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contents Principles of 50 Translation is the process by which a assembles from the .

The Rosetta stone. To translate from one language to another, you need a set of comparative rules that act as a template. The Rosetta stone is concrete evidence of how languages were first translated in early human cultures. By displaying the same story in different languages, it served as a template for comparison, a reference for how to get from one language to another. In cells, microscopic structures called serve as key sites that support the translation of the mRNA language into the language. Archiv/Photo Researchers/Science Source.

Topics Covered in this Module

Translating DNA into Proteins

Major Objectives of this Module

Describe the molecular structures involved in translation. Explain the process of translation in detail. Explain how post-translational processes prepare proteins for their functions.

page 256 of 989 3 pages left in this module

contents Principles of Biology

50 Translation

How does a genetic code with only four provide the information needed to generate proteins containing up to 20 different amino acids? For this process to occur, many with specific structures and function are required.

Translating DNA into Proteins Translation is the process of converting the information stored in mRNA into protein (Figure 1). Proteins are made up of a series of amino acids. With the help of adapter molecules called transfer (tRNAs) the appropriate is added for each set of three adjacent nucleotides in the mRNA, called a codon (Figure 2). Transfer RNAs transfer amino acids from a pool of cytoplasmic amino acids to the growing polypeptide.

Figure 1: Translation of mRNA into protein. mRNA carries genetic information required for the synthesis of a specific protein as a series of three- units called codons. In the process of translation, a sequence of nucleotides in a molecule of mRNA is converted to a sequence of amino acids in a polypeptide. A strand of mRNA is translated into a linear protein strand. Translation occurs in the 5′ to 3′ direction on the mRNA strand. © 2014 Nature Education All rights reserved. Figure Detail Figure 2: Transfer RNA (tRNA) links codons in mRNA to amino acids in proteins. tRNAs are adaptor molecules that with codons in the mRNA. They bring the amino acids corresponding to each codon and facilitate their addition to the growing polypeptide. © 2014 Nature Education All rights reserved. Figure Detail Why is a codon made up of three nucleotides, and not one or two nucleotides? For expression to occur successfully, different arrangements of the four mRNA nucleotides A, G, C, and U must determine the sequence of the amino acids that make up a protein. How are 20 different amino acids encoded using only 4 nucleotides? Since there are 20 amino acids and only 4 possible nucleotides, the relationship between a nucleotide in the mRNA sequence and an amino acid in the protein sequence cannot be one-to-one. If two nucleotides coded for an amino acid, they would only encode a maximum of 16 amino acids (42=16). However, if three nucleotides code for an amino acid, 64 variations (43=64) are possible. Experiments with short stretches of synthetic mRNA demonstrated that a codon is made up of three adjacent nucleotides. Since there are 64 possible codons and only 20 amino acids, most amino acids are coded for by multiple codons (Figure 3). There are also three stop codons, UAA, UAG, and UGA, which do not code for any amino acid but instead signal the termination of translation. Just as there are stop codons, there is also a . The codon AUG codes for the amino acid (Met), but if encountered in conjunction with other signals, AUG also indicates the start of translation. Figure 3: Table of codons. Three-letter sequences of nucleotides, called codons, code for amino acids. To find a specific codon in the table, start by finding the first letter on the left side of the table. Then find the second letter along the top and the third letter along the right side of the table. © 2013 Nature Education All rights reserved. Figure Detail

Test Yourself

If a changed the third nucleotide of a codon where the first nucleotide is U and the second nucleotide is C, what do you expect the result would be for the polypeptide generated?

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The genetic code is nearly universal to all known species on Earth. There are a few exceptions such as mitochondria, and some . However, it is clear that the exceptions are very few and affect very few codons. Furthermore, all known genetic codes are more similar than different to each other, which supports the assertion that all started from a common ancestor.

