TRNA inosine

FIGURE 8-5 Some minor purine and pyrimidine bases shown as the nucleosides (a) Minor bases of DNA. 5-Methylcytidine occurs in the DNA of animals and higher plants, A/6-methyladenosine in bacterial DNA, and 5-hydroxymethylcytidine in the DNA of bacteria infected with certain bacteriophages (b) Sbme minor bases of tRNAs Inosine contains the basehypoxanthine. Note that pseudouridine, like uridine, contains uracil they are distinct in the point of attachment to the ribose—in uridine, uracil is attached through N-1, the usual attachment point for pyrimidines in pseudouridine, through C-5. 

Figure 12-1. Codon-anticodon base pairing. Special wobble base-pairing rules apply to the third (3 ) position of the codon. The first (S ) position of the tRNA anticodon is frequently inosine (I) to provide this flexibility in hydrogen bonding.

Transfer RNAs base-pair with mRNA codons at a three-base sequence on the tRNA called the anticodon. The first base of the codon in mRNA (read in the 5 —>3 direction) pairs with the third base of the anticodon (Fig. 27-8a). If the anticodon triplet of a tRNA recognized only one codon triplet through Watson-Criclc base pairing at all three positions, cells would have a different tRNA for each amino acid codon. This is not the case, however, because the anticodons in some tRNAs include the nucleotide inosinate (designated I), which contains the uncommon base hypoxanthine (see Fig. 8-5b). Inosinate can form hydrogen bonds with three different nucleotides (U, C, and A Fig. 27-8b), although... 

FIGURE 27-8 Pairing relationship of codon and anticodon, (a) Alignment of the two RNAs is antiparallel. The tRNA is shown in the traditional cloverleaf configuration, (b) Three different codon pairing relationships are possible when the tRNA anticodon contains inosinate. 

The first surprise was that these molecules are much longer than seems necessary for the formation of adapters. In addition, 10-20% of their bases are modified greatly from their original form.171 Another surprise was that the anticodons are not all made up of "standard" bases. Thus, hypoxanthine (whose nucleoside is inosine) occurs in some anticodons. Conventional "cloverleaf" representations of tRNA, which display their secondary structures, are shown in Figs. 5-30 and 29-7. However, the molecules usually have an L shape rather than a cloverleaf form (Figs. 5-31 and 29-6),172 and the L form is essential for functioning in protein synthesis as indicated by X-ray and other data.173 Three-dimensional structures, now determined for several different tRNAs,174 175 are all very similar. Structures in solution are also thought to be... 

The sugar specificity of RNase Tx appears to require a 2 -hydroxyl group for the substrate because DNA is not attacked by RNase Tx. This is consistent with the intermediary formation of 2, 3 -cyclic phosphate and also with the finding that 2 -0-methylated guanylyl bonds in tRNA is resistant to the enzyme (48)- Holy and Sorm (49) found that RNase Tx did not attack L-guanosine 2, 3 -cyclic phosphate and L-inosine 2, 3 -cyclic phosphate. They found further that RNase Tx split 9-(a-L-lyxo-furanosyl)-hypoxanthine 2, 3 -cyclic phosphate but not the D-lyxofura-nose derivative, and they concluded that the substrate molecule was fixed at least to three regions of RNase Tx (50). 

When the 5 base in the anticodon is an A it can pair only with a U in the codon. However, it is rare that an A is found in this position of the anticodon. An A in this position in eukaryotes is usually deaminated to an inosine (I) base, which has an expanded capacity for pairing. Base A can pair only with U, but I can pair with U, C, or A. In eubacteria, deamination is limited to the conversion of the ACG sequence to an ICG anticodon. When C is the 5 base in the anticodon it pairs with G only. The only known exception to this rule is found in eubacteria, in which the C is covalently modified in the tRNA that recognizes the AUA codon. Thus, in this one instance the modified C can pair with a 3 A in the codon. A U base in the 5 position of the anticodon shows the greatest variability. An unmodified U can pair with any of the four bases in the 3 position of the codon. 

