Molecular Biology of the Cell - Volume 1 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1994

Molecular Organization of Cells
Basic Genetic Mechanisms
RNA and Protein Synthesis

Proteins typically account for more than half of the dry mass of a Cell, and their synthesis plays a major role in processes such as cell growth and differentiation, and the maintenance of cell Structure and function. Protein Synthesis depends on the concerted action of several classes of RNA molecules and is preceded by A number of preparatory steps. First, copying of the DNA that carries the information for the protein to be synthesized yields a Messenger RNA (mRNA) molecule. Simultaneously, in the Cytoplasm, each of the 20 Amino Acids that make up proteins is attached to a specific Transfer RNA (tRNA) molecule, and several auxiliary protein factors associate with the subunits of the ribosome on which synthesis occurs. Protein synthesis is initiated when these components assemble in the cytoplasm to form a functional ribosome. As the mRNA molecule moves step-by-step through the ribosome, its nucleotide sequence is translated into the corresponding sequence of amino acids, resulting in a specific polypeptide chain. First, however, we must address the question of how the various RNA molecules are produced in The Cell.

5.1.1. RNA polymerase "transcribes" the information encoded in DNA into RNA: The process of METABOLISM/31.html">Transcription [1]

RNA Synthesis on a DNA template is called transcription. Transcription yields mRNA molecules, which carry information for protein synthesis, as well as transfer, ribosomal, and Other types of RNA molecules that perform structural and catalytic Functions. The synthesis of these RNA molecules—that is, the synthesis of RNA copies of nucleotide sequences from specific Regions of the DNA molecule—is catalyzed by Enzymes called RNA polymerases. In eukaryotes, Different types of RNA are synthesized by different RNA polymerases, whereas in prokaryotes, all RNA synthesis is carried out by a single enzyme of this type. Almost everything we know about RNA polymerases has been elucidated in Bacteria.

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Fig. 5-1. RNA synthesis catalyzed by RNA polymerase. The enzyme initiates synthesis at a specific start signal in DNA, called a promoter, and terminates it at a stop signal (transcription termination signal), after which the polymerase and the completed, newly synthesized RNA chain release from each other. The rate of polymerization at 37°C is approximately 30 NUCLEOTIDES per second, so the synthesis of an RNA chain 5000 nucleotides long takes about 3 minutes.

Fig. 5-2. The elongation reaction catalyzed by the enzyme RNA polymerase. At each step, an incoming ribonucleoside triphosphate is selected that can complementarily pair with the exposed DNA template strand; As a result, a ribonucleoside monophosphate is added to the growing 3'-OH end of the RNA chain (colored arrow), and pyrophosphate (highlighted in color) is released. Thus, the new RNA chain grows in the 5' → 3' direction and is complementary to the DNA template strand. The driving force for the reaction is the thermodynamically favorable change in Free energy accompanying the release of pyrophosphate and the subsequent Hydrolysis of pyrophosphate to inorganic phosphate. As RNA polymerase moves along the DNA double helix, it continuously unwinds the helix just ahead of the polymerization site and rewinds it behind this site, releasing the newly synthesized RNA chain. Consequently, a short region of RNA-DNA hybrid helix (about 17 nucleotide pairs for the bacterial enzyme) exists only transiently. The completed RNA product is released as a single-stranded copy of one of the two DNA strands.

Bacterial RNA polymerase is a large, multisubunit enzyme associated with a number of auxiliary protein subunits that join the polymerase-DNA complex at different Stages of Transcription and then dissociate from it (see Section 9.4.1). Free RNA polymerase molecules, colliding randomly with the chromosome, bind weakly to most regions of DNA. However, this binding becomes highly stable if the RNA polymerase binds to a specific DNA sequence called a promoter, which contains the start signal for RNA synthesis—that is, the site where synthesis is to begin. The reactions that follow are shown in Fig. 5-1. Upon binding to the promoter, RNA polymerase unwinds a specific region of The Double Helix, thereby exposing the nucleotides on a short stretch of each of the two DNA strands. One of these two separated strands must now serve as a template for complementary base-pairing between the DNA and the incoming ribonucleoside triphosphate monomers; the polymerase joins the first two incoming monomers, thereby initiating the Synthesis of the RNA chain. Then, moving step-by-step along the DNA, RNA polymerase unwinds the DNA helix ahead of it, exposing a new region of the template for complementary base-pairing at each step. In this way, by adding one nucleotide at a time to the growing RNA chain, it gradually elongates the chain in the 5' → 3' direction (Fig. 5-2). The process of RNA chain elongation continues until the enzyme encounters another specific nucleotide sequence in the DNA strand, namely, a transcription termination signal (stop signal). Upon reaching this point, the polymerase dissociates from both the DNA template and the newly synthesized RNA chain (see also Fig. 5-6, b).

Fig. 5-3. As RNA polymerase moves along the DNA double helix, it continuously unwinds the helix just ahead of the polymerization site and rewinds it behind this site, releasing the newly synthesized RNA chain. Consequently, a short region of RNA-DNA hybrid helix (about 17 nucleotide pairs for the bacterial enzyme) exists only transiently. The completed RNA product is released as a single-stranded copy of one of the two DNA strands.

As the enzyme moves along the template strand, an RNA-DNA hybrid helix is formed within its Active Site. This hybrid helix is very short because the DNA-DNA double helix immediately reforms behind the polymerase molecule, displacing the RNA (Fig. 5-3). Therefore, each completed RNA chain is released from the DNA template as a free, single-stranded molecule, typically ranging in length from 70 to 10,000 nucleotides.

5.1.2. The promoter sequence determines which DNA strand will be transcribed [2]

In principle, any region of DNA can be transcribed to produce two different mRNA molecules—one for each of the two strands of the DNA double helix. In reality, however, only one of the two strands is transcribed in any given region of DNA, because the resulting RNA corresponds in its nucleotide sequence to the other, non-template DNA strand. Which of the two strands is transcribed is determined by the promoter, whose nucleotide sequence is oriented in such a way as to direct the RNA polymerase along one path or the other. Since RNA strands grow only in the 5' → 3' direction, the choice of the DNA strand for transcription depends entirely on the promoter (Fig. 5-4). In two adjacent genes, different DNA strands are often transcribed, as can be seen in Fig. 5-5, which shows a small region of a chromosome.

