In this article we will discuss about the activation of amino acids and formation of aminoacyl-tRNA.
The first step of protein biosynthesis comprises two reactions (see fig. 6-43) catalyzed, for a given amino acid, by one and the same enzyme called activation enzyme, or aminoacyl-tRNA synthetase, or aminoacyl-tRNA ligase.
In a first step, the L-amino acid is activated by ATP and forms an aminoacyl- AMP, by a reaction analogous to that of the activation of fatty acids where an acyl-AMP is formed (see fig. 5-10). A mixed anhydride is formed between the carboxyl and a phosphoric acidic OH.
This compound remains bound to the enzyme; then, since it is very reactive, it will dissociate and thus allow the amino acid to bind (by its carboxyl, again) to a hydroxyl at the 3′ end of a particular RNA called transfer RNA or tRNA; an ester linkage is therefore formed and this compound is called aminoacyl-tRNA. Depending on the tRNA, the amino acid is bound either to the OH at position 2′ or to the OH at position 3′ of the ribose of the 3′ terminal nucleotide.
1. Transfer RNAs (tRNA):
The RNA present in the soluble part (non-particulate) of the cytoplasm was originally called soluble RNA (sRNA), by opposition to the ribosomal RNAs present in the ribo-nucleoprotein particles, called ribosomes which will be referred to in the following.
It was subsequently found that this sRNA is in reality a mixture containing a large number of different RNAs which were called transfer RNAs or tRNAs, in view of their role in the biosynthesis of proteins; the tRNA, after binding an amino acid, will indeed transfer it and permit its incorporation into a protein, by a mechanism.
Each tRNA is specific of an amino acid, i.e. it can bind (or “accept”) only that particular amino acid. Thus, tRNAala denotes a (RNA specific of alanine, capable of binding then transferring alanine, tRNAtyr denotes a (RNA specific of tyrosine, etc.
One might think that there are 20 types of tRNAs (as many as the constituent amino acids of proteins), but in fact there are many more: in a bacterial cell, there are more than seventy tRNAs, and in eucaryotic cells this number is even greater, because there are tRNAs specific of mitochondria and chloroplasts (which usually differ from the corresponding cytoplasmic tRNAs).
Therefore, there are generally several tRNAs specific of the same amino acid (sometimes up to four or five): they are called isoacceptor tRNAs.
The tRNAs are small-sized ribonucleic acids, consisting of 70 to 90 nucleotides only, which corresponds to a molecular weight of about 30 000.
They contain rare nucleotides (several tens of them are presently known), i.e. nucleotides which are unusual either by the nature of the base which is often a methylated base (see figs. 6-2 and 6-3), or even by the nature of the bond between the base and the sugar (in tRNAs one, for instance, finds pseudo- uridylic acid where C1 of ribose is bound to C5 of uracil and not to N1).
These modifications are catalyzed by various specific enzymes after the synthesis of the polyribonucleotide chain, as described in the study of processes of maturation of tRNAs.
The various RNAs differ by their nucleotide sequences. One presently knows several hundreds of complete sequence of tRNAs of very diverse origins (bacteria, yeast, plants, animals) and it is observed that there are common characteristics. It was found that in particular, all tRNAs can take a clover leaf secondary structure, having 4 pairing zones between complementary bases and 3 loops.
Figure 6-44 shows that a pCpCpA terminal trinucleotide sequence is present at the 3′ end; it is identical in all tRNAs and indispensable for the binding of the amino acid as the latter binds to the ribose of the terminal adenylic nucleotide; this sequence can also be degraded in vitro, then formed again (from CTP and ATP) by a specific enzyme called tRNA nucleotidyl transferase.
One of the loops comprises a nucleotide triplet called anticodon, which plays a vital role in the transfer of the amino acid (i.e. in the incorporation of the amino acid at the proper place in the protein), and which will be again referred to in the following.
Lastly, it must be indicated that a native three-dimensional conformation is necessary for the biological activity of the tRNA; recently several tRNAs could be crystallized and their conformation can now be studied by X-ray diffraction.
These studies revealed that the tRNA molecule has a L-shaped structure, due to the existence of 2 perpendicular segments in double helix having each about 10 base pairs, and the presence of tertiary interactions between the bases of the non-helicoidal regions.
