Human Biochemistry, Volume 2 - Murray R. 1993

Structure, Function, and Replication of Informational Macromolecules
Protein Synthesis and the Genetic Code
The Process of Protein Synthesis

The core Structural Features of Ribosomes and The Mechanism of their self-assembly were discussed in Chapter 39. This specialized Structure acts as a "molecular machine" that translates The nucleotide sequence of mRNA into the Amino Acid Sequence of a protein molecule. Translation of mRNA begins at the 5' end, resulting in The formation of the N-terminus of the growing polypeptide chain. Information is read in the 5'→3' direction and terminates with the Formation of the C-terminus of the protein. This establishes the previously stated Concept of the polarity (unidirectionality) of Genetic information. As shown in Chapter 39, Gene METABOLISM/31.html">Transcription into the corresponding mRNA begins at the 5' end of the mRNA molecule. In prokaryotes, this allows translation to begin even before transcription is complete. In eukaryotes, transcription takes place in The Nucleus, whereas mRNA Translation occurs in the Cytoplasm. This compartmentalization prevents the simultaneous occurrence of transcription and translation, making the Processing of primary hnRNA transcripts into mature mRNA molecules an absolute necessity.

Protein Synthesis, much like DNA Replication and gene transcription, is divided into three stages: initiation, elongation, and termination.

Initiation (Fig. 40.7)

In most eukaryotic mRNAs, the 5' end is "capped" (Chapter 39). The cap consists of a methylguanosine triphosphate residue and is thought to be involved in binding the RNA molecule to the 40S ribosomal subunit. As a rule, translation starts with the AUG codon. The 18S ribosomal RNA (rRNA) of the 40S subunit binds to the region of the mRNA just preceding the first codon. Binding of mRNA to the 40S subunit requires a protein factor—initiation factor 3 (IF-3).

The first aminoacyl-tRNA involved in translating the initial codon interacts with GTP and initiation factor 2 (IF-2). In the presence of initiation factor 1 (IF-1), the resulting complex attaches the tRNA to the first codon of the template and forms the initiation complex with the 40S ribosomal subunit. Upon the release of the initiation factors (IF-1, IF-2, and IF-3), the 60S subunit joins the complex, coupled with the Hydrolysis of a GTP molecule. This completes the assembly of the intact 80S ribosomal particle.

A fully assembled ribosome contains two functional sites for interacting with tRNA molecules. The peptidyl site (P site) holds the growing polypeptide chain as a peptidyl-tRNA complexed with the most recently translated mRNA codon. The aminoacyl site (A site) holds the aminoacyl-tRNA paired with the corresponding mRNA codon. Following the formation of the initiation complex at the first codon, the aminoacyl-tRNA enters the nascent P site, leaving the A site vacant. Thus, the reading frame is set by the attachment of the first tRNA to the first translated codon. Recognition of this initiation codon apparently depends on the Introduction/11.html">Secondary structure of the mRNA molecule. In addition, specific Translation initiation in prokaryotes—and possibly in eukaryotes as well—involves a nucleotide sequence within the mRNA that is complementary to a segment of the 16S (18S) ribosomal RNA.

In prokaryotes, the initiation of synthesis for most, if not all, Proteins involves a specialized aminoacyl-tRNA—N-formylmethionyl-tRNA. In eukaryotes, methionyl-tRNA is not formylated, although Methionine itself serves as the N-terminal amino acid for the majority of proteins.

It is possible that in prokaryotes, N-formylation of the methionine residue on the tRNA mimics a peptide bond, thereby facilitating the translocation of the initiation complex into the peptidyl site. Prokaryotes also possess an enzyme that cleaves off the formyl group or the entire N-terminal formylmethionyl or methionyl residue from the protein. In many cases, this occurs even before translation is fully completed.

Elongation (Fig. 40.8)

In the 80S ribosome fully assembled during the initiation stage, the A site is unoccupied. The recruitment of the appropriate aminoacyl-tRNA to the A site requires precise codon recognition. Elongation factor 1 (EF-1) forms a complex with GTP and an aminoacyl-tRNA molecule, enabling the aminoacyl-tRNA to bind to the ribosome. This binding triggers the release of the EF-1-GDP complex and inorganic phosphate. As illustrated in Fig. 40.8, the EF-1-GDP complex is subsequently recycled back into EF-1-GTP through the action of other free protein factors and GTP.

