LEHNINGER PRINCIPLES OF BIOCHEMISTRY - VOL 3. INFORMATION PATHWAYS - 2017

PART III. INFORMATION PATHWAYS

27. PROTEIN METABOLISM

27.2. Protein Synthesis

Earlier, when describing DNA and RNA Synthesis (Chapters 25 and 26), we showed that the synthesis of polymeric Biomolecules generally involves the stages of initiation, elongation, and termination. Typically, these core processes are supplemented by two additional stages: precursor activation prior to synthesis and post-synthetic Processing of the assembled polymers. Protein Synthesis follows the same pathway. The activation of Amino Acids prior to their incorporation into Polypeptides and the post-translational processing of the assembled polypeptide play critical roles in ensuring both the accuracy of synthesis and the normal functioning of the finished product. The cellular components involved in these five Stages of Protein Synthesis in E. coli and other Bacteria are listed in Table 27-5; Eukaryotic Cells have broadly similar requirements, although eukaryotes occasionally require additional components. First, let us examine the stages of protein synthesis as a whole, which will guide our subsequent Structure/133.html">Discussion.

Class="center">Table 27-5. Cellular components involved in the five major stages of Protein synthesis in E. coli

Protein synthesis occurs in five stages

Stage 1: Amino acid activation. The synthesis of a polypeptide with a specific sequence requires two conditions to be met: (1) the carboxyl group of each amino acid must be activated to facilitate peptide bond formation, and (2) there must be a precise correspondence between each incoming Amino Acid and the mRNA encoding it. Both requirements are met by attaching The amino acid to a tRNA During the first stage of protein synthesis. It is fundamentally important to couple the correct amino acid with the correct tRNA. This reaction takes place in the Cytoplasm, not on the ribosome. Each of the 20 amino acids is covalently linked to a specific tRNA at the expense of ATP energy, mediated by activating, Mg²⁺-dependent enzymes known as aminoacyl-tRNA synthetases. A tRNA linked to its corresponding amino acid (aminoacylated) is referred to as "charged".

Stage 2: Initiation. The polypeptide-encoding mRNA binds to the smaller of the two ribosomal subunits and to the initiator aminoacyl-tRNA. The larger ribosomal subunit then joins them, forming the initiation complex. The initiator aminoacyl-tRNA base-pairs with the AUG codon in the mRNA, which designates THE START OF the polypeptide. This process requires the consumption of GTP and is driven by cytoplasmic Proteins called initiation factors.

Stage 3: Elongation. The polypeptide is extended by the sequential covalent attachment of amino acids, which are delivered to the ribosome and precisely positioned by specific tRNA molecules via base-pairing with the corresponding codons in the mRNA. Elongation requires the participation of cytoplasmic proteins known as elongation factors. GTP Hydrolysis facilitates the binding of each incoming aminoacyl-tRNA molecule and the translocation of the ribosome along the mRNA.

Stage 4: Termination and release. The signal for the cessation of Polypeptide chain synthesis is a stop codon in the mRNA. The newly synthesized polypeptide is released from the ribosomal complex with the assistance of release factors, and the ribosome prepares for the Synthesis of the next protein.

Stage 5: Folding and post-translational processing. To attain its active form, the new polypeptide must fold into its specific three-dimensional conformation. Before or after proper folding, the polypeptide may undergo enzymatic modifications, such as the removal of one or more amino acids (typically from the N-terminus), the addition of acetyl, phosphoryl, methyl, carboxyl, or other groups to specific amino acid residues, proteolytic Cleavage, and/or the addition of Oligosaccharides or prosthetic groups.

Before proceeding to a detailed discussion of these five stages, two key elements in METABOLISM/35.html">Protein Biosynthesis deserve attention: Ribosomes and tRNA molecules.

The ribosome is a complex supramolecular machine

Each E. coli Cell contains 15,000 or more ribosomes, which account for up to a quarter of The Cell's dry mass. Bacterial ribosomes consist of approximately 65% rRNA and 35% protein; they are roughly 18 nm in diameter and comprise two unequal subunits with sedimentation coefficients of 30S and 50S (yielding a total sedimentation coefficient of 70S). Both subunits contain dozens of ribosomal proteins and at least one large rRNA molecule (Table 27-6).

Table 27-6. RNA and Protein Components of the E. coli ribosome

* The protein designations L1 through L36 do not correspond to 36 entirely different proteins. The protein originally designated L7 is actually a modified form of L12, and L8 is a complex of three other proteins. It has also been established that L26 and S20 are the same protein, and this protein is not a component of the 50S subunit. The large subunit contains 33 distinct proteins. Protein L7/L12 is present in four copies, which, together with three additional copies of other proteins, brings the total protein count to 36.

Ribosomes became the focus of intense study after Zamecnik established their role in PROTEIN SYNTHESIS AND following the deciphering of The Genetic Code. In the late 1960s, Masayasu Nomura and his colleagues demonstrated that both ribosomal subunits could be dissociated into their constituent RNA and protein components and subsequently reassembled in vitro. Under specific experimental conditions, spontaneous assembly of RNA and protein occurs, yielding 30S and 50S subunits that are virtually identical in structure and activity to native subunits. This breakthrough paved the way for decades of research into the function and structure of Ribosomal RNAs and proteins. Simultaneously, advances in structural biology Methods allowed the architecture of ribosomes to be studied in exquisite detail.

Box 27-2 From the RNA World to the Protein World

Currently known ribozymes typically catalyze one of Two Types of reactions: the hydrolytic cleavage of phosphodiester bonds or transphosphorylation (Chapter 26). In both cases, the substrates for these reactions are also RNA molecules. Ribosomal RNAs significantly expand the catalytic repertoire of ribozymes. As laboratory research into the catalytic Functions of RNA progresses (see Box 26-3), the hypothesis of an RNA world as a precursor to modern life forms is gaining increasing popularity.

A viable RNA world would need the capacity to replicate independently, possess a primitive metabolism to synthesize necessary ribonucleotide precursors, and maintain cellular boundaries to concentrate substrates and isolate them from the surrounding environment. Larger and more complex RNA catalysts may have emerged to mediate reactions among diverse metabolites and macromolecules. The high density of negatively charged phosphoryl groups in the RNA backbone decreases its stability, thereby imposing limits on the size of RNA molecules. In the RNA world, divalent cations or other positively charged groups may have been incorporated to maintain the stability of large molecules.

Certain Peptides could have facilitated the stabilization of large RNA molecules. For instance, many ribosomal proteins in modern Eukaryotic cells are long, structurally unstructured molecules that extend along rRNA molecules, thereby stabilizing them (Fig. 1). It is highly probable that The Emergence of ribozyme-catalyzed Peptide Synthesis originally served to stabilize The structure of large RNA molecules. Although peptide synthesis may have helped stabilize large ribozymes, this evolutionary refinement marked the beginning of the end for the RNA world. Once peptide synthesis became possible, the superior catalytic potential of proteins ensured an irreversible transition toward a protein-based metabolism.

Fig. 1. 50S subunit of the bacterial ribosome (PDB ID 1NKW). Blue strands represent protein molecules, while rRNA structural elements are shown transparent. Unstructured extended regions of many ribosomal proteins weave into the rRNA structures, helping to stabilize them

Eventually, proteins took over the majority of catalytic functions, though not all. In all modern organisms, the vital task of Protein synthesis is carried out with the help of ribozymes. Apparently, the active center of a ribozyme contains a single optimal combination of NUCLEOTIDES (or very few such combinations) that can participate in catalyzing peptide synthesis. Across all species of organisms, the rRNA residues of the large subunits—which are presumably involved in the peptidyl transferase activity of ribosomes—are highly conserved. Using the in vitro evolution method (SELEX; see Box 26-3), researchers have isolated artificial ribozymes capable of mediating peptide synthesis. Interestingly, most of these Biopolymers contain a highly conserved sequence of eight ribonucleotides (5') AUAACAGG (3'), which is also found in the peptidyl transferase center of ribosomes in all cells. It appears there is only one optimal solution to the general problem of chemical protein synthesis with a specific sequence using ribozymes. Evolution found this solution once, and it has not been improved upon in any life form.

At the turn of the new millennium, the structure of bacterial ribosomal subunits was investigated at very high resolution, bringing many surprises (Fig. 27-13). First, the traditional view regarding the predominant role of ribosomal protein components changed. Ribosomal subunits are represented by exceptionally large (giant) RNA molecules. The core of the 50S subunit is formed by the 5S and 23S rRNAs. In this complex, proteins are secondary, so to speak—they coat its surface. Second, and most importantly, at a distance of 18 Å from the active center, there is no protein capable of forming the peptide bond. High-resolution structural studies confirmed what had been hypothesized over a decade earlier: the ribosome is, in fact, a ribozyme. Furthermore, the elucidation of The Mechanism of protein synthesis (described below) and the Determination of the detailed STRUCTURE OF THE ribosome and its subunits have generated new Perspectives on the evolution of life (Box 27-2).

