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Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th edition. New York: W. H. Freeman; 2000.

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An Introduction to Genetic Analysis. 7th edition.

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Protein synthesis

We can regard protein synthesis as a chemical reaction, and we shall take this approach at first. Then we shall take a three-dimensional look at the physical interactions of the major components.

In protein synthesis as a chemical reaction:


Each amino acid is attached to a tRNA molecule specific to that amino acid by a high-energy bond derived from ATP. The process is catalyzed by a specific enzyme called a synthetase (the tRNA is said to be “charged” when the amino acid is attached):

Image ch10e15.jpg
There is a separate synthetase for each amino acid.


The energy of the charged tRNA is converted into a peptide bond linking the amino acid to another one on the ribosome:

Image ch10e16.jpg


New amino acids are linked by means of a peptide bond to the growing chain:

Image ch10e17.jpg


This process continues until aa n (the final amino acid) is added. The whole thing works only in the presence of mRNA, ribosomes, several additional protein factors, enzymes, and inorganic ions.


Ribosomes consist of two subunits that, in prokaryotes, sediment as 50S and 30S particles and associate to form a 70S particle, as seen in Figure 10-30a. The eukaryotic counterparts are 60S and 40S for the large and small subunits, and 80S for the complete ribosome (Figure 10-30b). Ribosomes contain specific sites that enable them to bind to the mRNA, the tRNAs, and specific protein factors required for protein synthesis. Let’s look at a general picture of protein synthesis on the ribosome and then examine each of the steps in the process in more detail.

Figure 10-30. A ribosome contains a large and a small subunit.

Figure 10-30

A ribosome contains a large and a small subunit. Each subunit contains both rRNA of varying lengths and a set of proteins (designated by different shapes and shading). There are two principal rRNA molecules in all ribosomes. (a) Ribosomes from prokaryotes (more...)

Figure 10-31 shows a polypeptide being synthesized on the ribosome. The mRNA binds to the 30S subunit. The tRNAs bind to two sites on the ribosome. These sites overlap the subunits. The A site is the entry site for an aminoacyl-tRNA (a tRNA carrying a single amino acid). The peptidyl-tRNA carrying the growing polypeptide chain binds at the P site. Each new amino acid is added by the transfer of the growing chain to the new aminoacyl-tRNA, forming a new peptide bond. The deacylated tRNA is then released from the P site, and the ribosome moves one codon farther along the message, transferring the new peptidyl-tRNA to the P site and leaving the A site vacant for the next incoming aminoacyl-tRNA.

Figure 10-31. The addition of a single amino acid to the growing polypeptide chain in the course of translation of mRNA.

Figure 10-31

The addition of a single amino acid to the growing polypeptide chain in the course of translation of mRNA.

We can separate the process of protein synthesis into three distinct steps. Initiation, elongation, and termination. Let’s examine each of these steps in detail, by using prokaryotes as an example.


Three steps of initiation.  

In addition to mRNA, ribosomes, and specific tRNA molecules, initiation requires the participation of several factors, termed initiation factors IF1, IF2, and IF3. In E. coli and in most other prokaryotic organisms, the first amino acid in any newly synthesized polypeptide is N-formylmethionine. It is inserted not by tRNAMet, however, but by an initiator tRNA called tRNAfMET. This initiator tRNA has the normal methionine anticodon but inserts N-formylmethionine rather than methionine (Figure 10-32). In E. coli, AUG and GUG, and on rare occasions UUG, serve as initiation codons. When one of these triplets is present in the initiation position, it is recognized by N-formylMet-tRNA, and methionine appears as the first amino acid in the chain. Let’s examine the steps in initiation in detail.

Figure 10-32. The structures of methionine (Met) and N-formylmethionine (fMet).

Figure 10-32

The structures of methionine (Met) and N-formylmethionine (fMet). A tRNA bearing fMet can initiate a polypeptide chain in prokaryotes but cannot be inserted in a growing chain; a tRNA bearing Met can be inserted in a growing chain but will not initiate (more...)