The $\alpha$-amino group of the incoming aminoacyl-tRNA in the A site performs a nucleophilic attack on the esterified carboxyl group of the peptidyl-tRNA residing in the P site. This reaction is catalyzed by peptidyl transferase, a protein component of the 60S ribosomal subunit. Because The amino acid on the aminoacyl-tRNA is already "activated," this reaction requires no additional energy input. As a result of this reaction, the growing polypeptide chain becomes attached to the tRNA located in the A site.

Once the peptidyl moiety is removed from the tRNA in the P site, the free tRNA molecule rapidly departs. A complex of GTP with elongation factor 2 (EF-2) drives the translocation of the newly formed peptidyl-tRNA from the A site to the P site. This process involves the hydrolysis of GTP—acting as a cofactor for EF-2—into GDP and phosphate. Translocation shifts the newly formed peptidyl-tRNA and its corresponding codon into the P site, thus freeing up the A site for a new cycle of codon recognition by the next aminoacyl-tRNA and subsequent elongation.

Attaching an amino acid residue to a tRNA requires the hydrolysis of ATP to AMP, which is energetically equivalent to hydrolyzing two ATP molecules to ADP and phosphate. The delivery of aminoacyl-tRNA to the A site is accompanied by the hydrolysis of GTP to GDP. Similarly, the translocation of peptidyl-tRNA from the A site to the P site requires the hydrolysis of GTP to GDP and phosphate. Consequently, the energy required to form a single peptide bond is supplied by the hydrolysis of two ATP molecules to ADP and two GTP molecules to GDP, representing a total consumption of four high-energy phosphate bonds.

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Fig. 40.7. Scheme of protein synthesis initiation on an mRNA containing a 5'-capping structure and a 3'-poly(A) tail. IF-1, IF-2, and IF-3 denote initiation factors 1, 2, and 3, respectively. The hairpin structure with a Met residue at the end represents methionyl-tRNA. The letters P and A designate the binding sites on the ribosome for peptidyl-tRNA and aminoacyl-tRNA, respectively.

Fig. 40.8. Diagram of the protein synthesis elongation process. Circles labeled n-1, n, n+1, etc., represent amino acid residues of the synthesized protein molecule. EF-1 and EF-2 stand for elongation factors 1 and 2, respectively.

Termination (Fig. 40.9)

After numerous cycles of elongation that yield the polypeptide chain of the protein, a termination or nonsense codon appears in the A site. Under normal conditions, there are no tRNA molecules capable of recognizing nonsense codons. The appearance of a termination codon in the A site is recognized by so-called release factors (R factors). In the presence of GTP and peptidyl transferase, R factors mediate the hydrolysis of the bond between the polypeptide and the tRNA residing in the P site. Following this hydrolysis and the release of the synthesized polypeptide and tRNA, the 80S ribosome dissociates into 40S and 60S subunits, which can then be recycled to translate new mRNA molecules. Thus, release factors are proteins that hydrolyze peptidyl-tRNA upon encountering a nonsense codon in the A site.

A single mRNA molecule can be simultaneously translated by multiple ribosomes. Due to their considerable physical size, ribosomes maintain a spacing of at least 80 NUCLEOTIDES along the mRNA. Ribosomes positioned along the same mRNA molecule form structures known as polyribosomes, or Polysomes. In the absence of steric constraints, the number of ribosomes attached to an mRNA (and hence the size of the polysome) correlates with the length of the mRNA chain. The mass of the mRNA molecule itself is significantly smaller than that of even a single ribosome.

A single mammalian ribosome is capable of synthesizing approximately 100 peptide bonds per minute.

Polysomes actively engaged in protein synthesis may exist either as free particles or attached to the intracellular membrane network known as The Endoplasmic reticulum (ER). The numerous polysomes studding the membranes of the endoplasmic reticulum give it the "rough" appearance observed under an Electron microscope. Proteins synthesized on these bound polysomes are discharged into the space between the cisternae of the rough ER for subsequent export. For this purpose, certain protein products are packaged into zymogen granules via the Golgi apparatus (see Chapter 42). Cytoplasmic polysomes synthesize proteins required for intracellular Functions.