Analysis (see Section 4.6.6) shows that when E. coli RNA polymerase binds to the promoter, it covers a fairly large region of DNA: approximately 40 nucleotides upstream and 20 nucleotides downstream from the start site, i.e., the Transcription initiation site. Comparison of many DNA sequences that act as strong promoters for this polymerase has shown that it primarily recognizes two strictly defined (conserved) hexanucleotide sequences located upstream of the start site and separated from each other by approximately 17 nucleotides (Fig. 5-6, A). Such conserved sequences, found in all Examples of a particular type of regulatory DNA region, are called consensus sequences. Comparison of many E. coli promoters revealed two consensus hexanucleotide sequences: T₈₂T₈₄G₇₈A₆₅C₅₄A₉₅ and T₈₀A₉₅T₄₅A₆₀A₅₀T₉₆ (the numbers indicate the expected frequency (in percent) with which a given nucleotide occurs at the specified position in the sequence: 100 means it is always present, and 25 means it occurs in one out of four promoters). The sequences of strong promoters are usually quite close to these two consensus sequences, whereas in weak promoters (associated with genes producing relatively little mRNA), this similarity is less pronounced.

Fig. 5-4. Because the DNA strand serving as the template must be read from the 3' end to the 5' end (see Fig. 5-2), the choice of one of the two DNA strands to act as the template for RNA synthesis is determined by the direction of RNA polymerase movement. In turn, the direction of RNA polymerase movement is established by the orientation of the promoter nucleotide sequence from which the RNA polymerase begins reading.

Fig. 5-5. A short region of a typical bacterial chromosome. The diagram shows how Introduction/24.html">DNA Transcription associated with the expression of several adjacent genes occurs.

Fig. 5-6. Start and stop signals for RNA polymerase during RNA synthesis in E. coli. Note that the bottom DNA strand serves as the template, while the top strand corresponds in its nucleotide sequence to the synthesized RNA (except that U in RNA replaces T in DNA). It is customary to write The nucleotide sequence with respect to the non-template strand. A. Polymerase initiates synthesis at the promoter nucleotide sequence. Polymerase binding is thought to be determined by two short regions (highlighted in red) located approximately 35 and 10 nucleotides upstream from THE START OF RNA synthesis; together with the transcription start site, these sequences form the promoter. Any significant modifications in either of them lead to a loss of promoter activity, whereas changes in other regions of the DNA strand have no such effect. B. Polymerase terminates synthesis after a region of several U residues (corresponding to several A residues on the template) and a self-complementary nucleotide sequence (highlighted in gray) have been synthesized. The corresponding combination of nucleotide sequences in DNA serves as a stop signal. The nucleotide sequence in the self-complementary region can vary; the crucial factor for transcription termination is the rapid formation of a double-stranded hairpin structure in this region of the newly synthesized RNA.

Eukaryotic Cells contain three different RNA polymerases. One of them catalyzes the synthesis of all protein-coding RNAs (i.e., mRNAs), while the other two synthesize RNA molecules that perform structural or catalytic functions (such as ribosomal and Transfer RNAs). All three RNA polymerases are large multimeric molecules resembling the bacterial enzyme, but the promoters they recognize are more complex in structure and are not yet as well understood (see Section 9.4.3). It remains unclear why both bacterial and eukaryotic RNA polymerases are so complex. These molecules consist of several subunits with a total mass exceeding 500,000 daltons. Meanwhile, some bacteriophage RNA polymerases, which consist of a single chain and are one-fifth the mass, are known to catalyze RNA synthesis no less efficiently than the corresponding host cell enzyme. It can be assumed that the multimeric structure of cellular RNA polymerases is related to regulatory aspects of cellular RNA synthesis that are not yet fully understood.

In the description of DNA transcription provided above, many details have been omitted; the synthesis of an mRNA molecule typically involves several other complex steps. For example, it is known that specific Gene regulatory proteins play a crucial role in determining which regions of DNA will be transcribed by RNA polymerase, meaning that they are primarily responsible for which proteins the cell will produce. Furthermore, while in prokaryotes mRNA molecules are formed directly by DNA transcription, in the cells of higher eukaryotes, most RNA transcripts undergo significant Processing—splicing—before leaving the Cell Nucleus and entering the cytoplasm as mRNA. We will discuss all these aspects of mRNA formation in Chapters 9 and 10, which cover the cell nucleus and the Regulation of Gene Expression. Here, we will simply assume that functional mRNA molecules are somehow produced in the cell, and we will examine how they direct protein synthesis.

5.1.3. Transfer RNA molecules serve as adapters that translate nucleotide sequences into Amino acid sequences [3]

All cells contain a set of transfer RNAs (tRNAs)—small molecules ranging in size from 70 to 90 nucleotides. By binding at one end to a specific mRNA codon and at the other end to The amino acid specified by that codon, these RNAs allow amino acids to line up in the order dictated by the mRNA nucleotide sequence. Each tRNA can carry only one of the 20 amino acids used in protein synthesis. The transfer RNA that carries Glycine is designated tRNA^Gly, and so on. There is at least one type of tRNA for each of the 20 amino acids, and for Most amino acids, there are several. Before being incorporated into the growing polypeptide chain, an amino acid is attached by its carboxyl end to the 3' end of the corresponding tRNA molecule. This serves two purposes. First, and most importantly, the amino acid is covalently linked to a tRNA containing the correct anticodon—a three-nucleotide sequence complementary to the three-nucleotide codon that specifies this amino acid in the mRNA molecule. Codon-anticodon pairing allows each amino acid to be incorporated into the growing protein chain in the order dictated by the nucleotide sequence of the mRNA, so that The Genetic Code is used for translation.

Figure 5-7. Structure of a typical tRNA molecule. The base-pairing mechanism in the corresponding regions of the molecule (the cloverleaf structure) is shown on the left, and the overall three-dimensional STRUCTURE OF THE molecule, determined by X-Ray Diffraction, is shown on the right.