Transfer RNA carries amino acids to the , the site of protein synthesis. How do transfer RNA molecules add the correct amino acid to the growing protein? The mRNA and tRNAs are brought together by a protein-RNA complex called a ribosome. Each tRNA molecule has a specific amino acid at one end and a nucleotide triplet, known as an anticodon, at the other (Figure 4). The nucleotides in the anticodon base pair with the complementary codons on the mRNA in an antiparallel orientation. For example, the codon 5′–GCU–3′ will pair with the anticodon 3′–CGA–5′ and bring the tRNA for (Ala) into the ribosome. Figure 4: Schematic of tRNA. tRNAs are short molecules that are around 80 nucleotides long. In this schematic, the anticodon is highlighted at the bottom, and the amino acid corresponding to the tRNA appears at the top. © 2014 Nature Education All rights reserved. tRNA molecules are created by transcribing information stored in DNA. In , tRNA is generated in the nucleus and then exported to the to carry out its role in translation. In both prokaryotes and eukaryotes, a tRNA molecule is used over and over. It picks up an amino acid, adds it to a growing polypeptide chain, leaves the ribosome and is ready to pick up another amino acid and begin the process again. The process of matching a specific tRNA with the appropriate amino acid is carried out by a family of enzymes called aminoacyl-tRNA synthetases (Figure 5). There is a different synthetase for each of the 20 amino acids. Each synthetase contains an active site that fits only a specific combination of tRNA and amino acid. In addition, each synthetase is able to bind all of the tRNAs that code for its amino acid. The covalent attachment of the tRNA and the amino acid requires the hydrolysis of ATP. A tRNA to which an amino acid has been added is called an aminoacyl-tRNA or a charged tRNA. After charging, the aminoacyl-tRNA is released from the synthetase and is ready for translation. Figure 5: Formation of an aminoacyl-tRNA. Enzymes called aminoacyl-tRNA synthetases facilitate the covalent bonding of the appropriate amino acid to each tRNA molecule. In the first step (upper left), the catalyzes the linkage of ATP to an amino acid specific to each aminoacyl-tRNA synthetase. After the release of a pyrophosphate group, an amino acid-AMP intermediate remains bound to the enzyme (upper right). In the next step, the uncharged tRNA corresponding to the specific amino acid enters the active site of the enzyme (lower right), where it is linked to the amino acid, producing the charged tRNA and releasing AMP in the process (bottom). The charged tRNA leaves the active site, and the enzyme becomes available again (lower left). © 2014 Nature Education All rights reserved.

Test Yourself

What would you expect the result to be if a cell contains a mutation that knocks out a particular aminoacyl-tRNA synthetase?

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A ribosome is made up of a small subunit and a large subunit. A ribosome has three sites that the tRNA moves through as translation occurs. The growing polypeptide chain is held by the tRNA in the P site (Peptidyl-tRNA binding site). The charged tRNA carrying the next amino acid to be added enters at the A site (Aminoacyl-tRNA binding site). A bond is formed between the growing chain and the next amino acid to be added. This reaction moves the growing polypeptide to the tRNA in the A site. The tRNA in the A site now moves to the P site, and the tRNA in the P site moves to the E site (Exit site), where it is released from the ribosome (Figure 6). Figure 6: The three sites in the ribosome. Charged tRNA enters the ribosome in the A site, moves to the P site as a new peptide bond is formed between its amino acid and the growing polypeptide chain, and finally exits through the E site after its amino acid has been transferred. © 2014 Nature Education All rights reserved. Figure Detail

The ribosome keeps the mRNA and tRNA close to each other and brings the next amino acid to the carboxyl end of the growing polypeptide. Without the ribosome, the hydrogen bonding between the tRNA and mRNA would be too weak to hold it there long enough for a peptide bond to form. The ribosome catalyzes the peptide bond formation that adds the amino acid to the polypeptide. Experiments have supported the hypothesis that it is the ribosomal RNA (rRNA) and not the ribosomal proteins that contains the catalytic site for the peptide bond formation function of ribosomes. While the function of ribosomes is the same in prokaryotes and eukaryotes, the proteins and RNAs that make up their ribosomes are different. Humans have taken advantage of some of the differences between bacterial and human ribosomes to develop that are safe for human consumption but kill harmful by disrupting bacterial protein synthesis. We have discovered these antibiotics in microbes, which use them to disrupt competing bacteria's protein synthesis. For example, , a family of antibiotics produced by Streptomyces bacteria, bind to the small ribosomal subunit in competing bacteria and alter its structure so that an aminoacyl-tRNA cannot bind to the A site. We now prescribe these antibiotics as a treatment for bacterial . Unfortunately, many strains of the bacteria targeted by tetracyclines have developed a resistance to by producing a protein that protects the bacteria's ribosomes from binding the . The factors that influence the action of antibiotics and the evolutionary counter-measures of the bacteria they target are areas of great interest and importance at the intersection of basic biology and human health.

