TRNA nucleotidyltransferase

Transfer RNA precursors may undergo further posttranscriptional processing. The 3 -terminal trinucleotide CCA(3 ) to which an amino acid will be attached during protein synthesis (Chapter 27) is absent from some bacterial and all eukaryotic tRNA precursors and is added during processing (Fig. 26-23). This addition is carried out by tRNA nucleotidyltransferase, an unusual enzyme that binds the three ribonucleoside triphosphate precursors in separate active sites and catalyzes formation of the phosphodiester bonds to produce the CCA(3 ) sequence. The creation of this defined sequence of nucleotides is therefore not dependent on a DNA or RNA template—the template is the binding site of the enzyme. 

The stereochemistries of tRNA nucleotidyltransferase, which adds the terminal adenylyl group at the -CCA end of tRNA, and DNA-dependent RNA polymerase were elucidated by Eckstein and associates in a two stage study. The E. coli DNA-dependent RNA polymerase study exemplifies their approach. Diastereomer A of ATPaS was copolymerized with UTP using poly(dA-dT)-poly(dA-dT) as the template. The resulting alternating polymer (see Equation 15) was hydrolyzed by ribonuclease A to a cyclic phosphorothioate ... 

The enzyme tRNA nucleotidyltransferase is capable of rebuilding the 3 -CpCpA terminus on to a tRNA molecule in which this end is incomplete, using CTP and ATP as substrates in fact, it will repair a fragment as small as CpC, and will also accept CTP and ATP analogues as substrates, allowing the construction of modified tRNA termini. Thus, 2 -amino-2 -deoxy-CTP can be incorporated as the 3 -... 

Eukaryotic pre-tRNA processing Many eukaryotic tRNA transcripts contain small introns, which do not dismpt the cloverleaf structure and are excised. Eukaryotic tRNA transcripts lack the obligatory -CCA sequence at their 3 end. This sequence is appended stepwise by the action of tRNA nucleotidyltransferase using CTP and ATP as substrates. 

All . coli tRNAs have a genetically encoded 3 CCA terminus. However, some tRNAs in gram-positive bacteria, e.g., B. subtilis, do not have the transcribed CCA terminus (194), and none of the eukaryotic tRNA genes encode the CCA terminus. In those tRNAs, the CCA terminus is added posttranscriptionally by tRNA nucleotidyltransferase. The 3 CCA terminus of the acylated tRNA specifically interacts with a characteristic set of 6 bases in 23S rRNA in the 50S ribosomal P-site (195). 

An intron (see below) must be removed from the anticodon loop, and sequences at both the 5 - and the 3 -ends of the molecule must be trimmed. Other posttranscriptional modifications include addition of a -CCA sequence by nucleotidyltransferase)to the 3 -terminal end of tRNAs, and modification oTbases at specific positions to produce "unusual bases (see p. 290). 

The final step in forming the mature tRNA is the addition of a CCA sequence at its 3 -end (see Fig 14.20, circle 4). These nucleotides are added one at a time by nucleotidyltransferase. The tRNA then migrates to the cytoplasm. The terminal adenosine at the 3 -end is the site at which the specific amino acid for each tRNA is bound and activated for incorporation into a protein. 

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