TRNA molecules

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. 

In recent year s, clinical studies on the role of uiinai y luodified nucleosides as the biochemical mai kers of various types of cancer have been actively undertaken. Most of the urinai y modified nucleosides ai e piimai ily originated by methylation of either the base part, the sugar hydroxyl par t, or in some cases, both par ts of the course of biodegradation of tRNA molecules. Hence, their isolation and identification plays a major role in biochemical analysis. 

All tRNA molecules contain four main arms. The acceptor arm terminates in the nucleotides CpCpAoH. These three nucleotides are added posttranscription-ally. The tRNA-appropriate amino acid is attached to the 3 -OH group of the A moiety of the acceptor arm. 

A ribosome is a cytoplasmic nucleoprotein stmcture that acts as the machinery for the synthesis of proteins from the mRNA templates. On the ribosomes, the mRNA and tRNA molecules interact to translate into a specific protein molecule information transcribed from the gene. In active protein synthesis, many ribosomes are associated with an mRNA molecule in an assembly called the polysome. 

As described in Chapters 35 and 38, the tRNA molecules serve as adapter molecules for the translation of... 

The regions of the tRNA molecule teferred to in Chapter 35 (and illustrated in Figure 35-11) now become important. The thymidine-pseudouridine-cyti-dine (T PC) arm is involved in binding of the amino-acyl-tRNA to the ribosomal surface at the site of protein synthesis. The D arm is one of the sites important for the proper recognition of a given tRNA species by its proper aminoacyl-tRNA synthetase. The acceptor arm, located at the 3 -hydroxyl adenosyl terminal, is the site of attachment of the specific 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. 

Figure 38-1. Formation of aminoacyl-tRNA. A two-step reaction, involving the enzyme aminoacyl-tRNA synthetase, results in the formation of aminoacyl-tRNA. The first reaction involves the formation of an AMP-amino acid-enzyme complex. This activated amino acid is next transferred to the corresponding tRNA molecule. The AMP and enzyme are released, and the latter can be reutilized. The charging reactions have an error rate of less than 10" and so are extremely accurate.

The charging of the tRNA molecule with the aminoacyl moiety requires the hydrolysis of an ATP to an AMP, equivalent to the hydrolysis of two ATPs to two ADPs and phosphates. The entry of the aminoacyl-tRNA into the A site results in the hydrolysis of one GTP to GDP. Translocation of the newly formed pep-tidyl-tRNA in the A site into the P site by EF2 similarly results in hydrolysis of GTP to GDP and phosphate. Thus, the energy requirements for the formation of one peptide bond include the equivalent of the hydrolysis of two ATP molecules to ADP and of two GTP molecules to GDP, or the hydrolysis of four high-energy phosphate bonds. A eukaryotic ribosome can incorporate as many as six amino acids per second prokaryotic ribosomes incorporate as many as 18 per second. Thus, the process of peptide synthesis occurs with great speed and accuracy until a termination codon is reached. 

Mutations (eg, point mutations, or in some cases deletions) in the genes (nuclear or mitochondrial) encoding various proteins, enzymes, or tRNA molecules are the fundamental causes of the inherited cardiomyopathies. Some conditions are mild, whereas others are severe and may be part of a syndrome affecting other tissues. 

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. 

Although only a few types of amino acids were available during the first few million years of the development of the code, it is likely that three-letter codes were used from the beginning. F. Crick also believes in a three-letter code, because of the continuity principle . It is possible that the third base had no function in the early days but was only necessary for steric reasons for example, because with a two-letter code (only two bases) in the mRNA strand, there would not have been enough room for two amino acid-bearing tRNA molecules. 

The assumption was that, at a later phase of development, amino acids would attach themselves to the RNA structures, which would reach the approximate length of today s tRNA molecules. The structures could be stabilized, for example, by Ca2+ ions in the space between the strands (with their negatively charged phosphate... 

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. 

Figure 1 Overview of specific use of seienium in bioiogical systems. Selenium can be incorporated into macromolecules in at least three separate pathways. From the reduced form of selenide, selenium is activated to selenophosphate by the action of the enzyme selenophosphate synthetase (SPS or SelD). This activated form is then used as a substrate for pathway-specific enzymes that lead to (1) insertion as selenocysteine into proteins during translation (selenoproteins), (2) incorporation into tRNA molecules as mnm Se U or Se U, and (3) insertion into a unique class of molybdoenzymes as a labile, but required, cofactor. The need for activation to selenophosphate has been demonstrated in all cases at the genetic and biochemical level, with the exception of the labile selenoenzymes, where activation of selenium has only been proposed based on proximity of genes within an operon encoding SPS and a molybdoenzyme. ...

The aminoacyl site (A site) binds each new incoming tRNA molecule carrying an activated amino acid. 

Mitochondrial diseases are often expressed as neuropathies and myopathies because brain and muscle are highly dependent on oxidative phosphorylation. Mitochondrial genes code for some of the components of the electron transport chain and oxidative phosphorylation, as well as some mitochondrial tRNA molecules. 

These bases are not the only ones that occur in nucleic acids. There are many rare bases that are occasionally found in these molecules, particularly in RNA. Perhaps 100 of these are known, in addition to the five common bases. We shall encounter a few of these rare bases in transfer RNA, tRNA, molecules. It is enough to know that they exist. We need not be concerned with their structure or function. 

Figure 12.5 The structures for four tRNA molecules of yeast, (a) Alanyl-tRNA (b) phenylalanyl-tRNA (c) seryl-tRNA (d) tyrosyl-tRNA. The single letter designations identify the sequence of bases along the single chain. Note that several of these are unusual bases, most of which are methylated (Me). Note also the ACC sequence at the 3 terminus of each tRNA. This is the site to which amino acids are attached in the process of protein synthesis, as indicated. These tRNA molecules have a substantial amount of secondary structure created by formation of Watson-Crick base pairs. Finally, note that the anticoding triplet in the bottom loop is shown.

In order that tRNA molecules can function as adaptors between amino acids and mRNA, two things must be true. First, there must be a mechanism for attaching each amino acid specifically to its cognate tRNA. Doing so will create molecules known as aminoacyl-tRNAs. Secondly, each aminoacyl-tRNA must recognize the correct triplet code for that amino acid on mRNA. 1 am going to worry about the former issue first. 

All tRNA molecules have the sequence -CCA at the 3 end. This three base sequence is termed the acceptor stem. The aminoacyl-tRNA synthetases catalyze the formation of an ester between the carboxyl group of the amino acid and the 3 -OH of the ribose of the terminal adenosine moiety ... 

The formation of aminoacyl-tRNA molecules is an energy-requiring process, which means that some sort of driving force is required to push the reaction to completion. 

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