The first step in initiation is the binding of the mRNA to the 30S subunit (Figure 10-33). The binding is stimulated by the initiation factor IF3. When not engaged in protein synthesis, the ribosomal subunits exist in the free form; they assemble into complete ribosomes as a result of the initiation process.


The initiation factor IF2 binds to GTP and to the initiator fMet-tRNA and stimulates the binding of fMet-tRNA to the initiation complex, leading the fMet-tRNA into the P site, as shown in the middle of Figure 10-33.


A ribosomal protein splits the GTP bound to IF2, helping to drive the assembly of the two ribosomal subunits (Figure 10-33, bottom). At this stage, the factors IF2 and IF3 are released. (The exact role of IF1 is not completely clear, although it seems to take part in the recycling of the ribosome.)

Figure 10-33. Steps in the initiation of translation (see text).

Figure 10-33

Steps in the initiation of translation (see text).

Ribosome-binding sites.  

How are the correct initiation codons selected from the many AUG and GUG codons in an mRNA molecule? John Shine and Lynn Dalgarno first noticed that true initiation codons were preceded by sequences that paired well with the 3′ end of 16S rRNA. Figure 10-34 shows some of these sequences. There is a short but variable separation between the Shine-Dalgarno sequence and the initiation codon. Figure 10-35 depicts the base pairing between idealized mRNA and the 16S rRNA that results in ribosome-mRNA complexes leading to protein initiation in the presence of fMet-tRNA.

Figure 10-34. Ribosomal binding-site sequences in E.

Figure 10-34

Ribosomal binding-site sequences in E. coli and its bacteriophages have certain features in common, which are shown in the colored regions. The initiation codon (color) is separated by several bases from a short sequence (color) that is complementary (more...)

Figure 10-35. Binding of the Shine-Dalgarno sequence on an mRNA to the 3′ end of 16S rRNA.

Figure 10-35

Binding of the Shine-Dalgarno sequence on an mRNA to the 3′ end of 16S rRNA. (After L. Stryer, Biochemistry, 4th ed. Copyright © 1995 by Lubert Stryer.)


Figure 10-36 details the steps in elongation, which are aided by three protein factors, EF-Tu, EF-Ts, and EF-G. The steps are as follows:

Figure 10-36. Steps in elongation (see text).

Figure 10-36

Steps in elongation (see text).


Elongation factor EF-Tu mediates the entry of amino-acyl-tRNAs into the A site. To do so, EF-Tu first binds to GTP. This activated EF-Tu–GTP complex binds to the tRNA. Next, hydrolysis of the GTP of the complex to GDP helps drive the binding of the aminoacyl-tRNA to the A site, at which point the EF-Tu is released (Figure 10-36a), leaving the new tRNA in the A site (Figure 10-36b).


Elongation factor EF-Ts mediates the release of EF-Tu–GDP from the ribosome and the regeneration of EF-Tu–GTP.


In the translocation step, the polypeptide chain on the peptidyl-tRNA is transferred to the aminoacyl-tRNA on the A site in a reaction catalyzed by the enzyme peptidyltransferase (Figure 10-36c). The ribosome then translocates by moving one codon farther along the mRNA, going in the 5′ → 3′ direction. This step is mediated by the elongation factor EF-G (Figure 10-36d) and is driven by splitting a GTP to GDP. This action releases the uncharged tRNA from the P site and transfers the newly formed peptidyl-tRNA from the A site to the P site (Figure 10-36e).


Release factors.  

In the earlier discussion of the genetic code, we described the three chain-termination codons UAG, UAA, and UGA. Interestingly, these three triplets are not recognized by a tRNA, but instead by protein factors, termed release factors, which are abbreviated RF1 and RF2. RF1 recognizes the triplets UAA and UAG, and RF2 recognizes UAA and UGA. A third factor, RF3, also helps to catalyze chain termination. When the peptidyl-tRNA is in the P site, the release factors, in response to the chainterminating codons, bind to the A site. The polypeptide is then released from the P site, and the ribosomes dissociate into two subunits in a reaction driven by the hydrolysis of a GTP molecule. Figure 10-37 provides a schematic view of this process.

Figure 10-37. Steps leading to termination of protein synthesis (see text).