Fig. 27-13. Bacterial ribosome. We have made tremendous progress in understanding ribosome structure thanks to numerous high-resolution images of the bacterial ribosome and its subunits obtained by several research groups. Some of these structures are presented here: (a) The 50S and 30S subunits of the bacterial ribosome are separated to show the surfaces that interact in the active ribosome. The left side shows the structure of the 50S subunit (based on PDB ID 20W8, 1VSA, and 1GIX) with tRNA molecules (green) bound to the E, P, and A sites (described later in the text); tRNA anticodons are highlighted in red. Proteins are depicted as blue helices; rRNA is shown in gray, revealing its surface structure. The right side shows the structure of the 30S subunit (based on PDB ID 20W8). Proteins are depicted as brown helices; the rRNA is beige, showing its surface relief. The portion of mRNA interacting with the tRNA anticodons is colored red. The rest of the mRNA (not shown) extends through grooves or channels On the surface of the 30S subunit. (b) Active bacterial ribosome; bottom view of the groove separating the subunits (based on PDB ID 20W8, 1VSA, and 1GIX). All elements are colored as in part (a). (c) The ribosome is shown in the same perspective as in part (b), but all components are rendered in volume. On the right (part e), tRNA molecules are omitted to reveal the cavity where protein synthesis takes place. (d) The 50S subunit of the bacterial ribosome (PDB ID 1Q7Y). It is shown from the side that attaches to the 30S subunit. The active center where peptide bond formation occurs (As a result of peptidyl transferase activity) is located deep within the groove and far from all proteins; the inhibitor puromycin (red) is bound to the active center.

The bacterial ribosome has a complex structure, with a total molecular mass of ≈2.7 million Da. Two irregularly shaped ribosomal subunits are joined in such a way that a cleft is formed between them, through which the mRNA molecule passes during translation (Fig. 27-13, b). Bacterial ribosomes contain 57 proteins that vary greatly in Size and Structure, with molecular masses ranging from 6,000 to 75,000. Most proteins possess globular domains on The surface of the ribosome. Some proteins also feature extended unstructured tails that penetrate the rRNA-based core of the ribosome, stabilizing its structure. The function of some of these proteins remains unclear, but many appear to play a structural role.

The sequences of rRNA molecules have been determined for many organisms. Each of the three single-stranded rRNAs of E. coli has a specific three-dimensional conformation with extensive stretches of base-pairing within the chain. The predicted Introduction/11.html">Secondary structure of rRNA molecules (Fig. 27-14) has been largely confirmed by models, but it cannot account for the complex interactions within the tertiary structure.

Fig. 27-14. Bacterial rRNAs. Secondary structure of the 16S and 5S rRNAs of E. coli. The first (at the 5' end) and last (at the 3' end) ribonucleotide residues are marked (and numbered in the 16S rRNA—numbers in parentheses).

Eukaryotic cellular ribosomes (which differ from Mitochondrial and Chloroplast ribosomes) are larger and more complex than bacterial ribosomes (Fig. 27-15), with a diameter of ~23 nm and a sedimentation coefficient of ~80S. They also consist of two subunits, which vary in size among different species but average 60S and 40S. In total, eukaryotic ribosomes contain over 80 different proteins. Mitochondrial and chloroplast ribosomes are smaller and simpler than bacterial ribosomes. However, across all organisms and in all Organelles, the Structure and function of ribosomes are remarkably similar.

Fig. 27-15. Mass and composition of bacterial and eukaryotic ribosomes. Ribosomal subunits are characterized by sedimentation coefficients in Svedberg units (S), which reflect their rate of sedimentation during centrifugation. S values for ribosomes are not always additive, because the sedimentation rate is determined not only by their mass but also by their shape.

Transfer RNAs Have a Specific Structure

To understand how tRNA molecules can translate information from the language of Nucleic Acids into the language of proteins, we need to examine their structure in more detail. Transfer RNA molecules are relatively small and consist of a single RNA chain with a strictly defined three-dimensional structure (see Fig. 8-25, a). Bacterial and eukaryotic cytosolic tRNAs contain from 73 to 93 nucleotide residues, with a total molecular mass ranging from 24,000 to 31,000. Mitochondrial and chloroplast tRNAs are smaller in size. At least one type of tRNA is present in the cell for each amino acid. Recognizing the codons for all amino acids requires at least 32 Different types of RNA (some recognize more than one codon), but cells frequently employ more than 32 types of tRNA.

Yeast Alanine tRNA (tRNA^Ala; the first nucleic acid whose sequence was completely sequenced; Fig. 27-16) consists of 76 nucleotide residues, 10 of which contain modified bases. Comparison of tRNAs from different species has revealed many common structural features (Fig. 27-17). At least eight nucleotide residues contain modified bases and sugars, and many nucleotides are methylated derivatives of standard nucleotides. Most tRNA molecules carry a guanylate residue (pG) at the 5' end, and all tRNAs have a CCA (3') sequence at the 3' end. In Two-Dimensional Representations, the hydrogen-bonded structure of all tRNAs resembles a cloverleaf with four arms, while longer tRNA molecules feature a short additional fifth arm (Fig. 27-17). In three-dimensional representations, tRNA takes the shape of a twisted L (Fig. 27-18).

Fig. 27-16. Nucleotide sequence of yeast tRNA^Ala. This structure was determined in 1965 by Robert Holley and colleagues; the molecule is depicted in the cloverleaf conformation that achieves maximum base pairing. The following Abbreviations are used for modified nucleotides (on a pink Background): Ψ — pseudouridine; I — inosine; T — ribothymidine; D — 5,6-dihydrouridine; m¹I — 1-methylinosine; m¹G — 1-methylguanosine; m²G — N2-dimethylguanosine (see Fig. 26-23). Light blue lines between parallel fragments denote Watson-Crick Base Pairs. In RNA molecules, guanosine frequently pairs with uridine, although the G=U pair is not as stable as the G=C pair (Ch. 8 in Vol. 1). The anticodon can recognize three alanine codons (GCA, GCU, and GCC). Other Structural elements of tRNA are shown in Figs. 27-17 and 27-18.

Fig. 27-17. Schematic secondary structure of tRNA in the cloverleaf form. Dots within the sequence represent nucleotide residues; light blue lines denote base pairs. Nucleotides on a pink background are characteristic and/or invariant across all tRNAs. Transfer RNAs range in length from 73 to 93 nucleotides. Additional nucleotides form the D arm. At the end of the anticodon arm, a loop is formed that always contains seven unpaired nucleotides. The D arm in different tRNAs contains two or three D residues (5,6-dihydrouridine). In some tRNAs, only three base pairs of the D arm are hydrogen-bonded. The main symbols are defined in Fig. 27-16; Pu — purine nucleotide; Py — pyrimidine nucleotide; G* — guanylate or 2'-O-methylguanylate.

Fig. 27-18. Three-dimensional structure of yeast tRNA^Phe determined by X-ray crystallography. The shape of the molecule resembles an inverted and twisted letter L. (a) Schematic diagram of the molecule; the arms (Fig. 27-17) are highlighted in different colors. (b) Space-filling model with the same color coding (PDB ID 4TRA). The GCA sequence at the 3' end (orange) is the amino acid attachment site.

Two arms of tRNA are of fundamental importance for performing its adapter function. The amino acid arm carries a specific amino acid, whose carboxyl group is linked via an ester bond to the 2'- or 3'-hydroxyl group of the A residue at the 3' end of the tRNA. The anticodon arm contains the anticodon. Other major arms include the D arm, which contains the unusual nucleotide dihydrouridine D, and the TΨC arm, which contains ribothymidine T (typically absent in RNA molecules) and pseudouridine Ψ with an unusual carbon-carbon bond between the base and ribose (see Fig. 26-23). The D and TΨC arms provide essential interactions for tRNA folding, with the TΨC arm interacting with the larger ribosomal subunit rRNA.

Having examined the structure of ribosomes and tRNAs, we will now proceed to a detailed discussion of the five stages of protein synthesis.

Stage 1: Aminoacyl-tRNA synthetases attach specific amino acids to their corresponding tRNA molecules

In The First stage of protein synthesis, which takes place in the cytoplasm, aminoacyl-tRNA synthetases catalyze the Esterification of 20 amino acids with their cognate tRNAs. Each enzyme exhibits Specificity for a single amino acid and one or more tRNAs. In most organisms, a single aminoacyl-tRNA synthetase is responsible for each amino acid. If an amino acid corresponds to two or more tRNAs, their aminoacylation is typically carried out by the same enzyme.

The structures of all E. coli aminoacyl-tRNA synthetases have been determined. Based on substantial differences in their primary and tertiary structures as well as their catalytic mechanisms, it has been proposed to divide them into two classes of synthetases (Table 27-7; Fig. 27-19); both enzyme classes are found in all organisms. There is currently no evidence for a common evolutionary precursor, and the biological, chemical, and evolutionary reasons for having two distinct classes of enzymes catalyzing virtually identical processes remain unclear.