Note that the molecule is L-shaped; one of its ends (the acceptor end) is designed for amino acid attachment, while the other contains an anticodon consisting of three nucleotides. The amino acid is attached to the A residue of the CCA sequence at the 3' end of the molecule (see Fig. 5-11).

nucleotide sequences of Nucleic Acids into amino acid sequences of proteins. This is The Essence of the important "adaptor" function of tRNA: by attaching to an amino acid at one end and pairing with a codon at the other, tRNA translates the nucleotide sequence into an Amino Acid Sequence.

The second goal achieved by attaching the amino acid to tRNA is that the amino acid is thereby activated—a high-energy bond is formed at its carboxyl end, enabling it to react with the amino group of an adjacent amino acid in the given amino acid sequence, i.e., to form a peptide bond. This activation process is a necessary step in protein synthesis, as unactivated amino acids cannot directly join the growing polypeptide chain. (Only the reverse process—hydrolytic Cleavage of peptide bonds—can occur spontaneously.)

The function of tRNA depends on the three-dimensional structure of its molecule. Several types of tRNA have been crystallized, allowing their precise structure to be determined by X-ray crystallography. Proper folding of the tRNA molecule requires complementary base pairing and interactions involving unusual bases (see Section 3.2.9, Fig. 3-16). Studies of the Secondary structure of tRNA molecules from many different organisms have shown that it has a "cloverleaf" shape; it is believed that the loops and helical stems of this structure then fold further, resulting in the L-shaped conformation revealed by crystallographic analysis (Fig. 5-7). An amino acid attaches to one end of this "L", while the anticodon is located at the other end (Fig. 5-8).

In a completed nucleic acid chain, nucleotides (like amino acids in proteins) can undergo covalent modification, leading to changes in The activity of the nucleic acid. Such post-transcriptional modifications are particularly characteristic of tRNA molecules, which contain many modified nucleotides (Fig. 5-9). Some of these modifications affect the conformation and base pairing of the anticodon, facilitating the recognition of the corresponding mRNA codon by the tRNA molecule.

Figure 5-8. Three-dimensional model of a tRNA molecule with an attached amino acid. There are many different types of tRNA—at least one for each amino acid. Although these tRNA molecules differ in their nucleotide sequence, they are all folded in a similar manner. The tRNA molecule shown here binds the amino acid phenylalanine, so it is designated tRNA^Phe. (Courtesy of Sung-Hou Kim.)

Figure 5-9. Some unusual nucleotides found in tRNA molecules. They arise from Covalent Modification of a standard nucleotide after it has been incorporated into the polynucleotide chain. In most tRNA molecules, approximately 10% of the nucleotides are modified in this way (see Fig. 5-7).

5.1.4. Each amino acid is attached to its corresponding tRNA molecule by a specific enzyme [4]

Exactly where a given amino acid will be incorporated into the growing polypeptide chain depends not on the amino acid itself, but on the tRNA molecule that carries it. This was demonstrated by an elegant experiment in which an amino acid attached to a specific tRNA was chemically converted into another amino acid (Cysteine to Alanine). When these hybrid molecules were subsequently used in Protein synthesis in a cell-free system, the incorrect amino acid was incorporated into the protein chain at all positions "served" by that tRNA. Successful decoding, therefore, depends crucially on the accuracy of the mechanism that normally links each activated amino acid to its corresponding tRNA molecule.

Why does a tRNA molecule covalently couple to the correct partner among all twenty standard amino acids? This mechanism involves enzymes called Aminoacyl-tRNA synthetases, which attach each amino acid to its appropriate set of tRNA molecules. There is a specific synthetase for each of the 20 amino acids: one couples glycine to tRNA^Gly, another couples alanine to tRNA^Ala, and so on. The attachment reaction occurs in two steps, as shown in Fig. 5-10, and leads to The formation of an aminoacyl-tRNA molecule. The Nature of the aminoacyl-tRNA linkage is clear from Fig. 5-11.

tRNA molecules act as the ultimate "adaptors" that translate the information contained in the nucleotide sequence of a nucleic acid into the language of proteins. However, an equally important role is played by a second set of molecules—the aminoacyl-tRNA synthetase enzymes. Thus, the genetic code is deciphered by two sequential adaptors, each of which performs a highly specific fit of one molecular surface to another; through the combined action of these adaptor molecules, each amino acid can be matched with a specific sequence of three nucleotides in the mRNA molecule, in other words, with its codon (Fig. 5-12).

Figure 5-10. The two-step process in which an amino acid is activated by an aminoacyl-tRNA synthetase for protein synthesis. The attachment of each amino acid to its corresponding tRNA molecule is driven, as shown here, by the energy of ATP hydrolysis, since the resulting linkage is a high-energy bond. First, the amino acid is activated by the direct linkage of its carboxyl group to AMP, forming an adenylated amino acid; the energy for this adenylation reaction, which is normally thermodynamically unfavorable, is provided by the hydrolysis of ATP (acting as an AMP donor). While remaining bound to the aminoacyl-tRNA synthetase, the adenylated carboxyl group of the amino acid is then transferred to a hydroxyl group of the sugar residue at the 3' end of the tRNA molecule. This transfer produces an aminoacyl-tRNA molecule, in which the amino acid is linked to the tRNA by an activated ester bond.

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5.1.5. Amino acids are added to the carboxyl end of the growing polypeptide chain

The fundamental reaction in protein synthesis is the formation of a peptide bond between the carboxyl group at the end of the growing polypeptide chain and the free amino group of an incoming amino acid. The protein chain is therefore synthesized by its gradual elongation from the amino to the carboxyl terminus. Throughout this process, the growing carboxyl end of the polypeptide chain remains activated by being covalently linked to a tRNA (forming a peptidyl-tRNA molecule). In each cycle of synthesis, this covalent bond is cleaved, but it is immediately replaced by an identical bond formed by the next amino acid added to the chain (Fig. 5-13). Thus, during protein synthesis, each incoming amino acid carries the activation energy required not for its own addition, but for The addition of the next amino acid. This is an example of the "HEAD growth" mechanism described in Chapter 2 (Fig. 2-34).

