Protein Synthesis

Proteins are the workhorses of the cell, controlling virtually every reaction within as well as providing structure and serving as signals to other cells. Proteins are long chains of amino acids , and the exact sequence of the amino acids determines the final structure and function of the protein. Instructions for that sequence are encoded in genes . To make a particular protein, a messenger ribonucleic acid (mRNA) copy is made from the gene (in the process called transcription ), and the mRNA is transported to the ribosome . Protein synthesis, also called translation , begins when the two ribosomal subunits link onto the mRNA. This step, called initiation, is followed by elongation, in which successive amino acids are added to the growing chain, brought in by transfer RNAs (tRNAs). In this step, the ribosome reads the nucleotides of mRNA three by three, in units called codons , and matches each to three nucleotides on the tRNA, called the anticodon. Finally, during termination, the ribosome unbinds from the mRNA, and the amino acid chain goes on to be processed and folded to make the final, functional protein.

Initiation

In the first step, initiation, the ribosome must bind the mRNA and find the appropriate place to start translating it to make the protein. If the ribosome starts translating the mRNA in the wrong place, the wrong protein will be synthesized. This is a particularly tricky problem because there are three different reading frames in which an mRNA can be read. Each unit of the genetic code , called a codon, is made up of three bases and codes for one amino acid. Completely different protein sequences will be read out by the ribosome if it starts translating with the start of the first codon at base 0, base 1, or base 2 (Figure 1). Thus, it is easy to see why the ribosome must have a way to find the correct starting point for translating each different mRNA.

In almost every known case, translation begins at the three-base codon that codes for the amino acid methionine. This codon has the sequence AUG. Ribosomes are made up of two parts, called subunits, that contain both protein and RNA components. It is the job of the smaller ribosomal subunit to locate the AUG codon that will be used as the starting point for translation (called the initiation codon). Although always starting at AUG helps solve the reading frame problem, finding the right AUG is not an entirely straightforward task. There is often more than one AUG codon in an mRNA, and the small ribosomal subunit must find the correct one if the right protein is to be made.

Initiation in Prokaryotes. In prokaryotes (bacteria) there is a nucleotide sequence on the upstream (5-prime, or 5′) side of the initiation codon that tells the ribosome that the next AUG sequence is the correct place to start translating the mRNA. This sequence is called the Shine-Delgarno sequence, after its discoverers. The Shine-Delgarno sequence forms base pairs with RNA in the small ribosomal subunit, thus binding the ribosomal subunit to the mRNA near the initiation codon.

Next, a special tRNA forms base pairs with the AUG sequence of the initiation codon. The tRNA contains the complementary sequence to AUG as its anticodon. This tRNA carries a modified version of the amino acid methionine (fMet-tRNA_i or formylmethionyl initiator tRNA) and is already bound to the small ribosomal subunit. The interaction of codon and anti-codon triggers a series of events that is not entirely understood but that results in the joining of the large ribosomal subunit to the small ribosomal subunit. The resulting complex is called an initiation complex; it is a whole ribosome bound to an mRNA and an initiator tRNA, positioned so as to make the correct protein from the mRNA.

Initiation in Eukaryotes. In eukaryotes (animals, plants, fungi, and protists), the Shine-Delgarno sequence is missing from the small ribosomal subunit's RNA, and thus a different mechanism is used for locating the initiation codon. The strategy employed by eukaryotes is more complex and less well understood than that used by prokaryotes. In eukaryotes, the small ribosomal subunit is thought to bind to the 5′ end of the mRNA. This binding is mediated by a special structure on the 5′ end of eukaryotic mRNAs called a 7-methylguanosine cap and is also aided by a special tail of adenosine bases (the poly-A tail) on the 3′ end, both of which are added during RNA processing. A group of proteins called initiation factors binds to the 7-methyl-guanosine cap and poly(A) tail and appears to direct the binding of the small ribosomal subunit to the mRNA near the cap structure.

Once this has happened, the small ribosomal subunit can read along the mRNA and look for an AUG codon, a process called scanning. Recognition of the initiation codon is largely mediated by base-pairing interactions between the AUG codon and the anticodon sequence in a methionyl initiator tRNA (Met-tRNA_i; the methionine is not modified with a formyl group in eukaryotes as it is in prokaryotes ). As in prokaryotes, this Met-tRNA is already bound to the small ribosomal subunit.

