The C nucleotide at the mature 5' end of the Escherichia coli proline tRNAs is required for the RNase E cleavage specificity at the 3' terminus as well as functionality.

Proline tRNA 3'-maturation in Escherichia coli occurs through a one-step RNase E endonucleolytic cleavage immediately after the CCA determinant. This processing pathway is distinct from the 3'-end maturation of the other tRNAs by avoiding the widespread use of 3' → 5' exonucleolytic processing, 3'-polyadenylation and subsequent degradation. Here, we show that the cytosine (C) at the mature 5'-terminus of the proK and proL tRNAs is required for both the RNase E cleavage immediately after the CCA determinant and their functionality. Thus, changing the C nucleotide at the mature 5'-terminus of the proL and proK tRNAs to the more common G nucleotide led to RNase E cleavages 1-4 nucleotides downstream of the CCA determinant. Furthermore, the 5'-modified mutant tRNAs required RNase T and RNase PH for their 3'-maturation and became substrates for polyadenylation and degradation. Strikingly, the aminoacylation of the 5'-modified proline tRNAs was blocked due to the change in the recognition element for prolyl-tRNA-synthetase. An analogous modification of the pheV 5'-mature terminus from G to C nucleotide did not support cell viability. This result provides additional support for the importance of first nucleotide of the mature tRNAs in their processing and functionality.