Figure 5-11. The linkage between an Amino Acid and tRNA. The carboxyl group of the amino acid is attached to ribose by an ester bond. Because the hydrolytic cleavage of this ester bond is accompanied by a thermodynamically favorable change in free energy, the amino acid held by such a bond is activated. A. Schematic representation. B. Actual structure of the boxed region on the left. R in the amino acid portion of the molecule, as in Fig. 5-10, represents one of the 20 possible side chains.

Figure 5-12. Diagram showing how the Translation of the genetic code is accomplished by two cooperating "adaptors": the first is the aminoacyl-tRNA synthetase enzyme, which couples a given amino acid to its corresponding tRNA, and the second is the tRNA molecule itself, which then pairs with the appropriate nucleotide sequence in the mRNA.

Figure 5-13. A polypeptide chain grows by the stepwise addition of individual amino acids to its carboxyl end. The formation of each peptide bond is energetically favorable because the growing carboxyl end of the chain is activated by its covalent linkage to a tRNA molecule. The peptidyl-tRNA linkage that keeps the growing end of the polypeptide chain activated is regenerated in each cycle.

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5.1.6. The genetic code is degenerate [5]

During protein synthesis, the nucleotide sequence of an mRNA is read in groups of three nucleotides as the translation machinery moves along the mRNA molecule in the 5' → 3' direction. Each amino acid corresponds to a specific nucleotide triplet (codon) in the mRNA molecule, which pairs with a sequence of three complementary nucleotides in the anticodon loop of a specific tRNA molecule. Since only one of the many different types of cellular tRNA molecules can pair with a given codon, the choice of amino acid added at any given moment to the growing end of the polypeptide chain is determined by the codon (Fig. 5-14).

The number of possible combinations of three nucleotides of four types is 64 (4 x 4 x 4). Most of these combinations do indeed occur in almost all mRNA molecules. Three of the 64 codons do not code for any amino acids; these codons specify the termination of Polypeptide chain synthesis and are therefore called stop codons or termination codons. This leaves 61 codons, whereas the number of different amino acids found in proteins is only 20. It follows that most amino acids are represented by more than one codon. Therefore, the genetic code is said to be degenerate. For Two amino acids, Methionine and Tryptophan, there is only one codon each; these are the amino acids that occur least frequently in proteins.

The degeneracy of the genetic code can be interpreted in two ways: 1) there is more than one tRNA for each amino acid, and 2) each tRNA molecule can pair with more than one codon. In fact, both are true. For Some amino acids, there is more than one tRNA. Furthermore, some tRNAs require accurate pairing only at the first two positions of the codon, tolerating a mismatch (so-called wobble) at the third position. This explains why many alternative codons for an amino acid differ only in their third nucleotide (Fig. 5-15). Wobble pairing allows 20 amino acids to be matched with 61 codons using only 31 types of tRNA; in animal Mitochondria, wobble is even more pronounced, and here 22 types of tRNA are sufficient for protein synthesis (see Section 7.5.5).

Fig. 5-14. The choice of each amino acid added to the growing end of a polypeptide chain is determined by complementary base-pairing between the anticodon of the aminoacyl-tRNA and the next codon of the mRNA chain.

Fig. 5-15. The genetic code. Below the three-letter abbreviation for each amino acid, its standard single-letter designation is given. In this representation of codons, the 5'-terminal nucleotide is on the left. Note that Almost all amino acids are represented by more than one codon, and that differences affect mainly the third nucleotide (see also Fig. 3-15).

5.1.7. Protein synthesis reactions take place on Ribosomes [6]

The protein synthesis reactions we have just described require a complex catalytic apparatus. For example, the growing end of the polypeptide chain must be positioned relative to the mRNA molecule in such a way that each successive mRNA codon can pair precisely with the tRNA anticodon without skipping a single nucleotide, as this would cause a reading frame shift (see Section 3.2.8). These and other steps of protein synthesis are carried out by ribosomes—large complexes composed of Protein and RNA molecules. Eukaryotic and prokaryotic ribosomes are very similar in structure and function. Each consists of two subunits—large and small—which together form a complex with a mass of

Fig. 5-16. Three-dimensional model of the bacterial ribosome (viewed from two different sides). The locations of many ribosomal proteins in this structure have been determined by Electron Microscopy, which reveals the binding sites of specific Antibodies, and by neutron scattering from ribosomes containing one or more deuterated proteins. (From J. A. Lake, Ann. Rev. Biochem., 54, 507-530, 1985.)

Fig. 5-17. The complex arrangement of loops and paired helical regions in the three-dimensional structure of E. coli 16S rRNA (A) and Yeast (S. cerevisiae) 18S rRNA (B). In its main features, this structure is characteristic of all 16S-like rRNAs, including those of archaebacteria. Dots indicate postulated weak base-pairing interactions, such as G-U pairs. (From R. R. Gutell, B. Weiser, C. R. Woese, H. F. Noller, Prog. Nucleic Acid Res. Mol. Biol., 32, 155-216, 1985.)

several million daltons (Fig. 5-16). The small subunit holds the mRNA and tRNAs, while the large subunit catalyzes the Formation of the peptide bond.

More than half of the ribosome's mass is RNA, and there is a growing body of evidence indicating that ribosomal RNA (rRNA) plays a key role in the catalytic activity of the ribosome. The size of the rRNA molecules in the small ribosomal subunit varies among different organisms, but its complex structure remains highly conserved (Fig. 5-17); a high degree of Homology is also found among the rRNA molecules of the large ribosomal subunits in different organisms. The ribosome contains a significant number of proteins (Fig. 5-18), but their amino acid sequences have changed considerably over the course of evolution. Surprisingly, many of these proteins do not appear to be essential for ribosomal function. It can be suggested (as discussed in more detail below; see Section 5.1.16) that ribosomal proteins are needed primarily to enhance the function of the rRNA, and that RNA molecules, rather than protein molecules, catalyze many of the reactions occurring on ribosomes.