In most cases, the first AUG codon in a eukaryotic mRNA is used as the initiation codon, thus the small subunit locates the correct initiation codon simply by scanning along the mRNA starting at the 5′ end until it reaches the first AUG codon. However, the initiation AUG codon may be flanked by certain base sequences not found around other AUG codons not used for initiation. This preferred set of bases around the initiation codon is called the Kozak sequence, named after its discoverer, Marilyn Kozak. How the Kozak sequence helps direct the small ribosomal subunit to use one AUG codon instead of another is not known. As is the case in prokaryotes, once the correct AUG codon has been found, a complex series of steps takes place that results in the joining of the large ribosomal subunit to the small ribosomal subunit to produce an initiation complex: a complete ribosome assembled at the correct place on an mRNA with an initiator tRNA bound to it.

In both prokaryotes and eukaryotes there are proteins called initiation factors that are required for the correct assembly of an initiation complex. In prokaryotes there are three initiation factors, logically enough called IF1, IF2, and IF3. IF2 helps the fMet-tRNA_i bind to the small ribosomal subunit. IF3's main role appears to be to ensure that an AUG, and not another codon, is used as the starting site of translation. That is, IF3 monitors the fidelity of the selection of the initiation codon. IF1 appears to prevent the initiator tRNA from binding to the wrong place in the small ribosomal subunit. In eukaryotes, the situation is considerably more complex, with at least twenty-four protein components required for the initiation process.

The antibiotic tetracycline prevents tRNA from binding to the A sites.

Elongation

In the next phase of protein synthesis, elongation, the ribosome joins amino acids together in the sequence determined by the mRNA to make the corresponding protein. Amino acids are brought onto the ribosome attached to tRNAs. tRNAs are the adapter molecules that allow the ribosome to translate the information contained in the codon sequence of the mRNA into the amino acid sequence of a protein. This decoding happens by base pairing between the anticodon bases of the tRNA and the codon bases of the mRNA. When all three anticodon bases of the tRNA form base pairs with the next codon of the mRNA, the ribosome, with the aid of an elongation factor protein, recognizes that this tRNA has the correct amino acid attached to it and adds this amino acid to the growing protein chain. The process can then be repeated until the entire protein has been synthesized.

As just mentioned, elongation requires the help of elongation factor proteins. The tRNAs with attached amino acids (called aminoacyl tRNAs) are brought onto the ribosome by one such elongation factor. This factor is called EF-Tu in prokaryotes and EF1 in eukaryotes. Its job is to bring aminoacyl tRNAs onto the ribosome and then to help the ribosome make sure that this tRNA has the correct amino acid attached to it. The ribosome has three aminoacyl tRNA binding sites: the acceptor site (A), the peptidyl site (P), and the exit site (E). The tRNA that has the growing protein attached to it binds in the P site (hence the name peptidyl, for peptide). The incoming aminoacyl tRNA, containing the next amino acid to be added, binds in the A site. The A site is where decoding of the genetic code takes place; the correct aminoacyl tRNA is selected to match the next codon of the mRNA. Spent tRNAs that no longer have an amino acid or the growing peptide chain attached to them end up in the E site, from

which they fall off the ribosome back into the cytoplasm , where they can pick up new amino acids.

Once the A site is occupied by the correct tRNA, the ribosome links the new amino acid to the growing peptide chain. It does this by catalyzing the formation of a peptide (amide) bond between the amino (NH₂) group of the new amino acid in the A site and the carbonyl (CO) group that attaches the growing protein chain to the tRNA in the P site (Figure 2). This results in an intermediate state of the ribosome, called a hybrid state, in which the tRNA in the P site has lost the growing protein chain and moved partially into the E site, and the tRNA in the A site now has the growing protein chain attached to it and has moved partially into the P site.

To complete the round of elongation, a second elongation factor, called EF-G in prokaryotes and EF2 in eukaryotes, is needed. This elongation factor moves the tRNAs such that the spent tRNA that has lost the protein chain moves fully into the E site, and the tRNA with the growing protein chain moves fully into the P site. The mRNA is also shifted over one codon by EF-G, so that the next codon is in the A site. The A site is now empty of tRNAs and the next aminoacyl tRNA can be brought into it.