Aminoacyl-tRNA synthetase catalyzes the following reaction:

The reaction takes place at the Active Site of the enzyme in two stages. In stage ① (Fig. 27-19), an enzyme-bound intermediate, aminoacyl adenylate (aminoacyl-AMP), is formed. In the second stage, the aminoacyl group is transferred from the enzyme-bound aminoacyl-AMP to the corresponding specific tRNA. The pathway of the Second Stage depends on whether class I or class II synthetase is involved (reactions (2a) and (2b) in Fig. 27-19). The hydrolysis of the resulting ester bond between the amino acid and tRNA (Fig. 27-20) has a large negative standard free-energy change (∆G°′ = - 29 kJ/mol). The pyrophosphate produced in the activation reaction is subsequently hydrolyzed to inorganic phosphate by inorganic pyrophosphatase. Thus, two high-energy phosphate bonds are consumed for the activation of each amino acid, rendering the overall activation reaction irreversible:

Table 27-7. Two classes of aminoacyl-tRNA synthetases

Note. Abbreviations: Arg, arginyl-tRNA synthetase, etc. These two classes of enzymes have been found in all organisms studied; they possess distinct structures and operate via different catalytic mechanisms (see Fig. 27-19).

Figure 27-19. Reaction mechanism. Aminoacylation of tRNA by aminoacyl-tRNA synthetases. In stage ①, aminoacyl adenylate is formed and remains bound to the active site. In the second stage, the aminoacyl group is transferred to tRNA. The mechanism of this step differs slightly between the two classes of aminoacyl-tRNA synthetases (see Table 27-7). Class I aminoacyl-tRNA synthetase (2a) first transfers the aminoacyl group to the 2'-hydroxyl of the adenosine residue at the 3'-end of tRNA, followed by (3a) a transesterification to the 3'-hydroxyl group of the same residue. Class II aminoacyl-tRNA synthetase (2b) transfers the aminoacyl group directly to the 3'-hydroxyl group of the terminal adenylate.

Fig. 27-20. General structure of aminoacyl-tRNA molecules. The aminoacyl group is attached to the 3' position of the terminal A residue. The ester bond that activates the amino acid and links it to tRNA is highlighted in pink.

Proofreading function of aminoacyl-tRNA synthetases. Aminoacylation of tRNA serves two main purposes: (1) it activates the amino acid for peptide bond formation, and (2) it attaches the amino acid to its adapter tRNA, ensuring its correct positioning in the growing polypeptide chain. Because the attached amino acid is not recognized by the ribosome itself, ensuring that tRNA is linked to the correct amino acid is crucial for synthesizing a specific protein sequence.

As we discussed in Chapter 6 (Vol. 1), Enzyme Specificity is largely driven by enzyme-substrate binding energy. The ability to distinguish between two structurally similar amino acids has been extensively studied using Ile-tRNA synthetase, an enzyme that recognizes both valine and isoleucine—molecules that differ by a single methylene group (-CH₂-).

It appears that the methylene group (in Ile) enhances substrate binding (promoting Ile-AMP formation), allowing isoleucine to bind in the enzyme's active site and form Ile-tRNA synthetase roughly 200 times more frequently than valine. However, valine is mistakenly incorporated in place of isoleucine only about once in every 3,000 cycles. What boosts the enzyme's fidelity more than tenfold? Like several other aminoacyl-tRNA synthetases, Ile-tRNA synthetase possesses a proofreading (editing) activity.

Recall the general principle we explored when evaluating the proofreading activity of DNA polymerases (Table 25-1): when enzyme-substrate interactions with two different substrates are not sufficient for the enzyme to reliably differentiate between them, the required specificity can be achieved through a two-step mechanism. The Amplification of effect from using two sequential filters is multiplicative. In the case of Ile-tRNA synthetase, the first filter is the initial binding of the amino acid to the enzyme and its activation to aminoacyl-AMP. The second filter involves the binding of any incorrectly activated amino acids to a secondary active site on the enzyme, where the bound substrate undergoes hydrolysis. Because the R-group of valine is slightly smaller than that of isoleucine, Val-AMP can fit into the hydrolytic (editing) site of Ile-tRNA synthetase, whereas Ile-AMP cannot. As a result, Val-AMP bound at the editing site is cleaved into valine and AMP, preventing the tRNA attached to the enzyme from being misacylated with the incorrect amino acid.

In addition to proofreading activity, most aminoacyl-tRNA synthetases can hydrolyze the ester bond between the amino acid and tRNA within the aminoacyl-tRNA after aminoacyl-AMP has been formed. This hydrolysis occurs with particularly high efficiency on mischarged tRNA molecules, providing a third filter that further enhances the overall fidelity of the process. A few aminoacyl-tRNA synthetases (such as Cys-tRNA synthetase), which activate amino acids lacking close structural analogs, exhibit virtually no proofreading activity; in such cases, the aminoacylation active site alone is sufficiently precise to distinguish the correct substrate from any incorrect amino acid.

The overall error rate in protein synthesis (~1 error per 10⁴ incorporated amino acids) is significantly higher than that of DNA Replication. This is because damaged proteins are eventually degraded and do not pass on to subsequent generations. The fidelity of protein synthesis is high enough to ensure the correct assembly of most proteins without expending the massive amounts of energy that would otherwise be required. A single defective copy among many normal protein copies generally has negligible consequences.

Interaction between aminoacyl-tRNA synthetases and tRNA: The "second genetic code."

Each aminoacyl-tRNA synthetase is specific not only for a particular amino acid, but also for a specific set of tRNAs. Finding the correct tRNA among dozens of alternatives is just as crucial for the fidelity of protein synthesis as finding the correct amino acid. The interactions between aminoacyl-tRNA synthetases and tRNAs are often referred to as the "second genetic code," highlighting their vital role in ensuring translational accuracy. The rules governing this "second" code are more complex than those of the primary code.

Figure 27-21 summarizes data on the nucleotides essential for recognition by various aminoacyl-tRNA synthetases. Some nucleotides are conserved across all tRNAs and therefore do not play a role in selecting the correct sequences. By analyzing nucleotide variations in tRNAs that dictate the substrate specificity of aminoacyl-tRNA synthetases, researchers have identified the specific positions responsible for substrate discrimination. These positions are primarily clustered in the amino acid acceptor stem and the anticodon arm, including the anticodon sequence itself, though important determinants are also found in other Regions of the tRNA molecule. Determining the crystal structures of aminoacyl-tRNA synthetases has made an invaluable contribution to our understanding of the interactions involved in tRNA-ATP complex formation (Fig. 27-22).

Fig. 27-21. Nucleotide positions in tRNA molecules recognized by aminoacyl-tRNA synthetases. Certain positions (light blue dots) are conserved in all tRNA molecules and thus do not serve as distinguishing features. Other positions are recognized by a single synthetase (yellow) or by multiple synthetases (green). For some synthetases, recognition depends less on the specific tRNA sequence and more on other structural features.

Fig. 27-22. Aminoacyl-tRNA synthetases. Complexes of both synthetases with their corresponding tRNAs are shown (green backbone structures). Bound ATP (red) is located in the active site within the aminoacyl arm: (a) Gln-tRNA synthetase from E. coli, a typical example of a monomeric class I synthetase (PDB ID 1QRT); (b) Asp-tRNA synthetase from yeast, a typical example of a dimeric class II synthetase (PDB ID 1ASZ).

The recognition of a tRNA by its aminoacyl-tRNA synthetase involves 10 or more specific nucleotides. However, in some cases, the recognition mechanism is remarkably simple. Across all organisms, from bacteria to humans, a single base pair, G = U in the amino acid arm of tRNA^Ala, plays the primary role in tRNA recognition by Ala-tRNA synthetases (Fig. 27-23a). A short RNA consisting of just 7 bp that forms a simple hairpin ministem is successfully aminoacylated by Ala-tRNA synthetase as long as the key G = U base pair is present (Fig. 27-23b). Such a simple alanine recognition system likely dates back to a time when precursor RNA oligonucleotides underwent aminoacylation for protein synthesis in a primitive system.

The interaction of aminoacyl-tRNA synthetases with their cognate tRNAs plays a crucial role in the accurate reading of the genetic code. Any expansion of the code to include a new amino acid would inevitably require The formation of a new aminoacyl-tRNA synthetase/tRNA pair. In nature, expansion of the genetic code is rare and is more commonly achieved under laboratory conditions (Box 27-3).

Fig. 27-23. Structural elements of tRNA^Ala required for recognition by Ala-tRNA synthetase. (a) Structural elements of tRNA^Ala recognized by Ala-tRNA synthetase. The G = U base pair (highlighted in pink) is the sole element required for specific binding and aminoacylation. (b) A short RNA ministem containing the critical G = U base pair but lacking most of the tRNA Structure. This stem interacts specifically with Ala-tRNA synthetase almost as efficiently as the intact tRNA^Ala molecule.

Box 27-3. Natural and Artificial Expansion of the Genetic Code

As we have seen, the 20 Amino Acids Commonly Found in Proteins possess only limited chemical functionality. Living systems typically overcome these limitations by utilizing enzyme cofactors or modifying individual amino acids after their incorporation into proteins. Expanding the genetic code to incorporate novel amino acids offers a fundamentally different approach to altering protein functionality, though implementing this path is quite challenging. Such modifications could potentially inactivate thousands of cellular proteins.