5.1.8. The ribosome moves step by step along the mRNA chain [6, 7]

The ribosome has three distinct RNA-binding sites: one for mRNA and two for tRNA. Of the latter two, one site holds the tRNA molecule attached to the growing end of the polypeptide chain (hence called the peptidyl-tRNA-binding site, or P site), while the second serves to hold the newly arrived amino acid-loaded tRNA molecule, called the aminoacyl-tRNA-binding site, or A site. A tRNA molecule binds tightly to either site only if its anticodon pairs with the complementary codon of the mRNA. The A and P sites are positioned very close to each other, so that the two bound tRNA molecules pair with two adjacent codons in the mRNA molecule (Fig. 5-19).

Fig. 5-19. Three major binding sites where RNA molecules attach to the ribosome. An empty ribosome is shown on the left, and a loaded one on the right. In this figure, as well as in the next three, the ribosomes are shown schematically; a more accurate representation of their shape is given in Figs. 5-16 and 5-23.

The process of Polypeptide chain elongation on ribosomes can be viewed as a cycle consisting of three distinct steps (Fig. 5-20). In the first step, an aminoacyl-tRNA molecule binds to the vacant A site adjacent to the occupied P site. Binding occurs by pairing the anticodon nucleotides with the three mRNA nucleotides located in the A site. In the second step, the carboxyl end of the polypeptide chain is uncoupled from the tRNA molecule in the P site and forms a peptide bond with the amino acid attached to the tRNA molecule in the A site. This reaction is catalyzed by peptidyl transferase, an enzyme whose activity depends on the integrity of the ribosome and is also thought to reside in a specific region of the main rRNA molecule of the large ribosomal subunit. In the third step, the new peptidyl-tRNA is translocated to the P site of the ribosome, while the ribosome moves along the mRNA molecule by exactly three nucleotides. This step requires energy; the driving force is a series of Conformational changes induced in one of the ribosomal components by the hydrolysis of a bound GTP molecule (see Section 3.4.11).

The translocation process, which constitutes the third step, also includes the release of the free tRNA molecule (which was uncoupled from the polypeptide chain in the P site during the second step) back into the cytoplasmic tRNA pool. Therefore, after the third step is completed, the vacant A site can accept a new tRNA molecule loaded with the next amino acid, allowing the cycle to begin anew. In a bacterial cell, under optimal conditions, a single polypeptide chain elongation cycle takes about 1/20 of a second, so that the synthesis of an average-sized protein of 400 amino acids takes approximately 20 seconds. Ribosomes move along the mRNA molecule in the 5' → 3' direction, which is the same direction in which RNA synthesis occurs (see Fig. 5-2).

In most cells, protein synthesis is the most energy-consuming of all biosynthetic processes. The formation of each new peptide bond is accompanied by the cleavage of at least four high-energy phosphate bonds. Two of these are consumed in charging the tRNA molecule with an amino acid (see Fig. 5-10), and two are used for the synthesis itself during the reaction cycle on the ribosome: during aminoacyl-tRNA binding in the first step of the cycle, and during ribosomal translocation in the third step.

Figure 5-20. The elongation phase of protein synthesis occurring on the ribosome. The three-step cycle shown here is repeated many times during the synthesis of a protein chain. In the first step, an aminoacyl-tRNA molecule binds to the ribosomal A-site; the second step is characterized by the formation of a new peptide bond; in the third step, the ribosome translocates along the mRNA chain by a distance of three nucleotides, releasing the previous tRNA molecule, thereby positioning itself so that the cycle can repeat from the beginning.

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5.1.9 The protein chain is released from the ribosome as soon as it reaches one of the three termination codons [6, 8]

Of the 64 possible mRNA codons, three—namely UAA, UAG, and UGA—are termination or stop codons: they halt translation. Specific cytoplasmic proteins called release factors bind directly to any stop codon that reaches the ribosomal A-site. This binding alters the activity of peptidyl transferase. The enzyme with altered activity now adds a Water molecule, rather than an amino acid, to the peptidyl-tRNA. As a result, the carboxyl terminus of the growing polypeptide chain is cleaved from the tRNA molecule. Since the growing polypeptide is held on the ribosome only through its linkage to the tRNA molecule, the completed protein chain is freed and, upon detaching from the ribosome, immediately enters the cytoplasm (Fig. 5-21). The ribosome then releases the mRNA and dissociates into its two subunits; these subunits can then reassemble on another mRNA molecule to begin a new cycle of protein synthesis through a process described below.

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5.1.10. The reading frame of the template is established at the initiation of polypeptide chain synthesis [6, 9]

Theoretically, an RNA nucleotide sequence can be decoded in any of three different reading frames, and the resulting polypeptide chains would be entirely different in each case (see Fig. 3-14). How translation actually proceeds is determined at the moment the ribosome binds to the mRNA molecule. During the initiation phase of protein synthesis, the assembly of the ribosome from its two subunits on the mRNA molecule occurs at the precise site where polypeptide chain synthesis is to begin.

The initiation process is complex. It consists of a series of steps catalyzed by proteins called initiation factors (IFs), many of which are themselves composed of several polypeptide chains. Because of this complexity, many details of initiation remain to be fully elucidated. It is known, however, that the assembly of each ribosome on the mRNA chain occurs in two steps: first, the small ribosomal subunit, loaded with initiation factors, finds the start codon on the mRNA, and then the large subunit joins it.

Before a ribosome can begin synthesizing a new polypeptide chain, an aminoacyl-tRNA molecule must bind to its P-site, which normally holds the peptidyl-tRNA (Fig. 5-22). This requires a special tRNA molecule called the initiator tRNA. The initiator tRNA provides the amino acid that must start the polypeptide chain. This amino acid is always methionine or, in prokaryotes, its formylated derivative. In eukaryotes, the small ribosomal subunit is loaded with the initiator tRNA before it binds to the mRNA. An important initiation factor called eukaryotic initiation factor 2 (eIF-2) binds tightly to the initiator tRNA; it is required for the initiator tRNA to position itself correctly on the small ribosomal subunit. In some cells, the overall Rate of protein synthesis depends on this factor (see below).

Figure 5.21. The final phase of protein synthesis. Binding of a release factor to the stop codon terminates translation, the completed polypeptide is released, and the ribosome dissociates into two separate subunits.