Enzymes mediate translation. Like , translation has three phases: initiation, elongation and termination. Each phase requires protein factors that aid in the process. And energy, provided by hydrolysis of (GTP), is required for initiation, elongation and termination. Initiation occurs when the small ribosomal subunit binds to the mRNA at the 5′ end and scans the mRNA in a 3′ direction until it reaches the start codon (Figure 7). A specialized initiator tRNA, with the anticodon 3′-UAC-5′, pairs with the start codon, 5′-AUG-3′, at the P site of the ribosome. In prokaryotes, an AUG must be preceded by a specific sequence in order to be interpreted as the start codon. This sequence is recognized by an mRNA binding site in the small ribosomal subunit and helps position the start codon in the P site. In eukaryotes, the first AUG encountered by the ribosome is the start codon, but translation initiation normally also requires the 5′ cap that is added during mRNA processing following transcription. The initiator tRNA is charged with methionine (Met) in eukaryotes and with a chemically modified methionine, N-formylmethionine (fMet), in prokaryotes. Once the start codon is found, proteins known as initiation factors facilitate the binding of the large ribosomal subunit to the small ribosomal subunit. GTP is hydrolyzed to provide energy for the assembly of the subunits to form the translation initiation complex.

Figure 7: Initiation of translation. The small ribosomal subunit binds the mRNA, and the initiator tRNA binds the start codon. The recognition of the start codon is facilitated by an protein. Two other initiation factors prevent the large ribosomal subunit from binding before the arrival of the initiator tRNA. Upon binding of the initiator tRNA to the mRNA, GTP is hydrolyzed, and the large subunit binds to the small subunit to form the translation initiation complex. © 2014 Nature Education All rights reserved.

Synthesis of the polypeptide always occurs in the same direction, with the initial methionine at the amino end, known as the N-terminus. Each amino acid is added at the carboxyl end of the previous amino acid. The end of a polypeptide sequence is thus known as the C-terminus. During elongation, amino acids are added to the C-terminus of the growing polypeptide. Elongation is a three-step process that requires the participation of GTP and proteins called elongation factors. First, the aminoacyl-tRNA with the anticodon matching the next codon on the mRNA is brought to the A site by an . Many tRNAs are present around the ribosome, but only the aminoacyl-tRNA with the appropriate anticodon will remain at the A site (Figure 8). When correct base pairing between the mRNA codon and aminoacyl-tRNA anticodon occurs, the elongation factor hydrolyzes GTP, which releases the aminoacyl-tRNA into the ribosome. Next, the large ribosomal subunit catalyzes the formation of a peptide bond between the carboxyl end of the growing polypeptide in the P site and the amino group of the amino acid in the A site. This moves the growing polypeptide to the tRNA in the A site and unlinks the polypeptide from the tRNA currently in the P site. Energy from GTP hydrolysis is not required in this step. Finally, the tRNA with the growing polypeptide chain is translocated from the A site to the P site. The uncharged tRNA that was in the P site is translocated to the E site, where it is released from the ribosome. The ribosome moves along the mRNA with the attached tRNAs, and the next codon is brought into the A site. This step is facilitated by another elongation factor, which requires the hydrolysis of GTP each time a translocation event occurs.