Expanding the genetic code to include a new amino acid requires several cellular adaptations. Specifically, it necessitates a novel aminoacyl-tRNA synthetase along with a corresponding tRNA. Both components must be highly specific, interacting exclusively with each other and with the new amino acid. A sufficient concentration of the novel amino acid must also be present within the cell, which may require the emergence of new metabolic pathways. As highlighted in Box 27-1, the anticodon on the tRNA would most likely pair with a codon that normally signals Translation termination. While fulfilling all these conditions in a living cell seems unlikely, such scenarios do occur in nature and can be artificially engineered in the laboratory.

Currently, the genetic code is known to specify not 20, but 22 amino acids. The two additional Amino acids are selenocysteine and pyrrolysine; both are found in only a few proteins, yet they provide unique insights into the Evolution of the genetic code.

In all known cells, only a handful of proteins (such as bacterial formate dehydrogenase and mammalian Glutathione peroxidase) require selenocysteine for catalytic (enzymatic) activity. In E. coli cells, selenocysteine is co-translationally incorporated into formate dehydrogenase at THE POSITION OF an in-frame UGA codon. A specialized Ser-tRNA, present in lower amounts than other Ser-tRNAs, exclusively recognizes the UGA codon. This tRNA is charged with Serine by the standard serine aminoacyl-tRNA synthetase, and another enzyme subsequently converts the serine to selenocysteine prior to peptide bond formation on the ribosome. This charged tRNA does not recognize all UGA codons; yet-to-be-fully-understood signals within the mRNA direct this tRNA to recognize UGA codons in only specific genes, treating them as selenocysteine codons. In effect, UGA functions simultaneously as a stop codon and (very rarely) as a selenocysteine codon. This expansion of the genetic code required the evolution of a dedicated tRNA, though not a specific aminoacyl-tRNA synthetase. Given that selenocysteine is formed in this manner, one might speculate that this entire process represents an intermediate stage toward the evolution of an entirely new codon.

Pyrrolysine is found in a group of anaerobic archaea known as methanogens (see Box 22-1). The metabolic product of these microorganisms is methane, and some of them, belonging to the family Methanosarcinaceae, utilize methylamines as substrates for methanogenesis. The production of methane from monomethylamine requires a specific methyltransferase. The reading frame of this enzyme's Gene contains a UAG stop codon. The structure of this methyltransferase, elucidated in 2002, revealed that it contains the novel amino acid pyrrolysine encoded by this UAG codon. Subsequent experiments demonstrated that, unlike selenocysteine, pyrrolysine is directly attached to its cognate tRNA by pyrrolysyl-tRNA synthetase. The metabolic pathway for pyrrolysine synthesis in these cells remains to be fully elucidated. In this case, all the prerequisites for assigning a new function to the UAG codon are present, yet the UAG codon is translated as pyrrolysine in only one specific gene. As with selenocysteine, specific signals likely direct this tRNA to the correct UAG codon.

Can scientists replicate this evolutionary pathway artificially? Modifying proteins with diverse functional groups can significantly broaden our understanding of their activity and structure. However, protein modification often presents substantial challenges. For instance, if one wishes to attach a novel chemical group to a specific Cysteine residue, all other Cys residues in the protein must somehow be protected. But if the genetic code could be adapted to direct a cell to incorporate a modified residue at a precise position within a protein, working with such a system would become vastly more convenient. Peter Schultz and his colleagues pursued precisely this approach.

Creating a codon for a novel amino acid requires a new aminoacyl-tRNA synthetase and a corresponding new tRNA engineered to function exclusively with that novel amino acid. Researchers attempted to establish such an "artificial code" in E. coli cells. The UAG codon was selected to encode the new amino acid. This codon is used as a stop codon less frequently than the other two, and strains bearing tRNAs that suppress this codon (see Box 27-4) grow normally. To generate the new tRNA and tRNA synthetase, genes for tyrosyl-tRNA and its cognate tyrosyl-tRNA synthetase were taken from the archaeon Methanococcus jannaschii (Mj-tRNA^Tyr and Mj-tRNA^Tyr synthetase). Mj-tRNA^Tyr synthetase does not bind to the anticodon loop of Mj-tRNA^Tyr, which allowed the anticodon to be mutated to CUA (complementary to UAG) without disrupting the interaction. Archaeal and bacterial translation systems are orthologous, meaning the modified archaeal components can be introduced into E. coli cells without disrupting the bacteria's own translational machinery.

First, the Mj-tRNA^Tyr gene had to be modified to create an ideal tRNA that would not be recognized by endogenous E. coli aminoacyl-tRNA synthetases, yet would still be aminoacylated by Mj-tRNA^Tyr synthetase. To identify such a variant, a series of positive and negative Selection cycles was undertaken, enabling an efficient screening of tRNA gene variants (Fig. 1). The Mj-tRNA^Tyr gene was subjected to random mutagenesis to generate a library of clones expressing various tRNA versions. Additionally, the gene for barnase (a Ribonuclease toxic to E. coli), whose mRNA contained multiple UAG codons, was introduced into the bacterial cells on a plasmid. If a Mj-tRNA^Tyr variant expressed in a given cell from the library was aminoacylated by endogenous tRNA synthetases, it would also express the barnase gene, causing the cells to die (negative selection). Surviving cells were expected to harbor tRNA variants that escaped aminoacylation by endogenous tRNA synthetases while potentially remaining substrates for Mj-tRNA^Tyr synthetase. For positive selection (Fig. 1), an engineered β-lactamase gene (conferring resistance to the antibiotic ampicillin) containing multiple UAG codons in its transcript was used. This gene was introduced into the cells along with the Mj-tRNA^Tyr synthetase gene. Those Mj-tRNA^Tyr variants capable of being aminoacylated by Mj-tRNA^Tyr synthetase supported growth on ampicillin-containing media only when Mj-tRNA^Tyr synthetase was co-expressed. Several rounds of negative and positive selection led to the identification of a new Mj-tRNA^Tyr variant that was ignored by endogenous enzymes, was efficiently aminoacylated by Mj-tRNA^Tyr synthetase, and functioned properly in translation.

Fig. 1. Selection of Mj-tRNA^Tyr variants that interact exclusively with the specific tyrosyl-tRNA synthetase (Mj-tRNA^Tyr synthetase). Eleven point Mutations were introduced into non-interacting regions of the plasmid-borne Mj-tRNA^Tyr gene (red dots) without disrupting its interaction with Mj-tRNA^Tyr synthetase. Plasmids carrying the mutant gene were introduced into E. coli cells to create a library containing millions of Mj-tRNA^Tyr variants, represented here by six cells. For negative selection, a second plasmid encoding the cell-toxic barnase gene was introduced. A TAG sequence was engineered into this gene so that its transcript contained a UAG codon. Expression of the barnase gene leads to cell death. This gene could only be expressed if the Mj-tRNA^Tyr variant expressed by a given cell was aminoacylated by endogenous (i.e., E. coli) aminoacyl-tRNA synthetases, resulting in amino acid incorporation rather than translational termination. Furthermore, a modified β-lactamase gene—also containing a TAG sequence that generates a UAG stop codon upon Transcription—was introduced on yet another plasmid, which additionally carried the Mj-tRNA^Tyr synthetase gene. This system was used for the positive selection of surviving Mj-tRNA^Tyr variants. Variants that were successfully aminoacylated by Mj-tRNA^Tyr synthetase expressed the β-lactamase gene, enabling the cells to grow on ampicillin plates. After several cycles of positive and negative selection, the best Mj-tRNA^Tyr variants were isolated; these were aminoacylated exclusively by Mj-tRNA^Tyr synthetase and functioned efficiently in translation.

Second, the Mj-tRNA^Tyr synthetase had to be modified to recognize the novel amino acid. To generate a large library of variants, the Mj-tRNA^Tyr synthetase gene was subjected to mutagenesis. Variants that aminoacylated the new Mj-tRNA^Tyr variant using endogenous amino acids were eliminated through the barnase-based negative selection scheme. A secondary positive selection (similar to the ampicillin resistance screen) was then performed, ensuring that cells could survive only if the new Mj-tRNA^Tyr variant was aminoacylated exclusively in the presence of the non-natural amino acid. Following multiple cycles of negative and positive selection, a tRNA/synthetase pair was isolated that recognized only the unnatural amino acid. These molecules were renamed to reflect the non-natural Water/144.html">Origin of the amino acid used in their selection.

Using this approach, numerous E. coli strains have been engineered, each interpreting the UAG codon as a signal to incorporate a single, specific non-natural amino acid. The same strategy has been successfully applied to artificially expand the genetic code in yeast and even mammalian cells. Over 30 different Amino acids have been incorporated at designated positions within cloned proteins (Fig. 2). These achievements provide a highly valuable and versatile toolkit for probing Protein Structure and function in unprecedented detail.

Fig. 2. Structures of non-Natural Amino Acids whose codons have been added to the genetic code. These non-natural amino acids feature distinct chemical moieties: (a) a keto group; (b) an azide group; (c) a photo-cross-linking functional group capable of forming covalent bonds with neighboring groups upon light exposure; (d) a fluorescent amino acid; (e) an amino acid containing a heavy bromine atom for crystallographic studies; (f) a long-chain cysteine analog capable of forming extended disulfide bridges.