Figure 5-22. The initiation phase of protein synthesis. The sequence of events shown here is characteristic of eukaryotes, but a very similar process occurs in bacteria. Steps 1 and 2 belong to the elongation phase (see Fig. 5-20).

Figure 5-23. Three-dimensional model of a functional bacterial ribosome. The small (red) subunit and the large (gray) subunit form a complex through which the mRNA strand is threaded. The exact path of the mRNA and the growing polypeptide chain is unknown, but the site where amino acid addition occurs is indicated correctly here. (Modified from J. A. Lake, Annu. Rev. Biochem., 54, 507-530, 1985.)

In the next section, we will describe how the small ribosomal subunit helps its bound initiator tRNA locate a single specific AUG codon (the start codon) among all the AUG codons encountered in the mRNA molecule. As soon as this occurs, several initiation factors previously bound to the small subunit dissociate from it, making room for the large ribosomal subunit to assemble. Because the initiator tRNA molecule binds to the ribosomal P-site, polypeptide chain synthesis can begin directly with the binding of a second aminoacyl-tRNA molecule to the A-site (Fig. 5-22). This completes the assembly of a functional ribosome with the mRNA strand threaded through it (Fig. 5-23). This is followed by the subsequent steps of the elongation phase of protein synthesis described above (see step 2 in Fig. 5-20). Since the initiator tRNA always carries the amino acid methionine or, in prokaryotes, its formylated derivative, all newly synthesized protein chains have a methionine residue at their N-terminus. This methionine is often removed shortly after incorporation by a specific aminopeptidase, which is highly significant because the amino-terminal amino acid can determine the lifetime of cellular proteins by affecting the ubiquitin-dependent degradation pathway (see Section 8.2.5).

The Selection of the correct initiation site on the mRNA molecule is clearly determined by the small subunit acting in concert with initiation factors (but in the absence of the large subunit); this is likely why all ribosomes consist of two subunits. We will now examine how this selection is made.

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5.1.11. In eukaryotes, only one type of polypeptide chain is synthesized from each mRNA molecule [10]

An mRNA molecule typically contains many AUG triplets, each of which codes for methionine. In eukaryotes, however, only one of these AUG triplets is recognized by the initiator tRNA, thereby serving as the start codon. How does the ribosome recognize this start codon?

The mechanism for selecting the start codon differs between eukaryotes and prokaryotes. Eukaryotic mRNA molecules (excluding those synthesized in Mitochondria and Chloroplasts) undergo significant modifications in the cell nucleus immediately upon completion of transcription (see Section 9.4.8). Two major modifications of this type are the addition of a special structure to the 5' end, the so-called cap, consisting of a 7-methylguanosine residue linked to a triphosphate (Fig. 5-24), and a polyadenylic acid tail of approximately 200 residues (polyA) at the 3' end. Whether polyA plays any role in translation is not yet known. As for the 5' cap, it is essential for efficient protein synthesis. Experiments with Eukaryotic Cell extracts have shown that the small ribosomal subunit binds to the 5' end of the mRNA chain, a process facilitated by its recognition of the 5' cap (Fig. 5-22). This small subunit, carrying its bound initiator tRNA, then migrates along the mRNA chain in search of the AUG start codon. The requirements for the start codon do not appear to be overly stringent: only a few additional nucleotides besides the AUG triplet itself are needed. In most RNA species, the first suitable AUG codon near the 5' end is used, and none of the many other AUG triplets in the mRNA chain can then serve as an initiation site for the polypeptide chain. Consequently, as a rule, only one type of polypeptide chain is synthesized from a given mRNA molecule. In all these respects, prokaryotic mRNAs are completely different from eukaryotic ones (Fig. 5-25). Bacterial templates lack a 5' cap. Instead, they contain specific ribosome-binding sequences of about six nucleotides that occur repeatedly at various positions along the same mRNA chain. These sequences are usually located upstream of each AUG triplet, separated from it by a few (4 to 7) nucleotides; they pair with a specific region of the ribosomal rRNA, which serves as a signal to initiate protein synthesis at the nearest start codon. Furthermore, although bacterial ribosomes recognize termination codons as signals to end the synthesis of a polypeptide chain, they can "slide" further along the template. Therefore, bacterial mRNAs are typically polycistronic, meaning they encode multiple proteins synthesized from the same mRNA molecule. In contrast, eukaryotic mRNAs are generally monocistronic; in other words, only one type of polypeptide chain can be synthesized from a single mRNA molecule (Fig. 5-25).

Figure 5-24. The 5' cap found on eukaryotic mRNA molecules. Note the unusual 5' → 5' linkage to the positively charged 7-methylguanosine residue and the methylation of the 2'-hydroxyl of the first ribose residue in the RNA. (The second ribose residue is not always methylated.)

Figure 5-25. Comparison of the structures of prokaryotic and eukaryotic mRNAs. At the completion of synthesis, both of these mRNAs have a triphosphate at their 5' end, but the eukaryotic mRNA immediately acquires a 5' cap. In eukaryotes, the small ribosomal subunit recognizes the 5' end of the mRNA specifically by its 5' cap. Protein synthesis therefore begins at the start codon closest to the 5' end (see Figure 5-22). In contrast, in prokaryotes, the 5' end has no special significance, and ribosomes can bind to many sites along the mRNA strand, initiating the synthesis of a different protein in each case.

Figure 5-26. Schematic representation of a polyribosome, showing how a series of ribosomes simultaneously translate the same mRNA molecule. In eukaryotic cells, the synthesis of each polypeptide chain begins with the binding of the small ribosomal subunit to the single appropriate site on the mRNA molecule, and translation proceeds along this molecule in the 5' → 3' direction. Upon completion of the polypeptide chain, both ribosomal subunits dissociate from the mRNA molecule.