Many antibiotics (drugs that kill bacteria) affect the elongation phase of prokaryotic translation. Some decrease the fidelity (accuracy) with which the ribosome decodes the mRNA and the wrong amino acids get put into the proteins. This decrease in fidelity leads to an accumulation of proteins that do not work, which eventually kills the bacterium. Other antibiotics prevent the formation of the peptide bond or the movement of the tRNAs by EF-G after the peptide bond has been formed. The reason these drugs are effective on bacteria without killing the patient is that prokaryotic ribosomes have some different structural features than eukaryotic ribosomes, and thus these drugs can bind to the prokaryotic (bacterial) ribosomes but not the eukaryotic (that is, human) ribosomes. Since viruses use human ribosomes to reproduce, these antibiotics are not effective against them.

Termination

The end of the code for the protein in the mRNA is signaled by one of three special codons called stop codons. These stop codons have the sequences UAA, UAG, and UGA. In prokaryotes, the stop codons are bound by one of two release factor proteins (RFs) in prokaryotes: RF1 or RF2. These release factors cause the ribosome to cleave the finished protein off the tRNA in the P site. A third release factor, RF3, is responsible for releasing RF1 and RF2 from the ribosome after they have recognized the stop codon and caused the protein to be cleaved off the tRNA. Eukaryotes appear to have one protein, eRF1, that performs the functions of RF1 and RF2, and a second protein, eRF3, that performs the function of RF3. Once released, the protein can then go on to perform its function in the cell.

After the protein has been cleaved off the tRNA, the two ribosomal subunits must be dissociated from one another so that the ribosome can start translating another mRNA. This process is called recycling. In prokaryotes, recycling requires three proteins: one initiation factor (IF3), one elongation factor (EF-G), and a ribosome recycling factor called RRF. Once the subunits are dissociated from each other the whole process of translation can begin again.

Protein Folding

A functional protein is not a long, stretched-out chain of amino acids but rather a complex, three-dimensional structure. That is, each protein must fold up into a particular shape, or conformation , in order to perform its function in the cell. The evidence strongly suggests that all of the information required for the protein to fold into its correct three-dimensional structure is contained in the amino acid sequence of the protein (rather than, say, being determined by some other factor in the cell). However, as the protein is being synthesized on the ribosome there is a danger that the unfinished protein will begin to fold up incorrectly because the rest of the protein has not yet been made. It is also possible that the unfinished protein will interact with other unfinished proteins being made on other ribosomes and form what is called an aggregate : a network of partially folded proteins that have interacted with each other rather than with themselves, thus producing a mess inside the cell. Such protein aggregates can be fatal for the cell. It is the job of a class of proteins called chaperones to bind to the growing protein chains as they are synthesized by ribosomes and prevent aggregates from forming or the proteins from folding incorrectly before they have been fully synthesized. Chaperones may also help proteins efficiently fold up into the correct three-dimensional structure once translation is complete.

Protein Modification

While the mRNA encodes the complete amino acid sequence of the corresponding protein, some proteins are altered after they are translated. This process is called post-translational modification. For example, some proteases (proteins that digest other proteins) are synthesized by the ribosome as precursor proteins (pro-proteins) that contain an extra sequence of amino acids at one end that prevents them from digesting any proteins until they get to the right place (usually outside of the cell). Once the proteases reach their destination, the amino acid sequences that prevent them from being active (called pro-sequences) are removed (by another protein), and the proteases can begin digesting other proteins. If these pro-sequences did not exist, the proteases would digest all of the useful proteins inside the cells that made them—which would not be a good thing.

Many proteins made by eukaryotic cells are modified by having sugars attached to various amino acids, a process called glycosylation. Proteins that are destined to be exported from the cell or are going to be inserted into the cell's membrane enter the endoplasmic reticulum (ER) as they are synthesized by ribosomes that bind to the surface of the ER and feed the new proteins into the ER through small pores. Inside the ER, sugars are added to the protein, which is then sent to the Golgi apparatus where some of the sugars are removed and additional sugars are added. The role of protein glycosylation is not well understood, but because many euykaryotic proteins are glycosylated, it is clearly important.