Figure 8: Elongation. Elongation requires the hydrolysis of two GTP molecules and involves matching an amino acid-charged tRNA to a codon in the mRNA, forming a new peptide bond between the new amino acid and the previous one, and translocating the ribosome to the next codon. © 2014 Nature Education All rights reserved. Elongation continues until a is reached. The codons UAG, UAA and UGA do not code for amino acids, and there are normally no tRNAs with anticodons corresponding to the stop codons; instead, these three codons are signals to terminate translation. When a stop codon appears at the A site, a protein shaped like an aminoacyl-tRNA called a enters the A site and binds to the stop codon. The release factor causes a water molecule to be added to the polypeptide chain instead of an amino acid. This breaks the bond between the tRNA and the polypeptide in the P site. The polypeptide is released through the exit tunnel of the ribosome's large subunit. The translation assembly breaks apart in a process that requires the hydrolysis of a GTP molecule (Figure 9). Figure 10 summarizes the steps of translation. There are several notable differences in the translation process between prokaryotes and eukaryotes. In prokaryotes, a single mRNA may have multiple start and stop codons and can therefore direct the production of multiple polypeptides. Furthermore, prokaryotic translation may start as soon as a start codon is transcribed from the DNA since there is no mRNA processing or separation of nucleus and cytoplasm. In eukaryotes, mRNA is transcribed and processed in the nucleus and then exported to the cytoplasm for translation. As a result, transcription and translation are separated in space and time and cannot occur simultaneously as in prokaryotes. In addition, translation initiation requires the 5′ cap, so only the AUG nearest the 5′ cap is used as the start codon even if there are multiple start and stop codons in the mRNA. As a result, a single mRNA usually directs the production of only one polypeptide in eukaryotes.

Figure 9: Termination of translation. Translation terminates when the ribosome encounters a stop codon, and a release factor (instead of a tRNA) binds to the stop codon in the A site. The release factor helps separate the complete polypeptide from the last tRNA. The complete polypeptide is released from the ribosome, and the ribosomal subunits are disassembled and recycled for translating other mRNAs. Termination requires the hydrolysis of a GTP molecule. © 2014 Nature Education All rights reserved. Figure 10: Putting the steps of translation together. This figure summarizes the three phases of translation: initiation, elongation, and termination. Each phase involves a specific set of protein factors, as well as chemical energy largely supplied by GTP hydrolysis. © 2014 Nature Education All rights reserved.

Test Yourself

What steps of translation require energy to occur?

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It takes less than a minute for a ribosome to translate an average-size polypeptide. And multiple ribosomes are able to translate an mRNA molecule at the same time. Once a ribosome has cleared the start codon, another ribosome is able to attach to the start site and begin translation. A string of ribosomes along an mRNA is called a polyribosome (Figure 11). Polyribosomes allow a cell to make large quantities of protein very quickly. Figure 11: Polyribosomes. Multiple ribosomes can translate an mRNA molecule at the same time. In this figure, each ribosome is synthesizing a polypeptide independently of the others, resulting in a large number of polypeptide molecules synthesized using the same mRNA molecule. © 2014 Nature Education All rights reserved.

Post-translational modifications prepare proteins for their functions. As translation occurs, the polypeptide folds spontaneously due to its primary sequence. Frequently, a chaperone protein called chaperonin also aids in proper folding. However, many proteins require additional modifications after translation to become fully functional. There are many types of post-translational modification. is the cleavage of a regulatory subunit from a polypeptide to convert an inactive precursor to its active form. is the addition of carbohydrates to the polypeptide chain to aid in targeting and recognition. is the addition of phosphate groups to the polypeptide to alter the shape and therefore the activity of the protein. And other proteins may not be functional alone but require assembly with other polypeptides to form a functional multi- subunit protein complex. In addition to post-translational modifications, proteins sometimes must be targeted to specific locations in the cell to be functional. For example, a sodium-potassium pump serves no purpose in the but must be inserted into the plasma membrane to move ions in and out of the cell. Other proteins, such as peptide hormones, are ultimately secreted out of the cell. Instead of remaining in the cytosol, such proteins must pass through the secretory pathway — (ER), Golgi apparatus and plasma membrane. Regardless of a polypeptide's final destination, translation always begins with free ribosomes in the cytosol and will continue there unless the growing polypeptide contains a signal that causes the ribosome to attach to the ER. Polypeptides that are destined to be integral membrane proteins or secreted proteins contain a signal peptide, an approximately 20 amino acid sequence near the N-terminus. This sequence is recognized as it emerges from the ribosome by a protein-RNA complex known as the signal- recognition particle (SRP). The SRP escorts the ribosome to a receptor protein in the ER membrane, and the polypeptide is translocated across the ER membrane into the ER lumen as translation continues (Figure 12). From there, the polypeptide passes through the Golgi apparatus to the plasma membrane, where it is exocytosed (for a secreted protein) or remains embedded in the membrane (for an integral membrane protein).