These interactions are often based on unusual hydrogen bonds between some bases (AC, AA, GG), between some bases and the ribose-phosphate chain, or between different regions of this chain (the 2’OH groups of riboses of the chain often participate in these hydrogen bonds). The anticodon and the binding site of the amino acid (i.e. the CCA 3′ terminal triplet) are at the ends of the L, at a distance of about 80 Å.
2. Activation Enzyme or Aminoacyl-tRNA Synthetase:
These two names recall the two reactions catalyzed by this enzyme (see fig. 6-43) which besides ATP must recognize, on one hand the corresponding amino acid (it can catalyze the activation of the latter only), and on the other hand, the corresponding tRNA (it can allow the binding of the amino acid only to a tRNA specific of this amino acid).
A very high specificity is indeed necessary during this step in order to avoid errors in the biosynthesis of proteins, because once the aminoacyl-tRNA is formed, there is no longer any control mechanism in the cell to verify the nature of the amino acid and it is therefore no longer possible to reject an amino acid which would have been incorrectly bound; as will be seen in the following, the next steps of protein synthesis involve only the polynucleotide part of the aminoacyl-tRNA, with the result that an amino acid which would be bound to a tRNA other than the one corresponding to it, would be erroneously incorporated in the protein.
This was shown by Chapeville and co-workers in the following manner: they bound cysteine to tRNAcys thanks to the corresponding activation enzyme, then with the help of Nickel Raney they transformed cysteine into alanine without breaking the ester linkage and therefore obtained an alanyl-tRNAcys (a compound which cannot normally form in the cell); they observed that from this compound alanine was incorporated in the polypeptides in place of cysteine (see fig. 6-45) in a cell-free system of protein synthesis programmed either by poly UG (where the ratio U/G was 5) permitting normally the incorporation of cysteine (codon UGU) but not that of alanine (GCN), or by globin mRNA (in this case, addition of 14C-alanyl-tRNAcys led to the formation of a radioactive trypsic peptide normally comprising cysteine).
In vivo, the aminoacyl-tRNA synthetases must therefore, either not make any errors or be able to correct them. They must, in particular be able to distinguish between two amino acids of very similar structures.
Thus, tryosyl tRNA synthetase binds tyrosine 1000 times more effectively than phenylalanine (which differs only by one hydroxyl less). But the discrimination by isoleucyl-tRNA synthetase between valine and isoleucine (which has only one — CH2— more implies a correction mechanism or “proof-reading”.
This additional — CH2— favours the activation of isoleucine compared to valine by a factor of about 200, but since in general, there is 5 times more valine than isoleucine in vivo, one would expect that valine is incorporated by error once out of 40 times in place of isoleucine.
In fact, the frequency of such an error is only 1/3 000 because isoleucyl-tRNA synthetase is capable of correcting its own errors, i.e. in presence of tRNAile, the valyl-AMP formed is hydrolyzed (but not isoleucyl-AMP), thus preventing an erroneous aminoacylation (i.e. a mis-acyla- tion) of tRNAile.
Most aminoacyl-tRNA synthetases probably have a synthetic site which does not accept amino acids larger than the corresponding amino acid, and a hydrolytic site which degrades the aminoacyl-AMPs smaller than the correct aminoacyl-AMP.
The number of sub-units and molecular weight vary widely from one aminoacyl-tRNA synthetase to another even within the same organism.
One thus finds:
1. Monomers (α) of Molecular Weight between 50 000 and 60 000 d (dal- tons):
Cys RS, Glu Rs, Gin Rs, Arg Rs
or a molecular weight between 110 000 and 130 000 d:
Leu RS, lie RS, Val RS
2. Dimers of the Type α2, whose Monomer has:
i. A molecular weight between 35 000 and 40 000 d:
His RS, Trp RS, Tyr RS, Ser RS, Pro RS
ii. or a molecular weight between 75 000 and 80 000 d:
Thr RS, Lys RS, Met RS
3. Tetramers:
i. Either of type α4 whose monomer has a molecular weight of about 95 000 d: Ala RS
ii. or of type α2 β2.
In Phe RS of E. Coli, α has a molecular weight of 80 000 to 90 000d;β has a molecular weight of about 30 000 d.
In cytoplasmic Phe RS of yeast, α has a molecular weight of 70 000 d and β has a molecular weight of 60 000 d.