There are a number of additional ways that proteins can be modified after they are made. For example, many proteins can have one or more phosphate groups added to them by enzymes called kinases . These phosphorylations are often used by the cell to regulate the activity of specific proteins; the phosphorylated form of the protein often has different properties than the unphosphorylated form.

Protein Degradation

When a protein has outlived its usefulness or become damaged, it is degraded by the cell. In eukaryotes, a protein that is to be degraded has a number of copies of the small protein ubiquitin attached to it by a series of ubiquitin-adding enzymes. Ubiquitin serves as a tag that marks the protein for degradation. A tagged protein is then sucked into a large cellular machine called the proteasome, which itself is made up of a number of protein components and looks something like a trash can. Inside the proteasome, the tagged protein is digested into small peptide fragments that are released into the cytoplasm where they can be further digested into free amino acids by other proteases. The life of a protein begins in one cellular machine called the ribosome and ends in another called the proteasome.

see also Endoplasmic Reticulum; Genetic Code; Golgi; Protein Structure; Ribosome; RNA; RNA Processing

Jon Lorsch

Bibliography

Lewin, Benjamin. Genes VI. Oxford: Oxford University Press, 1997.

Merrick, William C., and John W. B. Hershey. "The Pathway and Mechanism of Eukaryotic Protein Synthesis." In Translational Control. Plainview, NY: Cold Spring Harbor Laboratory Press, 1996.

Stryer, Lubert. Biochemistry, 4th ed. New York: W. H. Freeman and Company, 1995.

Protein Synthesis

There is no task more important to the function of living cells than the synthesis of proteins. Because proteins carry out so many different tasks, the mechanism to synthesize them is intricate. There are several stages involved in the synthesis process, including transcription and translation.

Transcription

The primary role of deoxyribonucleic acid (DNA ) is to direct the synthesis of proteins. DNA, however, is located in the nucleus of the cell, and

protein synthesis occurs in cellular structures called ribosomes , found out-side the nucleus. The process by which genetic information is transferred from the nucleus to the ribosomes is called transcription. During transcription, a strand of ribonucleic acid (RNA) is synthesized. This messenger RNA (mRNA) is complementary to the portion of DNA that directed it—it has a complementary nucleotide at each point in the chain.

A specialized protein called an enzyme controls when transcription occurs. The enzyme called RNA polymerase is present in all cells; eukaryotic cells have three types of this enzyme. DNA has a section called the promoter region that identifies the sites where transcription starts and must be recognized by one subunit of the RNA polymerase called the sigma (σ) factor. Recognition between the promoter and the σ-factor helps to regulate how often a particular gene is transcribed. Once bound, the polymerase initiates the construction of mRNA (or other RNA molecules).

Initiation of the synthesis of a new RNA molecule does not always lead to a complete synthesis. After roughly ten nucleotides have been strung together, the continued addition of complementary base pairs takes place more readily in a process called elongation. The speed of addition of new nucleotides is remarkable—between twenty and fifty nucleotides per second can be added at body temperature.

Eventually the elongation process must stop. There are certain sequences of nucleotides that stop elongation, a process called termination. Often, termination occurs when the newly formed section of RNA loops back on itself in a tight formation called a hairpin. Once the hairpin structure has formed, the last component is then a string of uracil residues.

After transcription has taken place, the mRNA produced is not necessarily ready to direct the subsequent protein synthesis. Depending on the type of cell, segments of nucleotides may be removed or appended before the actual synthesis process takes place. This type of post-transcriptional processing often occurs in human cells.

Translation

Once the mRNA has been synthesized, and perhaps modified, the next step of protein synthesis, translation, takes place. For this stage, additional forms of RNA are needed.

Transfer RNA (tRNA) plays the role of carrying an amino acid to the synthesis site at the ribosome. tRNA molecules are relatively small, with around seventy-five nucleotides in a single strand. They form several loops, one of which is an anti-codon, a three-residue series that is complementary to the codon present in the mRNA (Figure 2). The opposite end of the tRNA is where an amino acid is bound. The correct binding of an amino acid to a specific tRNA is every bit as important as the anti-codon in ensuring that the correct amino acid is incorporated in the polypeptide that is synthesized. There are different tRNA molecules for each of the twenty amino acids that are present in living systems; some amino acids have more than one tRNA that carry them to the synthesis site.

