In this article we will discuss about the processing of pre-tRNA in E.coli and yeast.
In prokaryotes, there are many operons (e.g. 7 genes in E. coli) which are separated by spacer sequences. Mature tRNA molecules are generated by processing longer pre-tRNA transcripts. Through a specific step, RNases D, E, F and P generate these mature tRNA molecules by exo-and endo-nucleolytic cleavage (Fig. 10.11 A).
When the primary transcripts have folded and formed stem and loop, an endonuclease (RNase or F) cuts off a flaking sequence at 3′ end at the base of stem and generates a precursor with nine extra nucleotides. Then RNase D removes seven of these 3′- nucleotides one at a time.
RNase P cleaves to produce the mature 5′ end of the tRNA followed by RNase D trimming of remaining 2 nucleotides from 3′ end. Lastly, the tRNA undergoes a chain of base modification i.e. modification in 20% of bases to form unusual bases such as ribothymidine (rT), pseudouridine (Ψ), dihydrouridine (DHU) and inosine (I).
Many eukaryotes, pre-tRNA molecules are synthesised with a 16 nucleotide 5′-leader, a 14 nucleotide intron and extra 5′- and 3′- nucleotides. However, these are removed during processing. The primary transcript is converted into secondary structure having stems and loops.
These allow the endonucleases to recognise and cut the 5′- leader and two 3′-nucleotides. In contrast to prokaryotic tRNA, the 3’terminal 5′ CCA-3′ is added by the enzyme tRNA nucleotidyl transferase. At each end introns are removed by endo-catalytic cleavage followed by ligation of the half molecules of tRNA. (Fig. 10.11B).
