TRNA molecules anticodons

Cellular protein biosynthesis involves the following steps. One strand of double-stranded DNA serves as a template strand for the synthesis of a complementary single-stranded messenger ribonucleic acid (mRNA) in a process called transcription. This mRNA in turn serves as a template to direct the synthesis of the protein in a process called translation. The codons of the mRNA are read sequentially by transfer RNA (tRNA) molecules, which bind specifically to the mRNA via triplets of nucleotides that are complementary to the particular codon, called an anticodon. Protein synthesis occurs on a ribosome, a complex consisting of more than 50 different proteins and several stmctural RNA molecules, which moves along the mRNA and mediates the binding of the tRNA molecules and the formation of the nascent peptide chain. The tRNA molecule carries an activated form of the specific amino acid to the ribosome where it is added to the end of the growing peptide chain. There is at least one tRNA for each amino acid. 

The anticodon region consists of seven nucleotides, and it recognizes the three-letter codon in mRNA (Figure 38-2). The sequence read from the 3 to 5 direction in that anticodon loop consists of a variable base-modified purine-XYZ-pyrimidine-pyrimidine-5h Note that this direction of reading the anticodon is 3 " to 5 whereas the genetic code in Table 38—1 is read 5 to 3 since the codon and the anticodon loop of the mRNA and tRNA molecules, respectively, are antipar-allel in their complementarity just like all other inter-molecular interactions between nucleic acid strands. 

The information contained in the DNA (i.e., the order of the nucleotides) is first transcribed into RNA. The messenger RNA thus formed interacts with the amino-acid-charged tRNA molecules at specific cell organelles, the ribosomes. The loading of the tRNA with the necessary amino acids is carried out with the help of aminoacyl-tRNA synthetases (see Sect. 5.3.2). Each separate amino acid has its own tRNA species, i.e., there must be at least 20 different tRNA molecules in the cells. The tRNAs contain a nucleotide triplet (the anticodon), which interacts with the codon of the mRNA in a Watson-Crick manner. It is clear from the genetic code that the different amino acids have different numbers of codons thus, serine, leucine and arginine each have 6 codewords, while methionine and tryptophan are defined by only one single nucleotide triplet. 

Another hypothesis was provided by Mikio Shimitso (1982) on the basis of studies of steric effects in molecular models. It had been noted years previously that the fourth nucleotide at the 3 end of the tRNA molecules (referred to as the discrimination base) might have a recognition function. In the case of certain amino acids (i.e., their tRNA-amino acid complexes) this base pair, in combination with the anticodon of the tRNA molecule, can select the amino acid corresponding to the tRNA species in question this is done on the basis of the stereochemical properties of the molecule. Since the anticodon of a tRNA molecule and the fourth nucleotide of the acceptor stem are far apart in space, two tRNA molecules must complex in a head-to-tail manner. The pocket thus formed can then fit specifically to the corresponding amino acid. 

There are 64 different three-letter codons, but we don t have to have 64 different tRNA molecules. Some of the anticodon loops of some of the tRNAs can recognize (bind to) more than one condon in the mRNA. The anticodon loops of the various tRNAs may also contain modified bases that can read (pair with) multiple normal bases in the RNA. This turns out to be the reason for the wobble hypothesis, in which the first two letters of a codon are more significant than the last letter. Look in a codon table and you ll see that changing the last base in a codon often doesn t change the identity of the amino acid. A tRNA that could recognize any base in codon position 3 would translate all four codons as the same amino acid. If you ve actually bothered to look over a codon table, you realize that it s not quite so simple. Some amino acids have single codons (such as AUG for Met), some amino acids have only two codons, and some have four. 

Frameshift suppression is also possible. This can be achieved by a second mutation in a tRNA gene such that the anticodon of a tRNA molecule consists of 4 bases rather than 3, for example, an extra C residue in the CCC anticodon sequence of a glycine tRNA gene. This change will allow correction of a +1 frameshift involving the GGG codon for glycine (Bossi, 1985). 

How, in turn, does the synthetase recognize its specific tRNA From extensive mutagenesis studies, it appears that the aminoacyl-tRNA synthetases recognize particular regions of the tRNA molecule, most often in their anticodon loops and/or in their acceptor stems. 

A tRNA molecule is specific for a particular amino acid, though there may be several different forms for each amino acid. Although relatively small, the polynucleotide chain may show several loops or arms because of base pairing along the chain. One arm always ends in the sequence cytosine-cytosine-adenosine. The 3 -hydroxyl of this terminal adenosine unit is used to attach the amino acid via an ester linkage. However, it is now a section of the nucleotide sequence that identifies the tRNA-amino acid combination, and not the amino acid itself. A loop in the RNA molecule contains a specific sequence of bases, termed an anticodon, and this sequence allows the tRNA to bind to a complementary sequence of bases, a codon, on mRNA. The synthesis of a protein from the message carried in mRNA is called translation, and a simplified representation of the process as characterized in the bacterium Escherichia coli is shown below. 