5.1.12. Multiple ribosomes bound to a single mRNA molecule form a polysome [11]

The synthesis of a single protein takes on average from 20 to 560 seconds. However, even during this very short period, initiation of synthesis usually occurs multiple times on each mRNA molecule undergoing translation. A new ribosome binds to the 5' end of the mRNA molecule as soon as the preceding one has linked enough amino acids together to clear the site. In this case, mRNA molecules are part of polyribosomes (or Polysomes)—structures in which many ribosomes are strung along a single mRNA molecule, spaced approximately 80 nucleotides apart (Figures 5-26 and 5-27). In prokaryotes (unlike eukaryotes), ribosomes can bind to mRNA as soon as it is formed. They initiate protein synthesis at the 5' end of the nascent mRNA molecule and move immediately behind the RNA polymerase that is elongating the mRNA chain.

Polyribosomes are highly characteristic of cells. They are separated from free ribosomes in the Cytosol by ultracentrifugation after cell lysis (Figure 5-28). The mRNA isolated from polyribosomes can be used to confirm that the protein encoded by a given DNA sequence is being actively synthesized by the cells from which the polyribosomes were obtained. It can also serve as Starting Material for constructing specialized cDNA libraries (see Section 5.6.3).

Figure 5-27. Cytology/cytology/93.html">ELECTRON MICROGRAPHS OF typical polyribosomes translating protein in a eukaryotic cell. A. Deep-etching. (Courtesy of John Heuser.) B. Thin section. The cytoplasm of a cell is typically packed with such polyribosomes, either lying free in the cytosol or attached to membranes. (Courtesy of George Palade.)

Figure 5-28. Separation of polyribosomes from free ribosomes (and their subunits) by centrifugation. The method is based on the fact that large molecular aggregates move faster in a strong gravitational field than smaller ones. Sedimentation is usually carried out in a sucrose gradient to stabilize the solution and prevent mixing due to convection.

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5.1.13. The overall rate of protein synthesis in eukaryotes is regulated by initiation factors [12]

It is known (for details, see Chapter 13) that cells of a multicellular Organism proliferate only when they are in an appropriate environment and are acted upon by specific growth factors. The MECHANISM OF ACTION of these growth factors is not entirely clear, but undoubtedly one of the main effects must be an increase in the overall rate of protein synthesis (see Section 13.3.4). What determines this rate? Direct studies on Tissues are extremely difficult, but if cells in culture do not receive sufficient nutrients, the rate of initiation of polypeptide chain synthesis drops sharply, and this inhibition can be shown to result from the inactivation of one of the protein synthesis initiation factors, namely IF-2. It has been shown that in at least one cell type (immature erythrocytes), the activity of IF-2 is decreased in a controlled manner by the phosphorylation of one of its three protein subunits. It can therefore be assumed that the rate of protein synthesis in eukaryotes is regulated to some extent by specific protein Kinases that, in their active form, inhibit its initiation. It is possible that growth factors act through regulatory substances that inactive these protein kinases or neutralize their effect.

In eukaryotes, the initiation factors required for protein synthesis are more numerous and complex than in prokaryotes, although they perform the same basic functions in both. The numerous additional components may represent regulatory proteins that respond to various growth factors and coordinate cell growth and proliferation in Multicellular Organisms. Bacteria have no need for such regulation: they grow at whatever rate the availability of nutrients in the medium allows.

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5.1.14. The accuracy of protein synthesis is maintained by two distinct mechanisms [13]

The error rate in protein synthesis can be estimated by determining how often an amino acid that is normally absent from a given protein is incorporated into it. Observations show that, on average, one 'incorrect' amino acid is incorporated for every 10⁴ amino acids, meaning that only one error occurs for every 25 synthesized proteins of average size (400 amino acids). The accuracy of the decoding process depends on the reliability of the two adaptor mechanisms discussed above: the coupling of each amino acid to its appropriate tRNA molecule and the pairing of codons in mRNA with tRNA anticodons (see Figure 5-12). Not surprisingly, cells have evolved mechanisms to minimize errors at these two key Stages of Protein Synthesis.

The two mechanisms operating at these two stages are completely different; each reflects a strategy used by the cell in other processes. Both mechanisms, however, require the expenditure of free energy, because, as noted in Chapter 2 (see Section 2.2), any increase in order comes at a cost. The fidelity of amino acid binding to tRNA is ensured by a relatively simple mechanism. Many aminoacyl-tRNA synthetases have two separate active sites: one responsible for the reaction that charges the tRNA with an amino acid (Figure 5-10), and another that recognizes an incorrect amino acid attached to the tRNA and removes it by hydrolysis. Such a proofreading process is costly, as it can function effectively only if it also discards a significant number of correctly attached amino acids. A similarly costly two-step proofreading process is used in DNA Replication (see Section 5.3.3).

Figure 5-29. A more detailed view of the first step of the elongation phase of protein synthesis, showing how the correct tRNA is selected on the ribosome. During the initial binding stage, an aminoacyl-tRNA molecule associated with an elongation factor temporarily pairs with the codon in the A site. This pairing triggers GTP hydrolysis, which is induced by the elongation factor, allowing the elongation factor to dissociate from the aminoacyl-tRNA molecule. The tRNA is then positioned precisely in the A site, ready to play its designated role in polypeptide chain elongation (see Figure 5-20). Only tRNAs with the correct anticodon remain paired with the mRNA long enough to participate in chain elongation. The elongation factor (which is a protein) is designated EF-Tu in prokaryotes and EF-1 in eukaryotes.

The accuracy of codon-anticodon pairing is ensured by a more sophisticated mechanism of 'kinetic proofreading.' Previously, we limited ourselves to a simplified description of this pairing. In reality, once tRNA molecules are charged with their appropriate amino acids, they form a complex with a specific protein called an elongation factor (EF), which binds tightly to the aminoacyl end of the tRNA molecule and to a GTP molecule. It is this complex, rather than a free tRNA molecule, that pairs with the appropriate codon in the mRNA molecule. The bound elongation factor facilitates correct codon-anticodon pairing but prevents the amino acid from being incorporated into the growing polypeptide chain. However, initial codon recognition signals the elongation factor to hydrolyze its bound GTP (to GDP and inorganic phosphate), after which the factor itself dissociates from the ribosome, leaving the tRNA behind so that protein synthesis can proceed. As shown in Figure 5-29, the elongation factor creates a short time delay between codon-anticodon pairing and polypeptide chain elongation, allowing the bound tRNA molecule to dissociate from the ribosome. An incorrect tRNA molecule forms fewer Hydrogen Bonds in the codon-anticodon pair than a correct one; it is therefore held more weakly on the ribosome and is more likely to dissociate during this time interval. Because the delay introduced by the elongation factor allows most incorrectly bound tRNA molecules to leave the ribosome before being used in protein synthesis, this factor clearly reduces the frequency of incorrect amino acids in the synthesized protein.

