Transfer RNA (tRNA) forms a cloverleaf

KEY TERMS:

• Transfer RNA (tRNA) is the intermediate in protein synthesis that interprets the genetic code. Each tRNA can be linked to an amino acid. The tRNA has an anticodon sequence that complementary to a triplet codon representing the amino acid.
• The anticodon is a trinucleotide sequence in tRNA which is complementary to the codon in mRNA and enables the tRNA to place the appropriate amino acid in response to the codon.
• The cloverleaf describes the structure of tRNA drawn in two dimensions, forming four distinct arm-loops.
• A stem is the base-paired segment of a hairpin structure in RNA.
• A loop is a single-stranded region at the end of a hairpin in RNA (or single-stranded DNA); it corresponds to the sequence between inverted repeats in duplex DNA.
• An arm of tRNA is one of the four (or in some cases five) stem-loop structures that make up the secondary structure.
• The acceptor arm of tRNA is a short duplex that terminates in the CCA sequence to which an amino acid is linked.
• The anticodon arm of tRNA is a stem loop structure that exposes the anticodon triplet at one end.
• The D arm of tRNA has a high content of the base dihydrouridine.
• The extra arm of tRNA lies between the TψC and anticodon arms. It is the most variable in length in tRNA, from 3-21 bases. tRNAs are called class 1 if they lack it, and class 2 if they have it.
• Invariant base positions in tRNA have the same nucleotide in virtually all (>95%) of tRNAs.
• Conserved positions are defined when many examples of a particular nucleic acid or protein are compared and the same individual bases or amino acids are always found at particular locations.
• A semiconserved (Semiinvariant) position is one where comparison of many individual sequences finds the same type of base (pyrimidine or purine) always present.
• An aminoacyl-tRNA is a tRNA linked to an amino acid. The COOH group of the amino acid is linked to the 3- or 2-OH group of the terminal base of the tRNA.
• Aminoacyl-tRNA synthetases are enzymes responsible for covalently linking amino acids to the 2- or 3-OH position of tRNA.

KEY CONCEPTS:

• A tRNA has a sequence of 74-95 bases that folds into a clover-leaf secondary structure with four constant arms (and an additional arm in the longer tRNAs).
• tRNA is charged to form aminoacyl-tRNA by forming an ester link from the 2 or 3 OH group of the adenylic acid at the end of the acceptor arm to the COOH group of the amino acid.
• The sequence of the anticodon is solely responsible for the specificity of the aminoacyl-tRNA.

Messenger RNA can be distinguished from the apparatus responsible for its translation by the use of in vitro cell-free systems to synthesize proteins. A protein-synthesizing system from one cell type can translate the mRNA from another, demonstrating that both the genetic code and the translation apparatus are universal.

Each nucleotide triplet in the mRNA represents an amino acid. The incongruity of structure between trinucleotide and amino acid immediately raises the question of how each codon is matched to its particular amino acid. The "adapter" is transfer RNA (tRNA). A tRNA has two crucial properties:

• It represents a single amino acid, to which it is covalently linked.
• It contains a trinucleotide sequence, the anticodon, which is complementary to the codon representing its amino acid. The anticodon enables the tRNA to recognize the codon via complementary base pairing (Hoagland et al., 1958; Holley et al., 1965).

All tRNAs have common secondary and tertiary structures. The tRNA secondary structure can be written in the form of a cloverleaf, illustrated in Figure 5.3, in which complementary base pairing forms stems for single-stranded loops. The stem-loop structures are called the arms of tRNA. Their sequences include "unusual" bases that are generated by modification of the 4 standard bases after synthesis of the polynucleotide chain (for review see Soll and RajBhandary, 1995). 

The construction of the cloverleaf is illustrated in more detail in Figure 5.4. The four major arms are named for their structure or function:

• The acceptor arm consists of a base-paired stem that ends in an unpaired sequence whose free 2– or 3–OH group can be linked to an amino acid.
• The TψC arm is named for the presence of this triplet sequence. (ψ stands for pseudouridine, a modified base.)
• The anticodon arm always contains the anticodon triplet in the center of the loop.
• The D arm is named for its content of the base dihydrouridine (another of the modified bases in tRNA).
• The extra arm lies between the TψC and anticodon arms and varies from 3-21 bases.

The numbering system for tRNA illustrates the constancy of the structure. Positions are numbered from 5 to 3 according to the most common tRNA structure, which has 76 residues. The overall range of tRNA lengths is 74-95 bases. The variation in length is caused by differences in the D arm and extra arm (for more details see 32.8 tRNA sequences).

The base pairing that maintains the secondary structure is shown in Figure 5.4. Within a given tRNA, most of the base pairings are conventional partnerships of A·U and G·C, but occasional G·U, G·ψ, or A·ψ pairs are found. The additional types of base pairs are less stable than the regular pairs, but still allow a double-helical structure to form in RNA.

When the sequences of tRNAs are compared, the bases found at some positions are invariant (or conserved); almost always a particular base is found at the position (see 32.8 tRNA sequences). Some positions are described as semiinvariant (or semiconserved) because they are restricted to one type of base (purine versus pyrimidine), but either base of that type may be present.

When a tRNA is charged with the amino acid corresponding to its anticodon, it is called aminoacyl-tRNA. The amino acid is linked by an ester bond from its carboxyl group to the 2 or 3 hydroxyl group of the ribose of the 3 terminal base of the tRNA (which is always adenine). The process of charging a tRNA is catalyzed by a specific enzyme, aminoacyl-tRNA synthetase. There are (at least) 20 aminoacyl-tRNA synthetases. Each recognizes a single amino acid and all the tRNAs on to which it can legitimately be placed.

There is at least one tRNA (but usually more) for each amino acid. A tRNA is named by using the three letter abbreviation for the amino acid as a superscript. If there is more than one tRNA for the same amino acid, subscript numerals are used to distinguish them. So two tRNAs for tyrosine would be described as tRNA₁^Tyr and tRNA₂^Tyr. A tRNA carrying an amino acid—that is, an aminoacyl-tRNA—is indicated by a prefix that identifies the amino acid. Ala-tRNA describes tRNA^Ala carrying its amino acid.

Does the anticodon sequence alone allow aminoacyl-tRNA to recognize the correct codon? A classic experiment to test this question is illustrated in Figure 5.5. Reductive desulfuration converts the amino acid of cysteinyl-tRNA into alanine, generating alanyl-tRNA^Cys. The tRNA has an anticodon that responds to the codon UGU. Modification of the amino acid does not influence the specificity of the anticodon-codon interaction, so the alanine residue is incorporated into protein in place of cysteine. Once a tRNA has been charged, the amino acid plays no further role in its specificity, which is determined exclusively by the anticodon (Chapeville et al., 1962).