A tRNA molecule displays a three-base anticodon that is complementary to the codon on one end of its folded structure and carries the corresponding amino acid on the opposite end (Figure 12-1). 

The translation of the mRNA into proteins is the final step in the biological flow of information (see Fig. 6.1). Similar to other macromolecular polymerizations, protein synthesis can be divided into initiation, chain elongation, and termination. Critical players in this process are the aminoacyl transfer RNAs (tRNAs). These molecules form the interface between the mRNA and the growing polypeptide. Activation of tRNA involves the addition of an amino acid to its acceptor stem, a reaction catalyzed by an aminoacyl-tRNA synthetase. Each aminoacyl-tRNA synthetase is highly specific for one amino acid and its corresponding tRNA molecule. The anticodon loop of each aminoacyl-tRNA interacts... 

On each of the tRNA molecules, one of the single-stranded loops contains a trinucleotide sequence that is complementary to the triplet codon sequence used in the genetic code to specify a particular amino acid. This loop on the tRNA is known as the anticodon loop, and it is used to match the tRNA with a complementary codon on the mRNA. In this way the amino acids carried by the tRNA molecules can be aligned in the proper sequence for polymerization into a functional protein. 

By observing changes in nucleotides that alter substrate specificity, researchers have identified nucleotide positions that are involved in discrimination by the amino-acyl-tRNA synthetases. These nucleotide positions seem to be concentrated in the amino acid arm and the anticodon arm, including the nucleotides of the anticodon itself, but are also located in other parts of the tRNA molecule. Determination of the crystal structures of aminoacyl-tRNA synthetases complexed with their cognate tRNAs and ATP has added a great deal to our understanding of these interactions (Fig. 27-17). 

Anticodon Each tRNA molecule also contains a three-base nucleotide sequence—the anticodon—that recognizes a specific codon on the mRNA (see Figure 31.6). This codon specifies Ihe insertion into the growing peptide chain of the amino acid carried... 

Correct answer = B. Once an amino acid is attached to a tRNA molecule, only the anticodon of that tRNA determines the specificity of incor poration. The mischarged alanine will, therefore, be incorporated in the protein at a position determined by a cysteine codon. 

Translation involves transfer RNA. (a) The structure of a transfer RNA molecule, with an anticodon at one end and an amino acid attachment site at the other end. (b) A highly simplified symbol for tRNA. The anticodon is a series of three nucleotides that complement the mRNA codon and code for a specific amino acid at the amino acid attachment site. 

As the mRJSlA leaves the cell nucleus in which it was created and enters the cytoplasm, it binds with specialized structures called ribosomes, as shown in Figure 13.36. Ribosomes are microscopic complexes of rRNA and proteins, and they are the site where proteins are built. As the mRNA is scrolled sequentially over the ribosome, the anticodon end of a free tRNA molecule binds to an mRNA codon. In this manner, tRNA molecules and their tag-along amino acids are placed adjacent to one another along the mRNA strand. The amino acids then chemically bond with one another, forming a long polypeptide chain that breaks away from the tRNA as it forms. This process continues until a stop mRNA codon, for which there are no tRNA anticodons, is encountered. At this point, the primary structure of a new protein has been built. 

Figure 5-30 Schematic cloverleaf structure of a phenylalanine-specific transfer RNA (tRNA he) of yeast. The dots represent pairs or triplets of hydrogen bonds. Nucleosides common to almost all tRNA molecules are circled. Other features common to most tRNA molecules are also marked. The manner in which the anticodon may be matched to a codon of mRNA is indicated at the bottom.

Selenium is found to a minor extent wherever sulfur exists in nature. This includes the sulfur-containing modified bases of tRNA molecules. In addition to a small amount of nonspecific incorporation of Se into all S-containing bases there are, at least in bacteria, specific Se-containing tRNAs. In E. coli one of these is specific for lysine and one for glutamate. One of the modified bases has been identified as 5-methyl-amino-methyl-2-selenouridine.570 It is present at the first position of the anticodon, the "wobble" position.571... 

The small 4S tRNA molecules have masses of 26 kDa and consist of 75 5 nucleotides (Figs. 5-30,5-31, and 29-6). The basic structures are similar in bacteria and eukaryotic cells. The need for "adapters," to carry amino acids to the proper positions along the mRNA template, had been predicted prior to the discovery of tRNA.169170 It had been expected that there would be a base sequence constituting an anticodon, which would fit against the proper codon at some binding site on the protein-synthesizing machinery. This is just what tRNA molecules do, but their chemistry contained many surprises. 

