- Modification of DNA or RNA includes all changes made to the nucleotides after their initial incorporation into the polynucleotide chain.
- tRNAs contain >50 modified bases.
- Modification usually involves direct alteration of the primary bases in tRNA, but there are some exceptions in which a base is removed and replaced by another base.
Transfer RNA is unique among nucleic acids in its content of "unusual" bases. An unusual base is any purine or pyrimidine ring except the usual A, G, C, and U from which all RNAs are synthesized. All other bases are produced by modification of one of the four bases after it has been incorporated into the polyribonucleotide chain.
All classes of RNA display some degree of modification, but in all cases except tRNA this is confined to rather simple events, such as the addition of methyl groups. In tRNA, there is a vast range of modifications, ranging from simple methylation to wholesale restructuring of the purine ring. Modifications occur in all parts of the tRNA molecule There are >50 different types of modified bases in tRNA.
Figure 7.7 shows some of the more common modified bases. Modifications of pyrimidines (C and U) are less complex than those of purines (A and G). In addition to the modifications of the bases themselves, methylation at the 2–O position of the ribose ring also occurs.
The most common modifications of uridine are straightforward. Methylation at position 5 creates ribothymidine (T). The base is the same commonly found in DNA; but here it is attached to ribose, not deoxyribose. In RNA, thymine constitutes an unusual base, originating by modification of U.
Dihydrouridine (D) is generated by the saturation of a double bond, changing the ring structure. Pseudouridine (ψ) interchanges the positions of N and C atoms (see Figure 24.40). And 4-thiouridine has sulfur substituted for oxygen.
The nucleoside inosine is found normally in the cell as an intermediate in the purine biosynthetic pathway. However, it is not incorporated directly into RNA, where instead its existence depends on modification of A to create I. Other modifications of A include the addition of complex groups.
Two complex series of nucleotides depend on modification of G. The Q bases, such as queuosine, have an additional pentenyl ring added via an NH linkage to the methyl group of 7-methylguanosine. The pentenyl ring may carry various further groups. The Y bases, such as wyosine, have an additional ring fused with the purine ring itself; the extra ring carries a long carbon chain, again to which further groups are added in different cases.
The modification reaction usually involves the alteration of, or addition to, existing bases in the tRNA. An exception is the synthesis of Q bases, where a special enzyme exchanges free queuosine with a guanosine residue in the tRNA. The reaction involves breaking and remaking bonds on either side of the nucleoside.
The modified nucleosides are synthesized by specific tRNA-modifying enzymes. The original nucleoside present at each position can be determined either by comparing the sequence of tRNA with that of its gene or (less efficiently) by isolating precursor molecules that lack some or all of the modifications. The sequences of precursors show that different modifications are introduced at different stages during the maturation of tRNA.
Some modifications are constant features of all tRNA molecules—for example, the D residues that give rise to the name of the D arm, and the ψ found in the TψC sequence. On the 3 side of the anticodon there is always a modified purine, although the modification varies widely.
Other modifications are specific for particular tRNAs or groups of tRNAs. For example, wyosine bases are characteristic of tRNAPhe in bacteria, yeast, and mammals. There are also some species-specific patterns.
The many tRNA-modifying enzymes (~60 in yeast) vary greatly in specificity (for review see Hopper and Phizicky, 2003). In some cases, a single enzyme acts to make a particular modification at a single position. In other cases, an enzyme can modify bases at several different target positions. Some enzymes undertake single reactions with individual tRNAs; others have a range of substrate molecules. The features recognized by the tRNA-modifying enzymes are unknown, but probably involve recognition of structural features surrounding the site of modification. Some modifications require the successive actions of more than one enzyme.