Novel pathways for transfer RNA processing and maturation in Escherichia coli
Abstract
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.