Stage 2: Protein synthesis is initiated by a specific amino acid

Protein synthesis begins at the N-terminus and proceeds by the sequential addition of amino acids to the C-terminus of the growing polypeptide chain, as established by Howard Dintzis in 1961 (Fig. 27-24). The initiation codon AUG corresponds to the N-terminal Methionine residue. Although there is only a single codon for methionine—(5') AUG—all organisms possess two distinct tRNAs for methionine. One is used exclusively for the (5') AUG initiation codon, while the other is used to incorporate internal Met residues throughout the polypeptide chain.

Fig. 27-24. Evidence that polypeptide chain growth proceeds by the addition of amino acid residues to the C-terminus: Dintzis's experiment. Reticulocytes (immature erythrocytes), which are cells actively synthesizing Hemoglobin, were incubated with radioactively labeled leucine (this amino acid was chosen because it occurs frequently in both α- and β-globins). At regular time intervals, fully formed α-chains were isolated from the reticulocyte preparation, and the radioactivity distribution within them was determined. The dark red areas indicate the fraction of fully formed α-globin chains containing radioactively labeled Leu residues. At 4 minutes, the label is found only in a few residues near the C-terminus of the α-globin chain, because after 4 min only those α-globins whose synthesis had already been completed by the time the label was added could have formed. As the incubation time increased, longer polypeptides appeared containing labeled residues, always located in the region toward the C-terminus of the chain. This demonstrated that the unlabeled end of the polypeptide (the N-terminus) is the initiation end, and the polypeptide chain grows by the sequential addition of amino acids to the C-terminus.

Distinguishing between the initiator and internal AUG codons is straightforward. Bacteria have two types of tRNA specific for methionine, designated as tRNA^Met and tRNA^fMet. The initiator (5') AUG codon corresponds to the amino acid N-formylmethionine (fMet). It is delivered to the ribosome as N-formylmethionyl-tRNA^fMet, which is formed via two consecutive reactions. First, methionine is attached to tRNA^fMet by the enzyme Met-tRNA synthetase (in E. coli cells, this enzyme catalyzes aminoacylation of both tRNA^Met and tRNA^fMet):

Methionine + tRNA^fMet + ATP —> Met-tRNA^fMet + AMP + PP_i

Next, a transformylase transfers the formyl group from N¹⁰-formyltetrahydrofolate to the amino group of the Met residue:

N¹⁰-formyltetrahydrofolate + Met-tRNA^fMet —> tetrahydrofolate + fMet-tRNA^fMet

Transformylase is more selective than Met-tRNA synthetase; this enzyme is specific for methionine residues attached to tRNA^fMet, apparently recognizing structural features specific to this tRNA. Met-tRNA^Met, in contrast, inserts methionine internally within polypeptide sequences.

The addition of the N-formyl group to the amino group of methionine by transformylase prevents the incorporation of fMet into internal positions of polypeptide molecules; however, fMet-tRNA^fMet can bind to the specific initiation site on the ribosome, which does not bind either Met-tRNA^Met or any other aminoacyl-tRNAs.

In eukaryotic cells, all polypeptides synthesized by cytoplasmic ribosomes begin with a Met residue (rather than fMet), yet these cells also utilize a specialized initiator tRNA that differs from the tRNA^Met used for internal AUG codons in mRNA. Polypeptides synthesized on mitochondrial and chloroplast ribosomes begin with N-formylmethionine. This strongly Supports the theory that Mitochondria and Chloroplasts originated from bacterial ancestors that were incorporated into the progenitor cells of modern eukaryotes at an early stage of evolution (see Fig. 1-36 in Vol. 1).

How does a single (5') AUG codon determine whether a starting N-formylmethionine (or methionine in eukaryotes) or an internal methionine residue will be incorporated at a given position? The answer lies in the mechanism of the initiation process.

Three stages of initiation.

In bacteria, initiation of polypeptide synthesis requires (1) the 30S ribosomal subunit, (2) the mRNA of the peptide to be synthesized, (3) initiator fMet-tRNA^fMet, (4) a set of three proteins called initiation factors (IF-1, IF-2, and IF-3), (5) GTP, (6) the 50S ribosomal subunit, and (7) Mg²⁺ ions. The Formation of the initiation complex occurs in three stages (Fig. 27-25).

Fig. 27-25. Formation of the initiation complex in bacteria. The complex forms in three stages (described in the text) using energy derived from the hydrolysis of GTP to GDP and P_i. IF-1, IF-2, and IF-3 are initiation factors. P stands for the peptidyl site, A for the aminoacyl site, and E for the exit site. The tRNA anticodon is oriented in the 3' —> 5' direction (from left to right), as in Fig. 27-8, but in the opposite direction compared to its orientation in Figs. 27-21 and 27-23.

In stage ①, the 30S ribosomal subunit binds two initiation factors, IF-1 and IF-2. Factor IF-3 prevents the premature association of the 30S and 50S subunits. Next, the mRNA binds to the 30S subunit. The initiator (5') AUG codon is positioned correctly thanks to the Shine-Dalgarno sequence (named after the Australian researchers John Shine and Lynn Dalgarno who identified it) in the mRNA. This consensus sequence of 4–9 purine residues is located 8–13 bp upstream (toward the 5' end) of the initiator codon (Fig. 27-26a). This sequence pairs with a complementary pyrimidine-rich sequence located near the 3' end of the 16S rRNA of the 30S ribosomal subunit (Fig. 27-26b). The interaction between mRNA and rRNA positions the initiator (5') AUG sequence of the mRNA precisely at a specific site on the 30S subunit. The (5') AUG sequence to which fMet-tRNA^fMet must bind is distinguished from other methionine codons by its proximity to the Shine-Dalgarno sequence in the mRNA.

Fig. 27-26. mRNA sequences that serve as signals for the initiation of protein synthesis in bacteria. (a) The correct positioning of the initiator AUG codon (highlighted in green) within the 30S ribosomal subunit depends in part on the upstream Shine-Dalgarno sequence (highlighted in pink). Fragments of mRNA transcripts from five bacterial genes are shown. Note that the E. coli LacI protein begins with a GUG (Val) codon (see Box 27-1). (b) The Shine-Dalgarno sequence in the mRNA base-pairs with a sequence near the 3' end of the 16S rRNA.

Bacterial ribosomes have three aminoacyl-tRNA binding sites: the aminoacyl (A) site, the peptidyl (P) site, and the exit (E) site. Aminoacyl-tRNA molecules bind to the A and P sites, whereas uncharged tRNA binds exclusively to the E site. The A and P sites are formed by both ribosomal subunits (30S and 50S), while the E site is located primarily on the 50S subunit. The initiator (5') AUG codon is situated in the peptidyl site—this is the only place where fMet-tRNA^fMet can bind (Fig. 27-25). The only aminoacyl-tRNA that binds initially to the P site is fMet-tRNA^fMet. During elongation, all other incoming aminoacyl-tRNA molecules (including Met-tRNA^Met, which binds only to internal AUG codons) bind first to the A site and only subsequently to the P and E sites. "Unloaded" tRNAs are released from the E site during the elongation phase. Factor IF-1 binds to the A site, preventing any tRNA from binding to this site during initiation.

During initiation stage ②, GTP and initiator fMet-tRNA^fMet join the complex consisting of the 30S ribosomal subunit, IF-3, and mRNA (Fig. 27-25). The anticodon of this tRNA then base-pairs with the initiator codon of the mRNA.

In stage ③, this large complex associates with the 50S ribosomal subunit; simultaneously, the GTP bound to IF-2 is hydrolyzed to GDP and P_i, which are released from the complex. At this point, all three initiation factors dissociate from the ribosome.

The completion of these steps (Fig. 27-25) results in the formation of a functional 70S ribosome, termed the initiation complex, which contains the mRNA and initiator fMet-tRNA^fMet. The correct binding of fMet-tRNA^fMet at the P site in the 70S initiation complex is ensured by at least three recognition points: codon-anticodon interaction involving the locking of the initiator AUG codon into the P site; base-pairing between the Shine-Dalgarno sequence in the mRNA and the 16S rRNA; and the interaction between the ribosomal P site and fMet-tRNA^fMet. The initiation complex is now ready for elongation.

Initiation in eukaryotic cells.

Translation in eukaryotic and bacterial cells proceeds in a similar manner; most differences lie in the initiation mechanism. Eukaryotic mRNAs interact with the ribosome as a complex with several specific binding proteins. Some of these proteins bring the 5' and 3' ends of the RNA template together. At the 3' end, mRNA is bound by a poly(A)-binding protein (PAB). Eukaryotic cells contain at least nine initiation factors. The eIF4F complex, which includes the proteins eIF4E, eIF4G, and eIF4A, binds to the 5' cap (see Fig. 26-13) via eIF4E. The eIF4G protein interacts with both eIF4E and PAB, effectively linking them together (Fig. 27-27). The eIF4A protein possesses RNA helicase activity. The eIF4F complex associates with another factor, eIF3, and with the 40S ribosomal subunit. Translation efficiency depends on many Properties of the mRNA and the proteins of this complex, including the length of the 3' poly(A) tail (in most cases, the longer it is, the better). The juxtaposition of the ends of eukaryotic mRNA facilitates the translational Regulation of Gene Expression (see Chapter 28).