Figure 12: Targeting polypeptides to the ER. A signal-recognition particle (SRP) directs the protein-synthesizing ribosome to the ER membrane. Translation of the polypeptide continues as the polypeptide is directly translocated across the membrane. © 2014 Nature Education All rights reserved.

There are other signal that bring polypeptides to other cellular structures including the nucleus, mitochondria, chloroplasts and other . However, for these structures, translation occurs in the cytosol and then the polypeptide is imported into the appropriate structure. Test Yourself

How does the sequence of a polypeptide target it to a specific structure within the cell?

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BIOSKILL Western Blot. How do scientists determine if a protein is being synthesized? One technique for detecting a specific protein from a cell sample or tissue is a western blot, also known as a protein immunoblot. Proteins are separated using gel electrophoresis. One type of gel electrophoresis is called SDS-PAGE, which separates proteins by size. Next, the proteins are transferred to a special blotting paper, generally made of nitrocellulose. The protein pattern from the gel is the same as the pattern on the blotting paper. The blot is then "blocked" with nonspecific proteins, commonly derived from cow's milk, which will cover any open protein-binding regions left on the paper. To identify the specific protein of interest, an antibody is added that will only bind to the specific protein. Once the primary antibody is bound, a secondary antibody is added that binds to the primary antibody. The secondary antibody is bound to an enzyme or chemical that becomes visible when the proper substrate is added (Figure 13).

Figure 13: Western blot. A specific protein can be visualized by isolating the protein by gel electrophoresis and using a specific antibody to identify the protein on a paper blot. In the first step, a mixture of protein is separated by size using SDS-PAGE electrophoresis. The proteins are then transferred to a nitrocellulose membrane, and the membrane is probed with an antibody specific to the protein of interest. The antibody itself may carry a label, or it may be recognized by a labeled secondary antibody (bottom). In both cases, the label results in the appearance of a dark band corresponding to the protein of interest when the membrane is exposed to film. © 2011 Nature Education All rights reserved. Figure Detail Test Yourself

Researchers have created a line of mutant cells that are missing a particular protein. A western blot is performed to look at a sample of wild-type cells and a sample of the mutant cells. What do you expect to see on the western blot?

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BIOSKILL

IN THIS MODULE

Translating DNA into Proteins Summary Test Your Knowledge

WHY DOES THIS TOPIC MATTER?

Stem Cells Stem cells are powerful tools in biology and . What can scientists do with these cells and their incredible potential?

Cancer: What's Old Is New Again Is ancient, or is it largely a product of modern times? Can cutting-edge research lead to prevention and treatment strategies that could make cancer obsolete?

Synthetic Biology: Making Life from Bits and Pieces Scientists are combining biology and engineering to change the world.

PRIMARY LITERATURE

Classic paper: How scientists discovered the enzyme that turns RNA into DNA (1970) RNA-dependent DNA polymerase in virions of RNA tumour . View | Download

SCIENCE ON THE WEB

How Small? See the difference between a coffee bean and a single atom.

page 257 of 989 2 pages left in this module

contents Principles of Biology

50 Translation Summary OBJECTIVE Describe the molecular structures involved in translation. Ribosomes are RNA-protein complexes that facilitate translation. Aminoacyl-tRNA synthetases covalently link amino acids to their appropriate tRNA molecule(s).

OBJECTIVE Explain the process of translation in detail. Translation occurs in three phases: initiation, elongation, and termination. During initiation, the ribosome forms and the initiator tRNA provides the first amino acid. During elongation, tRNA molecules bring the appropriate amino acids to the ribosome using base pairing between the codon on the mRNA and the anticodon on the tRNA. The amino acid is added to the growing polypeptide chain. Termination occurs when the stop codon is reached.