This serine tRNA has inosine at the 5 position of its anticodon, which can pair with U, C, or A. Thus the anticodon of this tRNA is most likely IGA (given in the correct 5 to 3 direction). 

Following synthesis, nucleotides in the tRNA molecule may undergo modification to create unusual nucleotides such as 1-methylguanosine (m G), pseudouridine (4/), dihydrouridine (D), inosine (I) and 4-thiouridine (S4U). 

Figure 10.29 Diagram of tRNA from yeast, specific for alanine. I, inosine V pseudouridine mG, methylguanosine m2G, dimethylguanosine T, ribothymidine hU, dihydrouridine ml, methylinosine.

Transfer RNA (Mr s= 25,000) functions as an adapter in polypeptide chain synthesis. It comprises 10-20 percent of the total RNA in a cell, and there is at least one type of tRNA for each type of amino acid. Transfer RNAs are unique in that they contain a relatively high proportion of nucleosides of unusual structure (e.g., pseudouridine, inosine, and 2 -0-methylnucleosides) and many types of modified bases (e.g., methylated or acetylated adenine, cytosine, guanine, and uracil). As examples, the structures of pseudouridine and inosine are shown below. Inosine has an important role in codon-anticodon pairing (Chap. 17). 

The first base of an anticodon determines whether a particular tRNA molecule reads one, two, or three kinds of codons C or A (one codon), U or G (two codons), or I (three codons). Thus, part of the degeneracy of the genetic code arises from imprecision (wobble) in the pairing of the third base of the codon with the first base of the anticodon. We see here a strong reason for the frequent appearance of inosine, one of the unusual nucleosides, in anticodons. Inosine maximizes the number of codons that can be read by a particular tRNA molecule. The inosines in tRNA are formed by deamination of adenosine after synthesis of the primary transcript. 

Robert Holley first determined the base sequence of a tRNA molecule in 1965, as the culmination ul 7 years of effort, Indeed, his study of yeast alanyl-tRNA provided the first complete sequence of any nucleic acid. This adapter molecule is a single chain of 76 ribonucleotides (Figure 30.2). The 5 terminus is phosphorylated (pCi), whereas the 3 terminus has a free hydroxyl group. T he amino acid-attachment site is the 3 -hydroxyl group of the adenosine residue at the 3 terminus of the molecule. The sequence 5 - IGC-3 in the middle of the molecule is the anticodon, where I is the purine base inosine. It is complementary to 5 -GCC-3, one of the codons for alanine. 

Figure 30.2 Alanyl-tRNA sequence. The base sequence of yeast alanyl-tRNA and the deduced cloverleaf secondary structure are shown. Modified nucleosides are abbreviated as follows methylinosine (ml), dihydroLindine (UHj). ribothymidine (T), pseudouridine methylguanosine (mG), and diniethylguanosine (m G. Inosine (l. another modified nucleoside, is part of the anticodon.

Although adenine rarely is found in the anticodon wobble position, many tRNAs in plants and animals contain inosine... 

A FIGURE 4-23 Nonstandard codon-anticodon base pairing at the wobble position. The base in the third (or wobbie) position of an mRNA codon often forms a nonstandard base pair with the base in the first (or wobbie) position of a tRNA anticodon. Wobbie pairing aiiows a tRNA to recognize more than one mRNA codon (top) converseiy, it aiiows a codon to be recognized by more than one kind of tRNA (bottom), aithough each tRNA wiii bear the same amino acid. Note that a tRNA with i (inosine) in the wobbie position can "read" (become paired with) three different codons, and a tRNA with G or U in the wobbie position can read two codons. Aithough A is theoreticaiiy possibie in the wobbie position of the anticodon, it is aimost never found in nature. 

The best way to conceptualize the principle of the Wobble Hypothesis is from the perspective of the tRNA anticodon, as this emphasizes the use of a single tRNA species to recognize more than one mRNA codon. The first base in the tRNA anticodon corresponds to the last base in the mRNA codon, which is referred to as the wobble position. For example, tRNAs with an inosine in the 50 position of the anticodon have the greatest flexibility and can bind to as many as three different codons (Fig. 26.6). The Wobble Hypothesis was confirmed experimentally... 

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