Figure 25-26 (a) Generalized representation of a tRNA molecule. Each segment represents a nucleotide, the actual number and sequence of nucleotides varies with the tRNA. There are regions of intrachain basepairing (dashed lines). The nucleotide at the long end has a ribose with a free 3 -OH. The nucleotide at the short end is phosphorylated at 5 -OH. The three nucleotides of the anticodon loop pair with the appropriate bases in mRNA. (b) Three-dimensional picture of a tRNA to show the manner in which the chain is coiled. An excellent review article on the determination of the structure of phenylalanine tRNA by x-ray diffraction has been published, J. L. Sussman and S.-H. Kim, Science 192, 853 (1976). 

Many tRNA sequences are known today, both for a given codon at a variety of phylogenic levels and for a given species and many different anticodons. Each such category is interesting13,14 for comparative analysis. Phylogenic analysis shows whether tRNA has retained information from prebiotic times, or this information has been lost in the course of evolution. The comparison of different tRNA molecules in a single species may then lead to a reasonably complete reconstruction of the early forms and allow statements about the early evolution of the translation apparatus. 

Transfer RNA (tRNA) transports the required amino acids from the cell s amino acid pool to the ribosome. Each type of amino acid can only be transported by its own specific tRNA molecule. The tRNA, together with its amino acid residue, binds to the mRNA already bound to the ribosome. It recognizes the point on the mRNA where it has to deliver its amino acid through the use of a consecutive sequence of three bases known as an anticodon, which is found on one of the loops of the tRNA (Figure 1.33(b)). This anticodon binds to the complementary codon of the mRNA. Consequently, the amino acids can only be delivered to specific points on the mRNA, which controls the order in which amino acid residues are added to the growing protein. This growth occurs from the N-terminal end of the protein. 

After synthesis, the pre-tRNA molecule folds up into the characteristic stem-loops structures (Fig. 1) and non-tRNA sequence is cleaved from the 5 and 3 ends by ribonucleases. In prokaryotes, the CCA sequence at the 3 end of the tRNA (which is the site of bonding to the amino acid) is enclosed by the tRNA gene but this is not the case in eukaryotes. Instead, the CCA is added to the 3 end after the trimming reactions by tRNA nucleotidyl transferase. Another difference between prokaryotes and eukaryotes is that eukaryotic pre-tRNA molecules often contain a short intron in the loop of the anticodon arm (Fig. 4). 

Codons that specify the same amino acid are called synonyms. Most synonyms differ only in the third base of the codon for example GUU, GUC, GUA and GUG all code for valine. During protein synthesis, each codon is recognized by a triplet of bases, called an anticodon, in a specific tRNA molecule (see Topics G10 and H2). Each base in the codon base pairs with its complementary base in the anticodon. However, the pairing of the third base of a codon is less stringent than for the first two bases (i.e. there is some wobble base-pairing ) so that in some cases a single tRNA may base-pair with more than one codon. For example, phenylalanine tRNA, which has the anticodon GAA, recognizes both of the codons UUU and UUC. The third position of the codon is therefore also called the wobble position. 

While RNA molecules usually exist as single chains, they often form hairpin loops consisting of double helices in the A conformation (Moore, 1999). The best-known forms of RNA are the low-molecular-weight tRNA molecules. In all of them the bases can be paired to form a cloverleaf structure with three hairpin loops and sometimes a fourth. The cloverleaf structure of tRNA is further folded into an T-shape conformation with the anticodon triplet and the aminoacyl attachment CCA forming the two ends. 

The tertiary structure of all tRNAs are likewise similar. All known tRNAs are roughly L-shaped, with the anticodon on one end of the L and the acceptor stem on the other. Each stem of the L is made up of two of the stems of the cloverleaf, arranged so that the base pairs of each stem are stacked on top of each other. The parts of the molecule that are not base-paired are involved in other types of interactions, termed tertiary interactions. The tertiary structures of tRNAs thus reflect the dual functions of the molecule The anticodons are well-separated from the acceptor stems. This feature allows two tRNA molecules to interact with two codons that are adjacent on an mRNA molecule. See Figure 10-4. 

Leucine has six codons, the largest number for any amino acid. According to the wobble hypothesis, a single tRNA can accommodate more than one codon. The wobble hypothesis allows for up to three different nucleotides, but only at the third position in the codon, to interact with a single nucleotide in the anticodon. The fact that there are six codons in leucine means that they must differ at positions other than the third, and they therefore could not be accommodated by a single anticodon in a particular tRNA molecule. 

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