Genes VII

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 7.6 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. 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 Xfor 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 tRNA^Phe in bacteria, yeast, and mammals. There are also some species-specific patterns.

The features recognized by the tRNA-modifying enzymes are unknown. When a particular modification is found at more than one position in a tRNA, the same enzyme does not necessarily make all the changes; for example, a different enzyme may be needed to synthesize the pseudouridine at each location. We do not know what controls the specificity of the modifying enzymes, but it is clear that there are many enzymes, with varying specificities. Some enzymes undertake single reactions with individual tRNAs; others have a range of substrate molecules. Some modifications require the successive actions of more than one enzyme.

The most direct effect of modification is seen in the anticodon, where change of sequence influences the ability to pair with the codon, thus determining the meaning of the tRNA. Modifications elsewhere in the vicinity of the anticodon also influence its pairing.

When bases in the anticodon are modified, further pairing patterns become possible in addition to those predicted by the regular and wobble pairing involving A, C, U, and G. Figure 7.7 shows the use of inosine (I), which is often present at the first position of the anticodon. Inosime can pair with any one of three bases, U, C, and A.

This ability is especially important in the isoleucine codons, where AUA codes for isoleucine, while AUG codes for methionine. Because with the usual bases it is not possible to recognize A alone in the third position, any tRNA with U starting its anticodon would have to recognize AUG as well as AUA. So AUA must be read together with AUU and AUC, a problem that is solved by the existence of tRNA with I in the anticodon.

Actually, some of the predicted regular combinations do not occur, because some bases are always modified. There seems to be an absolute ban on the employment of A; usually it is converted to I. And U at the first position of the anticodon is usually converted to a modified form that has altered pairing properties. Figure 7.8 shows that 2-thiouracil pairs only with A. .

Some modifications create preferential readings of some codons with respect to others. Anticodons with uridine-5-oxyacetic acid and 5-methoxyuridine in the first position recognize A and G efficiently as third bases of the codon, but recognize U less efficiently. Another case in which multiple pairings can occur, but with some preferred to others, is provided by the series of queuosine and its derivatives. These modified G bases continue to recognize both C and U, but pair with U more readily.

A restriction not allowed by the usual rules can be achieved by the employment of 2-thiouridine in the anticodon. This modification allows the base to continue to pair with A, but prevents it from indulging in wobble pairing with G (for review see 32).

These and other pairing relationships make the general point that there are multiple ways to construct a set of tRNAs able to recognize all the 61 codons representing amino acids. No particular pattern predominates in any given organism, although the absence of a certain pathway for modification can prevent the use of some recognition patterns. So a particular codon family is read by tRNAs with different anticodons in different organisms.

Often the tRNAs will have overlapping responses, so that a particular codon is read by more than one tRNA. In such cases there may be differences in the efficiencies of the alternative recognition reactions. (As a general rule, codons that are commonly used tend to be more efficiently read.) And in addition to the construction of a set of tRNAs able to recognize all the codons, there may be multiple tRNAs that respond to the same codons.

The predictions of wobble pairing accord very well with the observed abilities of almost all tRNAs. But there are exceptions in which the codons recognized by a tRNA differ from those predicted by the wobble rules. Such effects probably result from the influence of neighboring bases and/or the conformation of the anticodon loop in the overall tertiary structure of the tRNA. Indeed, the importance of the structure of the anticodon loop is inherent in the idea of the wobble hypothesis itself. Further support for the influence of the surrounding structure is provided by the isolation of occasional mutants in which a change in a base in some other region of the molecule alters the ability of the anticodon to recognize codons (see later).

Another unexpected pairing reaction is presented by the ability of the bacterial initiator, fMet-tRNA_f, to recognize both AUG and GUG. This misbehavior involves the third base of the anticodon.