BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 27. PROTEIN SYNTHESIS

As we learned in the preceding chapter, the Amino Acid Sequence in Proteins is determined by The sequence of codons in mRNA, which are read by tRNA molecules. Let us now turn to The Mechanism of Protein Synthesis. This process is called Translation because the information written in the four-letter language of Nucleic Acids is translated into the protein language consisting of 20 letters. As one might expect, translation is a more complex process than METABOLISM/36.html">DNA Replication or Transcription, which use the same base-pairing language. Translation is carried out through the coordinated interaction of more than a hundred types of macromolecules. In addition to Ribosomes, tRNA molecules, activating Enzymes, soluble factors, and mRNA are required.

Before delving into a detailed description of protein synthesis, let us examine the process in broad outline. Proteins are synthesized in the amino-to-carboxyl direction by the sequential addition of Amino Acids to the carboxyl terminus of the growing peptide chain. Aminoacyl-tRNAs, in which the carboxyl group of The amino acid is attached to the 3'-end of the tRNA, play The Role of activated precursors in this process. The attachment of an amino acid to its corresponding tRNA is catalyzed by aminoacyl-tRNA synthetase. This activation reaction, being analogous to the activation of Fatty acids, is also driven by the energy of ATP Hydrolysis. For each amino acid, there is at least one type of tRNA and a specific activating enzyme. Protein synthesis proceeds in three stages, called initiation, elongation, and termination.

Initiation leads to the binding of the initiator tRNA to the transcription start signal in the mRNA. The initiator tRNA occupies the P site (from peptidyl) of the ribosome, one of the two tRNA-binding sites. Elongation begins with the binding of an aminoacyl-tRNA to the other tRNA-binding site on the ribosome, known as the A site (from aminoacyl). A peptide bond is then formed between the amino group of the second aminoacyl-tRNA and the carboxyl group of fMet linked to the initiator tRNA. The resulting dipeptidyl-tRNA then moves from the A site to the P site, while the deacylated tRNA molecule leaves the ribosome. The binding of aminoacyl-tRNA, the translocation of peptidyl-tRNA from the A site to the P site, and the simultaneous movement of the ribosome to the next mRNA codon require the hydrolysis of GTP. Next, a new aminoacyl-tRNA binds to the vacant A site, and a new elongation cycle begins, similar to the one just described. Termination occurs when a stop codon (termination signal) in the mRNA is recognized by a protein release factor. This results in the release of the completed polypeptide chain from the ribosome. In this chapter, we will focus primarily on Protein synthesis in E. coli Cells, where this process is best understood. Certain differences between prokaryotic and eukaryotic protein synthesis will be discussed in Section 29.25.

27.1. Amino acids are activated and attached to transfer RNAs by specific synthetases

The formation of a peptide bond between the amino group of one Amino Acid and the COOH group of another is thermodynamically unfavorable. This thermodynamic barrier is overcome by activating the COOH group of the precursor amino acids. The activated intermediates in protein synthesis are amino acid esters in which the carboxyl group of the amino acid is linked to the 2'- or 3'-hydroxyl group of the ribose residue at the 3'-end of the tRNA. The aminoacyl group can rapidly migrate between the 2'- and 3'-positions. This activated intermediate is called aminoacyl-tRNA (Fig. 27.1).

Class="center">Fig. 27.1. In aminoacyl-tRNA, the amino acid is linked via an ester bond to the 2'- or 3'-hydroxyl group of the terminal adenosine

The attachment of an amino acid to a tRNA is significant not only because it activates its carboxyl group for peptide bond formation, but also because amino acids themselves are unable to recognize codons in mRNA. Amino Acids are delivered to ribosomes by specific tRNAs, which recognize the codons in mRNA. Thus, these tRNAs function as adapter molecules.

In 1957, Paul Zamecnik and Mahlon Hoagland established that Amino Acid Activation and their subsequent attachment to tRNA are catalyzed by specific Aminoacyl-tRNA synthetases, also called activating enzymes.

In the Reactions Catalyzed by certain synthetases, the first step consists of the formation of aminoacyl adenylate from an amino acid and ATP. This activated compound is a mixed anhydride in which the carboxyl group of the amino acid is attached to the phosphate group of AMP. Other synthetases catalyze the reaction of ATP, an amino acid, and a tRNA without The intermediate formation of a detectable aminoacyl adenylate.

The next step is The transfer of the aminoacyl group of aminoacyl-AMP to a tRNA molecule, yielding aminoacyl-tRNA, the activated intermediate in protein synthesis. Whether the aminoacyl group is transferred to the 2'- or 3'-hydroxyl group of the ribose residue at the 3'-end of the tRNA depends on the specific amino acid and aminoacyl-tRNA synthetase involved. The activated amino acid can rapidly migrate between the 2'- and 3'-positions.

Aminoacyl-AMP + tRNA ⇄ Aminoacyl-tRNA + AMP.

The overall reaction of the activation and transfer steps is described by the following equation:

Amino acid + ATP + tRNA ⇄ Aminoacyl-tRNA + AMP + PPi.

∆G' for this reaction is close to zero, because the Free energy of aminoacyl-tRNA ester bond hydrolysis corresponds to the free energy of hydrolysis of the terminal phos-

phoryl group of ATP. What then drives the synthesis of aminoacyl-tRNA? As might be expected, the reaction is driven by the hydrolysis of pyrophosphate. The overall reaction of these conversions is highly exergonic:

Amino acid + ATP + tRNA + H2O ⇄ Aminoacyl-tRNA + AMP + 2Pi.

Thus, the synthesis of aminoacyl-tRNA consumes two high-energy phosphate bonds. One is expended in forming the aminoacyl-tRNA ester bond, and the other pulls the reaction equilibrium toward product formation.

The activation and transfer steps for a given amino acid are catalyzed by the same aminoacyl-tRNA synthetase. In fact, aminoacyl-AMP does not dissociate from the synthetase complex; it is tightly bound to the Active Site of the enzyme through noncovalent interactions. Normally, the aminoacyl-AMP formed as an intermediate in aminoacyl-tRNA synthesis is short-lived, but it is quite stable and can be readily isolated if tRNA is absent from the reaction mixture.

We have already encountered the acyl adenylate intermediate in fatty acid activation (Section 17.6). It is interesting to note that Paul Berg was the first to discover this intermediate in the fatty acid activation reaction, and he later found that this product is also formed during amino acid activation. The main difference between these reactions is that in the former case, CoA acts as the acyl group acceptor, whereas in the latter, it is tRNA. The energetics of these biosynthetic reactions are very similar: both are rendered irreversible by the hydrolysis of inorganic pyrophosphate.

There is at least one aminoacyl-tRNA synthetase for each amino acid. These enzymes vary in size, subunit Structure, and Amino Acid Composition (Table 27.1).

Table 27.1. Properties of certain aminoacyl-tRNA synthetases