Protein Processing

Certain animal Viruses, particularly poliovirus (an RNA virus), synthesize large polycistronic proteins translated from a single extended mRNA transcript. These protein precursors are subsequently cleaved at specific sites to yield several distinct viral proteins. In animal Cells, many proteins are synthesized from RNA templates as precursor molecules that must undergo modification to become fully active. A prime example is Insulin, a low-molecular-weight protein composed of two polypeptide chains linked by intra- and interchain Disulfide Bonds. Insulin is synthesized as a single-chain precursor, preproinsulin/proinsulin. Subsequently, a specific protease excises the connecting peptide that links the two chains found in the mature, functionally active insulin molecule (see Figs. 51.4 and 51.5).

Many other Polypeptides are synthesized as proproteins that require further modification to acquire biological activity. Post-translational modification often involves the removal of N-terminal amino acid residues by specific aminopeptidases. Collagen—the primary Extracellular matrix protein in higher eukaryotes—is synthesized as procollagen. Three procollagen polypeptide chains (often non-identical in Primary Structure) align into a quaternary structure, a process guided by specific N-terminal Peptides. Specialized Enzymes direct the hydroxylation and oxidation of specific amino acid residues within the procollagen chains to form stabilizing cross-links. This is followed by the Cleavage of the amino-terminal peptides to yield the final product: a tough, insoluble collagen molecule (see Chapter 56). Numerous other post-translational protein modifications are also known; for instance, covalent modifications such as Acetylation, phosphorylation, and glycosylation are widespread.

Protein Synthesis Inhibitors

The ribosomes of Bacteria and of Cell/35.html">Mitochondria in higher Eukaryotic cells differ from the mammalian cell ribosomes described in Chapter 37. Bacterial ribosomes are smaller (70S instead of 80S) and contain a different, somewhat simpler set of RNAs and proteins. This distinction is widely exploited in clinical practice because many potent Antibiotics selectively interact with prokaryotic ribosomal proteins and inhibit bacterial protein synthesis. As a result, bacteria either perish or their growth is arrested. The best antibiotics of this class do not interact with the specific proteins of eukaryotic ribosomes and are thus non-toxic to eukaryotic organisms. Some of these are shown in bold in Table 40.2, which summarizes data on the effects of various antibiotics on protein synthesis.

Fig. 40.9. Scheme of the protein synthesis termination process. The letters P and A designate the binding sites on the ribosome for peptidyl-tRNA and aminoacyl-tRNA, respectively. The hydrolysis of the peptidyl-tRNA complex is illustrated as an attack by an H₂O molecule. To illustrate the directionality of the translation process, the N- and C-terminal Amino Acids are labeled with the letters N and C.

Table 40.2. Antibiotic Translation Inhibitors

+ — inhibition; - — no inhibition; * — stimulation; ? — unknown

There are antibiotics that inhibit protein synthesis across all ribosome types (such as puromycin) or exclusively in eukaryotic ribosomes (such as cycloheximide). Puromycin, whose structure is shown in Fig. 40.10, is a structural analog of tyrosyl-tRNA. It enters the ribosomal A site, incorporates into the C-terminal position of the growing polypeptide chain, and causes its premature dissociation, thereby terminating protein synthesis. As a tyrosyl-tRNA analog, puromycin effectively inhibits Protein synthesis in both PROKARYOTES AND EUKARYOTES.

Diphtheria toxin is an exotoxin produced by Corynebacterium diphtheriae cells lysogenic for a specific phage. This toxin catalyzes the ADP-ribosylation of EF-2 in mammalian cells. This modification inactivates EF-2 and consequently inhibits protein synthesis. Many animals (e.g., mice) are resistant to diphtheria toxin. This resistance is due to the inability of the toxin to penetrate The Cell membrane rather than to any resistance of murine EF-2 to toxin-catalyzed ADP-ribosylation.

Fig. 40.10. Comparison of the structures of the antibiotic puromycin and the 3'-terminal region of tyrosyl-tRNA.

Many of the aforementioned compounds, such as puromycin and cycloheximide, are not used in clinical practice. However, they have proven immensely valuable in experimental studies investigating The Role of protein synthesis in the Regulation of Metabolic processes, particularly hormone-induced enzyme induction.

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