Fig. 27-27. The Role of proteins in the formation of the eukaryotic initiation complex. The 3' and 5' ends of eukaryotic mRNA are bound to a protein complex consisting of several initiation factors and a poly(A)-binding protein (PAB). Factors eIF4E and eIF4G are part of a larger eIF4F complex. This complex binds to the 40S ribosomal subunit.

The initiation codon (5') AUG in mRNA is recognized not by its proximity to the Shine-Dalgarno sequence, but through scanning the mRNA from the 5' end until the first AUG codon is encountered, which designates the start of the reading frame. This process likely involves the eIFIF complex, which utilizes the RNA helicase activity of eIF4 to resolve secondary structures within the 5'-untranslated region of the mRNA. The eIF4B protein also facilitates this scanning process.

The functions of various bacterial and eukaryotic initiation factors are summarized in Table 27–8. The MECHANISM OF ACTION of these proteins is a subject of active investigation.

Table 27–8. Protein Factors Required for Translation initiation in Bacterial and Eukaryotic Cells

Stage 3: Peptide bonds are formed during the elongation phase

The Third Stage of protein synthesis is elongation. To begin with, we will once again focus on bacterial cells. Elongation requires (1) the initiation complex described above, (2) aminoacyl-tRNA, (3) three soluble cytoplasmic proteins termed elongation factors (EF-Tu, EF-Ts, and EF-G in bacteria), and (4) GTP. Within the cell, the addition of each amino acid occurs in three distinct steps, which are repeated for every amino acid residue added to the chain.

First step of elongation: Binding of aminoacyl-tRNA.

In the first step of the elongation cycle (Fig. 27–28), the appropriate aminoacyl-tRNA interacts with EF-Tu complexed with GTP. The aminoacyl-tRNA–EF-Tu–GTP complex binds to the A site of the 70S initiation complex. The GTP molecule is then hydrolyzed, and the EF-Tu–GDP complex is released from the 70S ribosome. Subsequently, the EF-Tu–GTP complex is regenerated through the action of the EF-Ts factor and GTP.

Figure 27–28. First step of bacterial elongation: binding of the second aminoacyl-tRNA. The second aminoacyl-tRNA binds to the ribosomal A site in association with EF-Tu (referred to here simply as Tu), which is bound to GTP. The binding of the second aminoacyl-tRNA at the A site is accompanied by the hydrolysis of GTP to GDP and P_i, as well as the release of the EF-Tu–GDP complex from the ribosome. The bound GDP is released when the EF-Tu–GDP complex interacts with EF-Ts, and EF-Ts is released when another GTP molecule binds to EF-Tu. As a result, the EF-Tu factor is primed for the next elongation cycle.

Second step of elongation: Peptide bond formation.

At this stage, a peptide bond is formed between Two amino acids linked via their respective tRNAs to the A and P sites of the ribosome. The initiator N-formylmethionyl group is transferred from its tRNA to the amino group of the second amino acid located in the A site (Fig. 27–29). In this reaction, the α-amino group of the amino acid in the A site acts as a nucleophile, displacing the tRNA from the P site and forming a peptide bond. Consequently, a dipeptidyl-tRNA is formed at the A site, while the "discharged" (deacylated) tRNA^fMet remains bound to the P site. The tRNA molecules then shift into a hybrid state, occupying positions where each is partially located in two ribosomal sites, as illustrated in Figure 27–29.

Figure 27–29. Second step of bacterial elongation: formation of the first peptide bond. The peptidyl transferase activity responsible for this reaction resides in the 23S rRNA ribozyme. During the reaction, the N-formylmethionyl group is transferred to the amino group of the second aminoacyl-tRNA, yielding a dipeptidyl-tRNA. Both tRNAs remain bound to the ribosome while shifting to occupy intermediate positions on the 50S ribosomal subunit. The discharged tRNA shifts such that its 3' and 5' ends reside in the E site. Similarly, the 3' and 5' ends of the peptidyl-tRNA move into the P site, while their anticodons remain in the A and P sites.

Historically, the enzymatic activity responsible for peptide bond formation was termed peptidyl transferase and was thought to reside in one or more Proteins of the large ribosomal subunit. We now know that this reaction is catalyzed by the 23S rRNA (Fig. 27–13d), making it yet another ribozyme. This discovery is particularly fascinating as it offers insight into evolutionary relationships (see Box 27–2).

Third step of elongation: Translocation.

In the final step of the elongation cycle, known as translocation, the ribosome moves exactly one codon toward the 3' end of the mRNA (Fig. 27–30a). Concurrently, the anticodon of the dipeptidyl-tRNA—which remains attached to the second mRNA codon—shifts from the A site to the P site, while the deacylated tRNA moves from the P site to the E site, from which it is released into the Cytosol. The third mRNA codon now occupies the A site, and the second codon occupies the P site. Movement of the ribosome along the mRNA requires the elongation factor EF-G (translocase) and The energy released from the hydrolysis of another GTP molecule. Alterations in the three-dimensional conformation of the ribosome drive its movement along the mRNA molecule. Because the structure of EF-G mimics that of the EF-Tu–tRNA complex (Fig. 27–30b), it is likely that EF-G binds to the A site and displaces the peptidyl-tRNA.

Figure 27–30. Third step of bacterial elongation: translocation. (a) The ribosome shifts by one codon toward the 3' end of the mRNA, utilizing the energy derived from the hydrolysis of GTP bound to EF-G (translocase). The dipeptidyl-tRNA is fully shifted to the P site, and the A site is vacated to receive the next (third) aminoacyl-tRNA. The discharged tRNA dissociates from the E site, and the elongation cycle begins anew. (b) The structure of EF-G is analogous to that of EF-Tu in complex with tRNA. Left: EF-Tu bound to tRNA (green) (PDB ID 1B23); right: EF-G bound to GDP (red) (PDB ID 1DAR). The C-terminal domain of EF-G (dark gray) resembles the anticodon arm of tRNA in both shape and charge distribution.

Following translocation, the ribosome—with its attached dipeptidyl-tRNA and mRNA—is primed for the next elongation cycle and the binding of the third amino acid residue. This process proceeds identically to the addition of the second residue (Figs. 27–28, 27–29, and 27–30), with the ribosome advancing codon by codon along the mRNA toward the 3' end. The addition of each amino acid residue to the growing polypeptide chain is coupled to the hydrolysis of two GTP molecules to GDP and P_i.

The polypeptide remains tethered to tRNA via its most recently added amino acid, thereby preserving The Link Between the Genetic information in the mRNA and the synthesized polypeptide. The ester bond between the tRNA and the C-terminus of the growing polypeptide is thus positioned for a nucleophilic attack by the carboxyl group of the incoming amino acid, resulting in the formation of a new peptide bond (Fig. 27–29). While peptide bond formation severs the ester linkage between the polypeptide and the tRNA, The connection between the polypeptide and the mRNA blueprint is maintained because each new amino acid is delivered by its specific tRNA.

The eukaryotic elongation cycle closely mirrors that of bacteria. Functionally, the three eukaryotic elongation factors—eEF1α, eEF1βγ, and eEF2—are homologous to the bacterial elongation factors EF-Tu, EF-Ts, and EF-G, respectively. Eukaryotic ribosomes lack a distinct E site; instead, discharged tRNA molecules dissociate directly from the P site.

Ribosomal proofreading.

During the first step of elongation in bacterial cells (Fig. 27–28), the GTPase activity of EF-Tu plays a major role in determining the speed and fidelity of the entire biosynthesis process. Both the EF-Tu–GTP and EF-Tu–GDP complexes persist for only a few milliseconds before dissociating; within this brief window, codon-anticodon recognition is double-checked. Typically, an incorrectly paired aminoacyl-tRNA is rejected from the A site during the initial check. If the nonhydrolyzable GTP analog guanosine-5'-O-(3-thiotriphosphate) (GTPγS) is substituted for GTP, hydrolysis is delayed, which increases translational accuracy (due to an extended proofreading window) at the expense of overall reaction velocity.

Over the course of evolution, protein synthesis (codon-anticodon pairing) has achieved an optimal balance between speed and fidelity (e.g., the accuracy of the Amino Acid Sequence or base pairing). High fidelity can slow down the speed, and high speed can compromise fidelity. Recall also that the proofreading mechanism on the ribosome checks only the accuracy of codon-anticodon pairing, not the choice of the amino acid attached to the tRNA. If a tRNA has been charged with the wrong amino acid (which can be done experimentally), that amino acid will be successfully incorporated into the protein sequence at the position specified by the codon recognized by that tRNA.

Stage 4: Polypeptide synthesis termination requires a specific signal

Elongation continues until the ribosome adds the final amino acid encoded by the mRNA. The transition to The final stage of protein biosynthesis, termination, is signaled by one of three stop codons (UAA, UAG, UGA) located immediately after the codon for the last amino acid. Mutations in the tRNA anticodon that allow an amino acid to be inserted in response to a stop codon are usually lethal to the cell (Box 27-4).

