase variants, ranging from temperature-sensitive pathogenic mutations to specifically mutagenized forms. In a second line of experiments, we investigated the integrity of CCA-ends in the tRNA pool of E. coli by deep sequencing. The obtained high read numbers allowed to detect even low abundant tRNA populations ending in CCACCA (tagged for degradation) or CC (RNase T-mediated end turnover, see above) as well as a range of tRNA precursor transcripts. Interestingly, the integrity of tRNAs also depends on the growth conditions of E. coli. In the exponential phase, tRNA integrity is lower than in stationary phase. In this transition, the discriminator base at position 73 seems to play a specific role, as tRNAs with G73 are almost unaffected. Taken together, our in vivo as well as in vitro results shed new light on the tRNA quality control in E. coli and indicate an important contribution of RNase T activity and its interplay with the CCA-adding enzyme in this regard. The RNase T-based in vivo test system that tracks the turnover of the terminal A residue will be used to address several aspects of tRNA nucleotidyltransferase activity in vivo, where currently only in vitro data are available.
Publications
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(2016) Einfluss variabler Translationsgeschwindigkeit auf die Zelle. Biospektrum 22(5), 452
Czech, A., Adamla, F., Ignatova Z.
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(2016) Exploiting tRNAs to Boost Virulence. Life 6(4), 4
Albers, S. & Czech, A.
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A tRNA’s fate is decided at its 3′ end: Collaborative actions of CCA-adding enzyme and RNases involved in tRNA processing and degradation. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Vol. 1861. 2018, Issue 4, pp. 433-441.
Wellner, K., Betat, H., Mörl, M.
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Examining tRNA 3′-ends in Escherichia coli : teamwork between CCA-adding enzyme, RNase T, and RNase R. RNA, Vol. 24. 2018, Issue 3, pp. 361–370.
Wellner, K., Czech, A., Ignatova, Z., Betat, H., & Mörl, M.
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Dual expression of CCA-adding enzyme and RNase T in Escherichia coli generates a distinct cca growth phenotype with diverse applications. Nucleic Acids Research, Vol. 47. 2019, Issue 7, pp. 3631–3639.
Wellner, K., Pöhler, M.-T., Betat, H., Mörl, M.
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Post-transcriptional regulation of tRNA pools to govern the Central Dogma - a Perspective. Biochemistry, Vol. 58.2019, Issue 5, pp. 299-304.
Wellner, K., Mörl, M.
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Deep sequencing of tRNA’s 3′-termini sheds light on CCA-tail integrity and maturation. RNA, Vol. 26. 2020, Issue 2, pp. 199-208.
Czech, A.