OBJECTIVE Explain how post-translational processes prepare proteins for their functions. There are a variety of post-translational processes that may be required for a polypeptide to become functional. Polypeptides may be cleaved or brought together. Glycosylation and phosphorylation add specific chemical groups to the polypeptide.

Key Terms aminoacyl-tRNA synthetase An enzyme that charges tRNA molecules with their appropriate amino acid; a different synthetase exists for each of the 20 amino acids.

anticodon The region of a tRNA that is complementary to a codon on mRNA.

elongation factor One of multiple proteins that facilitates the lengthening of a polypeptide during protein synthesis.

guanosine triphosphate (GTP) A nucleotide similar to ATP that provides energy during several steps of protein synthesis.

initiation factor One of several proteins that facilitate the recognition of the start codon and the subsequent assembly of the large and small subunits of the ribosome.

polyribosome String of ribosomes on a single mRNA molecule that allows the cell to make large quantities of protein rapidly. release factor One of several proteins that recognize stop codons and facilitate the detachment of the completed polypeptide from the ribosome at the end of translation. ribosome A protein-RNA complex that facilitates the interaction of mRNA and tRNA; site of protein synthesis. signal peptide A sequence of twenty amino acids near the N-terminus that redirects protein synthesis to the ER in conjunction with the signal-recognition particle. signal-recognition particle (SRP) A protein-RNA complex that recognizes and binds to signal peptides, facilitating the translocation of free ribosomes to the ER surface. transfer RNA (tRNA) A specialized adapter molecule that brings a specific amino acid to the ribosome during protein synthesis; contains an anticodon complementary to a specific codon in the mRNA.

IN THIS MODULE

Translating DNA into Proteins Summary Test Your Knowledge

WHY DOES THIS TOPIC MATTER?

Stem Cells Stem cells are powerful tools in biology and medicine. What can scientists do with these cells and their incredible potential?

Cancer: What's Old Is New Again Is cancer ancient, or is it largely a product of modern times? Can cutting-edge research lead to prevention and treatment strategies that could make cancer obsolete?

Synthetic Biology: Making Life from Bits and Pieces Scientists are combining biology and engineering to change the world.

PRIMARY LITERATURE

Classic paper: How scientists discovered the enzyme that turns RNA into DNA (1970) RNA-dependent DNA polymerase in virions of RNA tumour viruses. View | Download

SCIENCE ON THE WEB

How Small? See the difference between a coffee bean and a single atom.

page 258 of 989 1 pages left in this module

contents Principles of Biology

50 Translation

Test Your Knowledge

1. One codon for has the sequence AAC. What is the sequence of the anticodon on the aminoacyl-tRNA (in the 5′ to 3′ direction)?

CAA GUU GGA UUG TTG

2. All polypeptides in eukaryotes have which of the following in common?

All polypeptides start with the same amino acid. All polypeptides end with the same amino acid. All polypeptides have the same number of amino acids. All polypeptides are cleaved before they become active. None of the answers are correct.

3. Which of the following are post-translational modifications?

glycosylation phosphorylation protein cleavage multiple polypeptides binding together All answers are correct.

4. Which of the following first binds to the mRNA at the translation initiation site?

GTP aminoacyl-tRNA synthetase small ribosomal subunit large ribosomal subunit aminoacyl-tRNA

5. How many different aminoacyl-tRNA synthetases exist?

20 21 64 4 16

Submit

IN THIS MODULE

Translating DNA into Proteins Summary Test Your Knowledge

WHY DOES THIS TOPIC MATTER?

Stem Cells Stem cells are powerful tools in biology and medicine. What can scientists do with these cells and their incredible potential?

Cancer: What's Old Is New Again Is cancer ancient, or is it largely a product of modern times? Can cutting-edge research lead to prevention and treatment strategies that could make cancer obsolete?

Synthetic Biology: Making Life from Bits and Pieces Scientists are combining biology and engineering to change the world.

PRIMARY LITERATURE

Classic paper: How scientists discovered the enzyme that turns RNA into DNA (1970) RNA-dependent DNA polymerase in virions of RNA tumour viruses. View | Download

SCIENCE ON THE WEB

How Small? See the difference between a coffee bean and a single atom.

page 259 of 989