In bacteria, once a stop codon occupies the ribosomal A site, three termination factors, or release factors,—proteins RF-1, RF-2, and RF-3—come into play. They participate in (1) the hydrolysis of the terminal ester bond of peptidyl-tRNA; (2) the release of the free polypeptide and the last deacylated tRNA molecule from the P site; and (3) the dissociation of the 70S ribosome into 30S and 50S subunits, which are then ready to initiate a new cycle of polypeptide synthesis (Fig. 27-31). Factor RF-1 recognizes the stop codons UAG and UAA, whereas RF-2 recognizes UGA and UAA. Either RF-1 or RF-2 (depending on the stop codon) binds to the stop codon; then, catalyzed by peptidyl transferase, the growing polypeptide is linked to a water molecule rather than another amino acid. Apparently, termination factors contain domains that mimic the structure of tRNA, as shown in Figure 27-30b for the elongation factor EF-G. The specific function of RF-3 has not yet been definitively established, but it is thought to assist in the release of the ribosomal subunit. In eukaryotes, a single termination factor, eRF, recognizes all three stop codons.

The release and breakdown of ribosomes into subunits leads to the dissociation of the translational complex components. The termination factors leave the complex (along with the deacylated tRNA in the P site) and are replaced by EF-G and the so-called ribosome recycling factor (RRF; M_r 20,300). GTP hydrolysis driven by EF-G causes the 50S subunit to leave the 30S-tRNA-mRNA complex. EF-G and RRF are then replaced by IF-3, which promotes the release of the tRNA. Next, the mRNA dissociates. Following this, the IF-3–30S subunit complex is ready to initiate a new round of protein synthesis (Fig. 27-25).

Box 27-4. Induced Variations in the Genetic Code: Nonsense Suppression

If a mutation within a gene creates a stop codon, translation terminates prematurely, and the unfinished polypeptide is typically inactive. Such mutations are called nonsense mutations. The normal function of a gene can be restored by a secondary mutation that either (1) converts the erroneous stop codon back into an amino acid-coding codon, or (2) suppresses The Effect of the stop codon. Such compensatory mutations are known as nonsense suppressors; they typically occur in tRNA genes and result in the production of altered (suppressor) tRNA molecules capable of recognizing the stop codon and inserting an amino acid at that site. The anticodons of most known suppressor tRNA molecules carry a single-base substitution.

Suppressor tRNAs create experimentally induced variations in the genetic code by allowing readthrough of stop codons, analogous to the natural variations in the code described in Box 27-1. Nonsense suppressors do not completely block the normal flow of genetic information in the cell because cells typically contain multiple copies of each tRNA gene; some of these copies are weakly expressed and constitute only a minor fraction of the cellular pool for a given tRNA. Suppressor mutations usually affect these "minor" tRNA copies, while the major tRNAs continue to read the corresponding codon normally.

For example, E. coli has three identical genes for tRNA^Tyr, each of which synthesizes a tRNA with the (5′)GUA anticodon. One of these genes is expressed relatively intensely, and its product represents the major type of tRNA^Tyr; the other two Genes are transcribed very weakly. A mutation in the anticodon of one of these two minor products changes (5′)GUA to (5′)CUA, resulting in the synthesis of a minor tRNA^Tyr that inserts Tyrosine at UAG stop codons. Although the insertion of tyrosine at the UAG codon occurs with low efficiency, it produces a sufficient amount of full-length protein from the mutated gene to allow the cell to survive. The major tRNA^Tyr continues to translate the genetic code normally for the synthesis of most proteins.

A mutation that generates a suppressor tRNA does not always affect the anticodon. Suppression of UGA nonsense codons typically occurs in a tRNA^Tyr that normally recognizes UGG. The alteration that enables the reading of UGA (and the insertion of Trp residues at that position) is a G-to-A substitution at position 24 (in the distant stem-loop region of the tRNA); this modified tRNA can now recognize both UGG and UGA codons. A similar alteration has been found in tRNAs involved in the most common natural Modification of the genetic code (UGA = Trp; see Box 27-1).

Although suppression can lead to the production of abnormally long proteins, this does not always happen. Currently, only some details of the molecular events occurring during translation termination and nonsense suppression are known.

The Energetic Cost of Protein Synthesis Fidelity (Accuracy).

Protein synthesis based on the information encoded in mRNA requires an investment of energy. The formation of each aminoacyl-tRNA consumes two high-energy phosphate groups. Each time an incorrectly activated amino acid is hydrolyzed by the proofreading (deacylating) activity of aminoacyl-tRNA synthetase, an additional ATP molecule is consumed. One GTP molecule is cleaved to GDP and P_i during the first stage of elongation, and another during translocation. Thus, the formation of each peptide bond in a polypeptide requires the hydrolysis energy of at least four NTP molecules to NDP.

Fig. 27-31. Termination of protein synthesis in bacteria. Termination is triggered by a stop codon in the A site. First, a RF termination factor (RF-1 or RF-2, depending on the stop codon) binds to the A site. This leads to the hydrolysis of the ester bond between the polypeptide and the tRNA in the P site, releasing the completed polypeptide. At the end of mRNA, the deacylated tRNA and the termination factor leave the ribosome, which then dissociates into 30S and 50S subunits with the assistance of ribosome recycling factor (RRF), initiation factor IF-3, and the energy released by EF-G-mediated GTP hydrolysis. The complex of the 30S subunit with IF-3 is then ready to begin the next translation cycle (see Fig. 27-25).

This provides a more than adequate thermodynamic driving force for the synthesis process: at least the energy of four phosphodiester bonds goes into forming a single peptide bond, whose Standard Free energy of hydrolysis is only about -21 kJ/mol: 4 × 30.5 kJ/mol = 122 kJ/mol. Thus, the overall change in free energy for the peptide bond synthesis reaction is -101 kJ/mol. Proteins are informational biopolymers. The biochemical purpose of peptide synthesis reactions is not simply to form just any peptide bond, but to form a bond between two specific amino acids. Each of the energy-rich phosphate compounds consumed in this process plays a crucial role in ensuring the correct match between each new codon in the mRNA and the corresponding amino acid in the growing polypeptide. The energy expended ensures the exceptionally high fidelity of translating genetic information from mRNA into the amino acid sequence of a protein.

Rapid Translation of a Single Template by Polysomes.

Both bacterial and eukaryotic cells can yield large clusters of 10 to 100 ribosomes that exhibit high protein-synthesizing activity. Electron micrographs reveal that ribosomes form clusters—polysomes—in which adjacent ribosomes are connected by thin fibers (Fig. 27-32). These fibers are mRNA molecules that are being translated simultaneously by many closely spaced ribosomes, which significantly increases the efficiency of the process.

Fig. 27-32. Polysomes. (a) Four ribosomes simultaneously translating a single eukaryotic mRNA molecule, moving from the 5′ to the 3′ end and synthesizing the polypeptide from the N-terminus to the C-terminus. (b) Electron micrograph and accompanying interpretive diagram of a polysome from the silk gland of a silkworm larva. The mRNA is being translated simultaneously by multiple ribosomes. The growing polypeptide elongates as the ribosomes move toward the 3′ end of the mRNA. The ultimate product of this process is a silk fiber.

In bacteria, transcription and translation are closely coupled. mRNA molecules are synthesized and translated in the same direction (5′ → 3′). Ribosomes begin translating the 5′ end of the mRNA even before transcription is complete (Fig. 27-33). A completely different situation is observed in eukaryotic cells, where newly synthesized mRNA molecules must first leave The Nucleus before they can be used for protein synthesis.

Fig. 27-33. Coupling of transcription and translation in bacteria. Ribosomes begin translating the mRNA even before RNA polymerase has finished transcribing it from the DNA template. This is possible because bacterial mRNA does not need to be transported from the nucleus to the cytoplasm to encounter ribosomes. In the diagram, ribosomes are depicted as smaller than RNA polymerase. In reality, ribosomes (M_r = 2.7 × 10⁶) are an order of magnitude larger than RNA polymerase (M_r = 3.9 × 10⁵).

Bacterial mRNA molecules typically have a half-life of only a few minutes (Section 26.2) before they are degraded by Nucleases. To maintain high rates of protein synthesis, each mRNA must be continuously synthesized and translated with maximum efficiency. The short lifespan of bacterial mRNA allows the cell to rapidly shut down synthesis when a particular protein is no longer needed.

Stage 5: Newly synthesized polypeptides fold and undergo processing

At the final stage of protein synthesis, the newly formed polypeptide chain folds into its biologically active shape. During or after synthesis, the polypeptide gradually acquires its native conformation through the formation of Hydrogen Bonds, as well as Van der Waals, ionic, and hydrophobic interactions. As a result, the linear—i.e., one-dimensional—genetic information of mRNA is translated into the three-dimensional structure of a protein. Certain Newly synthesized proteins in bacteria, archaea, and eukaryotes do not assume their final biologically active conformation until they undergo specific processing reactions known as post-translational modifications.

N-terminal and C-terminal modifications.

The first amino acid residue in all polypeptides is N-formylmethionine (in bacteria) or methionine (in eukaryotes). However, during the formation of a functional protein, the formyl group, the N-terminal methionine residue, and several other N-terminal residues (and sometimes C-terminal residues) may be enzymatically removed. In approximately 50% of eukaryotic proteins, the amino group of the N-terminal residue undergoes post-translational N-acylation. C-terminal residues are also occasionally modified.

Removal of the signal sequence.