Figure 10-37

Steps leading to termination of protein synthesis (see text).

Nonsense suppressor mutations

It is interesting to consider the suppressors of the nonsense mutations that Brenner and co-workers defined. Many of these nonsense suppressor mutations are known to alter the anticodon loop of specific tRNAs in such a way as to allow recognition of a nonsense codon in mRNA. Thus, an amino acid is inserted in response to the nonsense codon, and translation continues past that triplet. In Figure 10-38, the amber mutation replaces a wild-type codon with the chain-terminating nonsense codon UAG. By itself, the UAG would result in prematurely cutting off the protein at the corresponding position. The suppressor mutation in this case produces a tRNATyr with an anticodon that recognizes the mutant UAG stop codon. The suppressed mutant thus contains tyrosine at that position in the protein.

Figure 10-38. (a) Termination of translation.

Figure 10-38

(a) Termination of translation. Here the translation apparatus cannot go past a nonsense codon (UAG in this case), because there is no tRNA that can recognize the UAG triplet. This leads to the termination of protein synthesis and to the subsequent release (more...)

What happens to normal termination signals at the ends of proteins in the presesnce of a suppressor? Many of the natural termination signals consist of two chain-termination signals in a row. Nonsense suppressors are sufficiently inefficient in translating through chain-terminating triplets, because of competition with release factors, that the probability of suppression at two codons in a row is small. Consequently, very few protein copies that carry many extraneous amino acids resulting from translation beyond the natural stop codon are produced.

Overview of protein synthesis

Figure 10-39 summarizes the steps in protein synthesis covered in this section. A direct visualization of protein synthesis can be seen in the electron micrograph shown in Figure 10-40, which shows the simultaneous transcription and translation of a gene in E. coli.

Figure 10-39. The transactions of the ribosome.

Figure 10-39

The transactions of the ribosome. At initiation, the ribosome recognizes the starting point in a segment of mRNA and binds a molecule of tRNA bearing a single amino acid. In all bacterial proteins, this first amino acid is N-formylmethionine. In elongation, (more...)

Figure 10-40. A gene of E.

Figure 10-40

A gene of E. coli being simultaneously transcribed and translated. (Electron micrograph by O. L. Miller, Jr., and Barbara A. Hamkalo.)

Protein processing

Even after mRNA has been successfully translated into its protein product, processing may continue. For example, membrane proteins or proteins that are secreted from the cell are synthesized with a short leader peptide, called a signal sequence, at the amino-terminal (N-terminal) end. This signal sequence is a stretch of 15 to 25 amino acids, most of which are hydrophobic. It allows for recognition by factors and protein receptors that mediate transport through the cell membrane; in this process, the signal sequence is cleaved by a peptidase (Figure 10-41). (A similar phenomenon exists for certain bacterial proteins that are secreted.) Moreover, several small peptide hormones, such as corticotropin (ACTH), result from the specific cleavage of a single, large polypeptide precursor.

Figure 10-41. Signal sequences.

Figure 10-41

Signal sequences. Proteins destined to be secreted from the cell have an amino-terminal sequence that is rich in hydrophobic residues. This signal sequence binds to the membrane and draws the remainder of the protein through the lipid bilayer. The signal (more...)

Protein splicing

An extraordinary process that splices out an internal segment of certain proteins has been described in a variety of organisms, including prokaryotes and eukaryotes. This internal segment is termed an intervening protein sequence, or IVPS. The essential facet of this process is the formation of a new peptide bond between the two sequences flanking the IVPS. This reaction is autocatalytic and can take place in vitro. Figure 10-42 depicts protein splicing in schematic form. Interestingly, all IVPS segments studied so far contain an endonuclease activity, although this activity is unrelated to the protein-splicing reaction.

Figure 10-42. Protein splicing results in the removal of an internal segment (IVPS) and the formation of a new peptide bond that links the two regions that originally flanked the IVPS.

Figure 10-42

Protein splicing results in the removal of an internal segment (IVPS) and the formation of a new peptide bond that links the two regions that originally flanked the IVPS.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK22022


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