5.1.15. Many inhibitors of prokaryotic protein synthesis are useful Antibiotics [14]

Many of the most effective antibiotics used in modern medicine act by inhibiting protein synthesis in bacterial cells. A number of these drugs have been designed to exploit structural and Functional differences between prokaryotic and eukaryotic ribosomes, so that they act preferentially on prokaryotic ribosomes. Because of this selectivity, these compounds can be administered to humans in relatively high concentrations without causing toxic side effects. Different antibiotics bind to different regions of bacterial ribosomes and therefore often inhibit different steps in the translation process. Table 5-1 lists the most common compounds in this group and describes their specific actions. The table also includes several other widely used inhibitors of protein synthesis, including some that act on eukaryotic cells; the latter, of course, cannot be used as antibiotics.

Table 5-1. Inhibitors of protein or RNA synthesis

¹⁾ Ribosomes in eukaryotic mitochondria (and chloroplasts) are often similar to prokaryotic ribosomes in their sensitivity to inhibitors.

Many of the compounds listed in Table 5-1 block highly specific steps in The transfer of Genetic information from DNA to protein, making them widely used in studying various cellular mechanisms. Among the drugs used for this purpose are chloramphenicol, cycloheximide, and puromycin. All of them inhibit protein synthesis in a specific manner. Chloramphenicol, for example, inhibits protein synthesis in eukaryotic cells only on ribosomes within mitochondria (and plant chloroplasts), which likely reflects the prokaryotic origin of these Organelles (see Section 7.5.16). Cycloheximide, conversely, acts only on cytosolic ribosomes. The differing sensitivity of protein synthesis to these two drugs provides a highly reliable way to determine in which cellular compartment a particular protein is being translated. Puromycin is of particular interest because its structure closely resembles the terminal aminoacyl-adenylate of an aminoacyl-tRNA; it therefore reacts with the C-terminus of the growing peptidyl-tRNA on the ribosome, just as an incoming amino acid would. However, further elongation is prevented as a result—premature chain termination occurs, and the peptidyl-puromycin leaves the ribosome. Consequently, puromycin inhibits all types of protein synthesis.

5.1.16. The Evolution of protein Synthesis [15]

The molecular processes underlying protein synthesis are inexplicably complex. Although we are now able to describe many of them, their rationale remains puzzling, unlike, for example, the processes of DNA transcription, repair, and replication. As we already know, protein synthesis in modern organisms occurs on a very large ribonucleoprotein complex—the ribosome, which consists of various proteins grouped around a core of rRNA molecules. Why are rRNA molecules needed at all, and how did they come to play a dominant role in ribosomal structure and function? Answering this question will undoubtedly help us better understand protein synthesis itself. Previously, before mRNA was discovered in the early 1960s, it was assumed that the large amounts of RNA in ribosomes served an informational function—transferring genetic information from DNA to proteins. Today, however, we know that all ribosomes in a cell share the same set of rRNA molecules and that these molecules do not play such an informational role. In bacterial ribosomes, it has been shown that certain small regions of rRNA perform catalytic functions in protein synthesis; for instance, the rRNA of the small subunit of prokaryotic ribosomes base-pairs with the initiation sequence of the mRNA molecule to form a short helix, helping to position the adjacent AUG start codon in the P-site. Similar base-pairing interactions may also occur between tRNA and rRNA molecules, though this has not yet been convincingly demonstrated.

A large number of different proteins associated with ribosomal rRNA also play an important role in protein synthesis. The extreme complexity of this process, involving so many components, has led many biologists to doubt that its evolutionary pathways will ever be understood. However, a recent discovery—the identification of RNA molecules capable of acting as enzymes (see Section 3.2.11)—has shed new light on this subject. As noted in Chapter 1, RNA molecules, rather than proteins, may have served as catalysts in the earliest biological reactions. It is possible that in the Early stages of the first cells, tRNA molecules themselves, without the aid of aminoacyl-tRNA synthetases, formed catalytic surfaces that allowed them to bind and activate amino acids. It is also possible that at that time, The Role of the entire "ribosome" was performed by rRNA molecules folding in such a way as to create a complex system of surfaces that ensured both the directed pairing of tRNAs with mRNA codons and the catalysis of polymerization of tRNA-bound amino acids (see Fig. 1-7). Over the course of evolution, individual proteins could have joined this apparatus, each making the process more reliable and efficient. The high proportion of RNA in modern ribosomes is likely a relic of those very early evolutionary stages when proteins did not yet play a dominant role in biological catalysis.

Conclusion

For the synthesis of a specific protein to begin, the corresponding mRNA must first be produced (by DNA transcription). The small ribosomal subunit binds to this mRNA at its start codon, which is recognized by a special initiator tRNA. The binding of the large subunit completes the assembly of the ribosome. This is followed by the elongation phase of protein synthesis. During this phase, different aminoacyl-tRNAs, each charged with its specific amino acid, sequentially bind to the corresponding codon on the mRNA by base-pairing between the codon and the tRNA anticodon. Each subsequent amino acid is added to the carboxyl terminus of the growing polypeptide in a cyclic process consisting of three successive steps: aminoacyl-tRNA binding, peptide bond formation, and ribosome translocation. The ribosome moves along the mRNA molecule in the 5' → 3' direction from one codon to the next until one of the three stop codons is reached. A release factor then binds to this stop codon, causing the completed polypeptide to dissociate from the ribosome.

Prokaryotic and eukaryotic ribosomes share a high degree of homology, despite significant differences in the number and size of both types of components—rRNA and proteins. The predominant role of rRNA in ribosomal structure and function likely reflects THE ORIGIN OF protein synthesis, which evolved early on in an environment where catalysis was carried out by RNA.