As we will see in Section 27.3, in some proteins an N-terminal sequence of 15–30 residues is required to direct the protein to its cellular destination. Such signal sequences are cleaved by specific peptidases.

Modification of specific amino acids.

The hydroxyl groups of certain Ser, Thr, and Tyr residues in specific proteins undergo enzymatic phosphorylation driven by ATP (Fig. 27-34, a); phosphate groups impart a negative charge to polypeptides. The Functional Significance of these modifications varies among different proteins. For instance, the milk protein casein contains numerous phosphate groups that bind Ca²⁺ ions. Calcium, phosphate, and amino acids are three essential nutritional components for newborns. Furthermore, as we have seen in numerous Examples, phosphorylation–dephosphorylation cycles regulate The activity of many enzymes and regulatory proteins.

Fig. 27-34. Several modified amino acid residues. a — Phosphorylated Amino Acids, b — carboxylated amino acid, c — certain methylated amino acids.

Additional carboxyl groups can be attached to Glu residues in certain proteins. For example, the Blood-clotting protein prothrombin contains several γ-carboxyglutamate residues in its N-terminal region (Fig. 27-34, b) attached by a vitamin K-dependent enzyme. These carboxyl groups bind the Ca²⁺ ions required to initiate the clotting mechanism.

Monomethyl- and dimethyllysine residues are present in certain Muscle Proteins and in cytochrome c (Fig. 27-34, c). In most species, the protein calmodulin contains a single trimethyllysine residue at a specific position in the polypeptide chain. In other proteins, the carboxyl groups of certain Glu residues undergo methylation, which neutralizes the negative charge of the proteins.

Addition of carbohydrate side chains.

Carbohydrate side chains of Glycoproteins are attached by covalent bonds during or after polypeptide synthesis. In some glycoproteins, carbohydrate side chains are attached enzymatically to Asn residues (N-linked oligosaccharides), whereas in others they are attached to Ser or Thr residues (O-linked oligosaccharides) (see Fig. 7-29 in Vol. 1). Many extracellular proteins, as well as Proteoglycans of mucosal cell membranes, contain oligosaccharide side chains (see Fig. 7-27).

Addition of isoprenyl groups.

Certain eukaryotic proteins are modified by the addition of isoprenyl groups (isoprene derivatives). A thioether bond is formed between the isoprenyl group and a Cys residue in the protein (see Fig. 11-14 in Vol. 1). Isoprenyl groups are derived from pyrophosphorylated intermediates of Cholesterol Biosynthesis (see Fig. 21-35 in Vol. 2), such as farnesyl pyrophosphate (Fig. 27-35). Such proteins include Ras proteins—the products of the ras oncogenes and Proto-oncogenes—and G proteins (both discussed in Chapter 12 of Vol. 1), as well as lamins, which are proteins of the nuclear lamina. The isoprenyl group helps anchor the protein to the membrane. The transforming (carcinogenic) activity of the ras oncogene is lost when Ras protein isoprenylation is defective; this discovery spurred the search for inhibitors for use in antitumor Chemotherapy.

Fig. 27-35. Farnesylation of a Cys residue. The thioether bond is highlighted in red. The Ras protein is the product of the ras oncogene.

Addition of prosthetic groups.

The activity of many bacterial and eukaryotic proteins depends on the presence of covalently attached prosthetic groups. Two characteristic examples are the biotin molecule in acetyl-CoA carboxylase and the heme group in hemoglobin or cytochrome c.

Proteolytic processing.

Many proteins are synthesized as large inactive precursors that are converted via proteolysis into shorter active forms. Such proteins include proinsulin, certain viral proteins, and proteases, including chymotrypsinogen and trypsinogen (see Fig. 6-38 in Vol. 1).

Formation of Disulfide Bonds.

In some proteins within their native conformation, intra- or intermolecular disulfide bridges are formed between Cys residues. In eukaryotes, disulfide bonds are frequently found in proteins that project outside the cell. These sulfide bridges protect the native protein conformation against Denaturation in the extracellular environment, which can differ significantly from the intracellular environment and is typically oxidizing.

Many Antibiotics and toxins inhibit protein synthesis

Protein synthesis is the most vital cellular process and a primary target for many natural antibiotics and toxins. With few exceptions, antibiotics inhibit protein synthesis in bacteria. Despite very minor differences in the pathways of protein synthesis between bacteria and eukaryotes, the compounds discussed below are harmless to eukaryotic cells—meaning they exhibit no toxic effects. It is remarkable that compounds synthesized by certain microorganisms prove toxic to others. Because protein synthesis at any stage can be specifically inhibited by particular antibiotics, these compounds serve as invaluable tools for studying the mechanism of protein biosynthesis.

Puromycin, produced by the actinomycete Streptomyces alboniger, is one of the most thoroughly studied inhibitory antibiotics. Its structure closely mimics the 3'-end of aminoacyl-tRNA, enabling it to bind to the ribosomal A-site and participate in peptide bond formation, thereby yielding peptidylpuromycin (Fig. 27-36). However, because puromycin resembles only the 3'-end of tRNA, it cannot participate in translocation and dissociates from the ribosome shortly after binding to the C-terminus of the peptide. This leads to premature termination of protein synthesis.

Fig. 27-36. Disruption of peptide bond formation in the presence of puromycin. (a) The structure of the antibiotic puromycin mimics the aminoacyl terminus of a charged tRNA, allowing it to bind to the ribosomal A-site and participate in peptide bond formation (see Fig. 27-13, d). However, the product of this reaction does not translocate to the P-site; instead, it leaves the ribosome, causing premature chain termination. (b) Peptidylpuromycin.

Tetracyclines inhibit bacterial protein synthesis by blocking the ribosomal A-site and preventing aminoacyl-tRNA binding. Chloramphenicol inhibits protein synthesis by bacterial ribosomes (as well as those of mitochondria and chloroplasts) by suppressing peptidyltransferase activity; it has no effect on cytoplasmic protein synthesis in eukaryotes. Cycloheximide blocks peptidyltransferase in eukaryotic 80S ribosomes, but not in bacterial 70S ribosomes (nor in mitochondrial and chloroplast ribosomes). The trisaccharide streptomycin induces genetic code misreading at low concentrations (in bacteria) and inhibits initiation at higher concentrations.

Some Protein Synthesis Inhibitors are toxic to humans and other mammals. Diphtheria toxin (M_r = 58,330) catalyzes the ADP-ribosylation of a diphthamide residue (a modified Histidine) in the eukaryotic elongation factor eEF2, thereby inactivating it. Ricin (M_r = 29,895), a highly toxic protein from the castor bean plant, inactivates the 60S subunit of eukaryotic ribosomes by depurinating a specific adenosine residue within the 23S rRNA.

Summary of Section 27.2 Protein Synthesis

■ Protein synthesis takes place on ribosomes, which are composed of protein and rRNA. Bacteria contain 70S ribosomes consisting of large (50S) and small (30S) subunits. Eukaryotic ribosomes are significantly larger (80S) and contain a greater number of proteins.

■ Transfer RNAs (tRNAs) range from 73 to 93 nucleotide residues in length and feature residues with modified bases. Each tRNA possesses an amino acid arm ending in a CCA (3') sequence—to which an amino acid is attached via an esterification reaction—along with an anticodon arm, a TΨC arm, and a D arm; some tRNAs also contain a fifth arm. The anticodon mediates the specific interaction between the aminoacyl-tRNA and the complementary mRNA codon.

■ Polypeptide chain growth on ribosomes initiates at the N-terminal amino acid and proceeds through the sequential addition of new residues to the C-terminus.

■ Protein synthesis occurs in five stages.

1. Amino acids are activated in the cytoplasm by specific aminoacyl-tRNA synthetases. These enzymes catalyze the formation of aminoacyl-tRNA coupled with the hydrolysis of ATP to AMP and PP_i. The overall fidelity of protein synthesis depends directly on the accuracy of this reaction, and certain of these

enzymes possess independent proofreading activities.

2. In all bacterial proteins, the initiator aminoacyl-tRNA is N-formylmethionyl-tRNA^fMet. Translation initiation involves the assembly of a complex comprising the ribosomal 30S subunit, mRNA, GTP, fMet-tRNA^fMet, three initiation factors, and the 50S subunit; GTP is hydrolyzed to GDP and P_i.

3. During elongation, the binding of each subsequent aminoacyl-tRNA to the ribosomal A-site requires GTP and elongation factors. In the initial peptidyltransferase reaction, the fMet residue is transferred to the amino group of the incoming aminoacyl-tRNA. Subsequent translocation of the ribosome along the mRNA molecule shifts the dipeptidyl-tRNA from the A-site to the P-site, coupled with GTP hydrolysis. Deacylated tRNAs leave the ribosome via the E-site.

4. Following repeated cycles of elongation, polypeptide synthesis is terminated through the action of release factors. Each peptide bond formed requires at least four high-energy phosphate equivalents (derived from ATP and GTP); this Energy Expenditure is essential to ensure high translational fidelity.

5. Newly synthesized polypeptides fold into their active three-dimensional Conformations. Many proteins undergo post-translational modifications following synthesis.

■ Numerous well-characterized antibiotics and toxins inhibit specific stages of protein synthesis.