When translation begins, mRNA forms a complex with a ribosome to form an assembly site. This complex requires the assistance of proteins called initiation factors, so the existence of an mRNA does not mean that a protein will always be synthesized. The first tRNA that takes part in the initiation always carries the same amino acid, methionine. When the protein is completely synthesized, this initial methionine is often removed.

With the initial methionine in place, another tRNA with its amino acid joins the assembly site as dictated by the codon on the mRNA. With two amino acids present, a peptide bond can be formed and the polypeptide can begin forming. The new amino acid is added to the carbon end of the polypeptide (the C-terminus) with the peptide bond forming between the C-O of the polypeptide and the amine of the new amino acid. This structural specificity is enforced by the nature of the binding between the amino acid and the tRNA. The portion of the amino acid that is unbound in the tRNA complex is the amine.

Elongation ultimately requires the repetition of several steps: (1) The tRNA–amino acid complexes must be made. (2) This complex must bind to the mRNA-ribosome assembly site. The correct amino acid is assured by the matching of the anti-codon on the tRNA to the codon on the mRNA.(3) A peptide bond is formed between the new amino acid and the growing polypeptide chain. (4) The amino acid is cleaved from the tRNA, which can be cycled back to form another complex with an amino acid for a later synthesis. (5) The growing polypeptide forms a fiber-like tendril. (6) The ribosome essentially moves along the mRNA, reopening the initiation site for additional protein synthesis. In this way, proteins are synthesized by several ribosomes acting on the same mRNA molecule.

The structure of the ribosome plays an important role in this elongation process. There must be two sites available for synthesis to occur. One site, called the P site (for peptide), is where the growing (or nascent) polypeptide is located. Adjacent to this location is another site where the tRNA with

its new amino acid can bind. This site is called the A site (for the amino acid that is delivered there along with the tRNA).

As was the case in the elongation of mRNA noted earlier, somehow the emerging polypeptide must stop adding amino acids. The termination is actually part of the coding present in the codons. Three specific codons are known as stop codes, and when they are present in mRNA, the elongation is stopped.

Despite the overall complexity of this process, it occurs with remarkable accuracy. The rate of error is roughly one in every 10,000 amino acids. Using the processes of transcription and translation, the body makes an amazing number and variety of proteins.

The transcription and translation processes provide the correct primary structure of the protein. The protein must fold to obtain the correct secondary and tertiary structures. Protein folding remains an active research area.

Medical Applications

Protein synthesis is critical to the growth of cells; medicines that work by killing cells often target this process. A majority of antibiotics work by disrupting the translation process. Tetracycline is an antibiotic that inhibits the binding of tRNA to the assembly site. Streptomycin works by causing the translation process to make more mistakes than usual—as high as one mistake for every 100 amino acids. Proteins with this many errors are not capable of performing their tasks, and the cells (in this case, bacteria) die. Streptomycin also inhibits the initiation of the synthesis process.

see also Peptide Bond, Proteins.

Thomas A. Holme

Bibliography

Adams, R. L. P.; Knowler, J. T.; and Leader, D. P. (1992). The Biochemistry of the Nucleic Acids. New York: Chapman and Hall.

Darnell, J. E., Jr. (1985). "RNA." Scientific American 253 (4): 68–78.

Lake, J. A. (1981). "The Ribosome." Scientific American 245 (2): 84–97.

Protein synthesis

Protein synthesis represents the final stage in the translation of genetic information from DNA , via messenger RNA (mRNA), to protein. It can be viewed as a four-stage process, consisting of amino acid activation, translation initiation, chain elongation, and termination. The events are similar in both prokaryotes, such as bacteria , and higher eukaryotic organisms, although in the latter there are more factors involved in the process.

