Novel pathways for transfer RNA processing and maturation in Escherichia coli
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Post-transcriptional processing of all tRNA primary transcripts constitutes an important cellular activity, thought to play a role in regulation of protein synthesis and overall cell fitness. Ribonucleases are the predominant effectors of these processing events. This dissertation research aimed to achieve greater understanding of the mechanisms involved in tRNA processing, with a focus on the biological role of RNase P through microbiological, genetic and biochemical analysis. We also attempted to discern the role of polyadenylation in tRNA maturation and identify physiological interactions between RNase P and poly(A) polymerase I, the primary polyadenylation enzyme in E. coli. Based on our analysis of the valU and lysT polycistronic operons, we have proposed a novel pathway for RNase P-mediated processing of tRNA primary transcripts. The enzyme works in an overall 3’ → 5’ direction, proficiently removing Rho-independent transcription terminators. Other endoribonucleases such as RNase E play only a minor role in the processing of the large polycistrons. Dramatic reductions in the amount of mature valine tRNA do not result in a similar reduction in the level of aminoacylated valine tRNA. Furthermore, deficient processing at the 5’-ends of tRNAs does not lead to reduced levels of aminoacylated tRNAs, suggesting that a few extra nucleotides at the 5’-termini of tRNAs might not completely inhibit aminoacylation, as previously believed. The work presented also discovered that inactivation of poly(A) polymerase I suppresses the temperature sensitivity of an RNase P mutant. There is significant polyadenylation of tRNA precursors in an RNase P mutant and removal of the polyadenylation enzyme, PAP I, results in improved processing of the tRNA precursor 3’-termini. Lack of PAP I leads to increased cellular levels of RNase T, the primary exoribonuclease involved in 3’-end processing of tRNA precursors.