To begin with, each of the 20 cellular amino acids are combined chemically with a transfer RNA (tRNA) molecule to create a specific aminoacyl-tRNA for each amino acid. The process is catalyzed by a group of enzymes called aminoacyltRNA synthetases, which are highly specific with respect to the amino acid that they activate. The initiation of translation starts with the binding of the small subunit of a ribosome, (30S in prokaryotes, 40S in eukaryotes ) to the initiation codon with the nucleotide sequence AUG, on the mRNA transcript. In prokaryotes, a sequence to the left of the AUG codon is recognized. This is the Shine-Delgrano sequence and is complementary to part of the small ribosome subunit. Eukaryotic ribosomes start with the AUG nearest the 5'-end of the mRNA, and recognize it by means of a "cap" of 7-methylguanosine triphosphate. After locating the cap, the small ribosome subunit moves along the mRNA until it meets the first AUG codon, where it combines with the large ribosomal subunit.

In both prokaryotes and eukaryotes, the initiation complex is prepared for the addition of the large ribosomal subunit at the AUG site, by the release of initiation factor (IF) 3. In bacteria, the large 50S ribosomal subunit appears simply to replace IF–3, with IF–1 and IF–2. In eukaryotes, another factor eIF–5 (eukaryotic initiation factor 5), catalyses the departure of the previous initiation factors and the joining of the large 60S ribosomal subunit. In both cases, the release of initiation factor 2 involves the hydrolysis of the GTP bound to it. At this stage, the first aminoacyl-tRNA, Met-tRNA, is bound to the ribosome. The ribosome can accommodate two tRNA molecules at once. One of these carries the Met-tRNA at initiation, or the peptide-tRNA complex during elongation and is thus called the P (peptide) site, while the other accepts incoming aminoacyl-tRNA and is therefore called the A (acceptor) site. What binds to the A site is usually a complex of GTP, elongation factor EF-TU, and aminoacyl-tRNA. The tRNA is aligned with the next codon on the mRNA, which is to be read and the elongation factor guides it to the correct nucleotide triplet. The energy providing GTP is then hydrolysed to GDP and the complex of EF-TU:GDP leaves the ribosome. The GDP is released from the complex when the EF-TU complexes with EF-TS, which is then replaced by GTP. The recycled EF-TU: GTP is then ready to pick up another aminoacyl-tRNA for addition to the growing polypeptide chain. On the ribosome, a reaction is catalysed between the carboxyl of the P site occupant and the free amino group of the A site occupant, linking the two together and promoting the growth of the polypeptide chain. The peptidyl transferase activity which catalyses this transfer is intrinsic to the ribosome. The final step of elongation is the movement of the ribosome relative to the mRNA accompanied by the translocation of the peptidyl-tRNA from the A to the P. Elongation factor EF-G is involved in this step and a complex of EF-G and GTP binds to the ribosome, GTP being hydrolysed in the course of the reaction. The de-acylated tRNA is also released at this time.

The end of polypeptide synthesis is signalled by a termination codon contacting the A site. Three prokaryotic release factors (RF) are known: RF–1 is specific for termination codons UAA and UAG, while RF–2 is specific for UAA and UGA. RF–3 stimulates RF–1 and RF–2, but does not in itself recognize the termination codons. RF–3 also has GTPase activity and appears to accelerate the termination at the expense of GTP. Only one eukaryotic release factor is known and it has GTPase activity.

At any one time, there can be several ribosomes positioned along the mRNA and thus initiation, elongation and termination proceed simultaneously on the same length of mRNA. The three dimensional structure of the final protein begins to appear during protein synthesis before translation is completed. In many cases, after the synthesis of the amino acid chain, proteins are subjected to further reactions which convert them to their biologically active forms, e.g., by the attachment of chemical groups or by removal of certain amino acids—a processes known as post-translational modification.

See also Deoxyribonucleic acid (DNA); Genetic code; Molecular biology and molecular genetics; Ribonucleic acid (RNA)

protein synthesis The process by which living cells manufacture proteins from their constituent amino acids, in accordance with the genetic information carried in the DNA of the chromosomes. This information is encoded in messenger RNA, which is transcribed from DNA in the nucleus of the cell (see genetic code; transcription): the sequence of amino acids in a particular protein is determined by the sequence of nucleotides in messenger RNA. At the ribosomes the information carried by messenger RNA is translated into the sequence of amino acids of the protein in the process of translation.