Molecular Biology and Cytogenetics - Translation (PDF)

Molecular Biology and Cytogenetics 8. Translation - from mRNA to protein Instructor: Dr. Mohammad Abukhalil Translation is the RNA-directed synthesis of a polypeptide ⚫ During translation, which is the second major step in gene expression, the mRNA is "read" according to t...

Molecular Biology and Cytogenetics 8. Translation - from mRNA to protein Instructor: Dr. Mohammad Abukhalil Translation is the RNA-directed synthesis of a polypeptide ⚫ During translation, which is the second major step in gene expression, the mRNA is "read" according to the genetic code, which relates the DNA sequence to the amino acid sequence in proteins. ⚫ Each group of three bases in mRNA constitutes a codon, and each codon specifies a particular amino acid (hence, it is a triplet code). The mRNA sequence is thus used as a template to assemble—in order—the chain of amino acids that form a protein. The Genetic Code ⚫ The genetic code is not information in itself but is a dictionary to translate the four-nucleotide sequence information in DNA to the 20–amino acid sequence information in proteins. ⚫ The genetic code is a dictionary that identifies the correspondence between a sequence of nucleotide bases and a sequence of amino acids. ⚫ Each individual word in the code is composed of three nucleotide bases. These genetic words are called codons.  Codons ⚫ Codons are presented in the messenger RNA (mRNA) language of adenine (A), guanine (G), cytosine (C), and uracil (U). ⚫ Their nucleotide sequences are always written from the 5′ end to the 3′ end. The four nucleotide bases are used to produce the three-base codons (a triplet code). ⚫ There are, therefore, 64 different combinations of bases, taken three at a time.  How to translate a codon ⚫ This table (or “dictionary”) can be used to translate any codon sequence and, thus, to determine which amino acids are coded for by an mRNA sequence. ⚫ For example, the codon 5′-AUG-3′ codes for methionine. Sixty- one of the 64 codons code for the 20 common amino acids. ⚫ Three of the codons, UAG, UGA, and UAA, do not code for amino acids but rather are termination codons. When one of these codons appears in an mRNA sequence, it signals that the synthesis of the protein coded for by that mRNA is complete. Characteristics of the genetic code ⚫ Specificity: The genetic code is specific (unambiguous), that is, a particular codon always codes for the same amino acid. ⚫ Degeneracy: The genetic code is degenerate (sometimes called redundant). Codon degeneracy refers to a single amino acid being encoded by more than one codon. For example, arginine is specified by six different codons. ⚫ Universality: the genetic code is nearly universal, with only rare variations reported. For instance, mitochondria have an alternative genetic code with slight variations. Characteristics of the genetic code ⚫ Nonoverlapping and commaless: the genetic code is nonoverlapping and commaless, that is, the code is read from a fixed starting point as a continuous sequence of bases, taken three at a time. ⚫ A genetic message is written with no spaces between the codons, the cell’s protein-synthesizing machinery reads the message as a series of nonoverlapping three-letter words. ⚫ For example, UGGUUU is read as UGG/UUU without any “punctuation” between the codons. Where does translation take place within a cell? ⚫ Within all cells, the translation machinery resides within a specialized organelle called the ribosome. ⚫ In eukaryotes, mature mRNA molecules must leave the nucleus and travel to the cytoplasm, where the ribosomes are located. ⚫ On the other hand, in prokaryotic organisms, ribosomes can attach to mRNA while it is still being transcribed. In this situation, translation begins at the 5' end of the mRNA while the 3' end is still attached to DNA. Components required for translation ⚫ A large number of components are required for the synthesis of a protein. ⚫ These include all the amino acids that are found in the finished product, the mRNA to be translated, transfer RNA (tRNA), functional ribosomes, energy sources, and enzymes, as well as protein factors needed for initiation, elongation, and termination of the polypeptide chain. A. Amino acids ⚫ All the amino acids that eventually appear in the finished protein must be present at the time of protein synthesis. ⚫ (Note: If one amino acid is missing [e.g., if the diet does not contain an essential amino acid], translation stops at the codon specifying that amino acid. This demonstrates the importance of having all the essential amino acids in sufficient quantities in the diet to ensure continued protein synthesis.). B. Transfer RNA ⚫ tRNAs are able to carry a specific amino acid and to recognize the codon for that amino acid. tRNA, therefore, functions as adaptor molecules. ⚫ At least one specific type of tRNA is required per amino acid. ⚫ In humans, there are at least 50 species of tRNA, whereas bacteria contain 30 to 40 species. ⚫ Because there are only 20 different amino acids commonly carried by tRNA, some amino acids have more than one specific tRNA molecule. This is particularly true of those amino acids that are coded for by several codons. Cloverleaf 1. Amino acid attachment site ⚫ Each tRNA molecule has an attachment site for a specific (cognate) amino acid at its 3′ end. ⚫ The carboxyl group of the amino acid is in an ester linkage with the 3′- hydroxyl group of the ribose moiety of the adenosine nucleotide in the – CCA sequence at the 3′ end of the tRNA. ⚫ (Note: When a tRNA has a covalently attached amino acid, it is said to be charged; when tRNA is not bound to an amino acid, it is described as being uncharged.). ⚫ The amino acid that is attached to the tRNA molecule is said to be activated. 2. Anticodon ⚫ Each tRNA molecule also contains a three-base nucleotide sequence—the anticodon—that recognizes a specific codon on the mRNA. ⚫ This codon specifies the insertion into the growing peptide chain of the amino acid carried by that tRNA. C. Aminoacyl-tRNA synthetases ⚫ The correct matching up of tRNA and amino acid is carried out by a family of related enzymes called aminoacyl-tRNA synthetases. ⚫ This family of enzymes is required for the attachment of amino acids to their corresponding tRNA. ⚫ The active site of each type of aminoacyl-tRNA synthetase fits only a specific combination of amino acid and tRNA. ⚫ There are 20 different synthetases, one for each amino acid; each synthetase is able to bind to all the different tRNAs that code for its particular amino acid. C. Aminoacyl-tRNA synthetases ⚫ Each aminoacyl-tRNA synthetase catalyzes a two-step reaction that results in the covalent attachment of the carboxyl group of an amino acid to the 3′ end of its corresponding tRNA. ⚫ The overall reaction requires adenosine triphosphate (ATP), which is cleaved to adenosine monophosphate (AMP) and inorganic pyrophosphate (PPi). D. Messenger RNA ⚫ The specific mRNA required as a template for the synthesis of the desired polypeptide chain must be present. E. Functionally competent ribosomes ⚫ They consist of two subunits—one large and one small—whose relative sizes are generally given in terms of their sedimentation coefficients, or S (Svedberg) values. ⚫ (Note: Because the S values are determined both by shape as well as molecular mass, their numeric values are not strictly additive. The eukaryotic 60S and 40S subunits form an 80S ribosome.) E. Functionally competent ribosomes ⚫ Prokaryotic and eukaryotic ribosomes are similar in structure and serve the same function, namely, as the “factories” in which the synthesis of proteins occurs. E. Functionally competent ribosomes ⚫ The larger ribosomal subunit catalyzes the formation of the peptide bonds that link amino acid residues in a protein. ⚫ The smaller subunit binds mRNA and is responsible for the accuracy of translation by ensuring correct base-pairing between the codon in the mRNA and the anticodon of the tRNA. E. Functionally competent ribosomes 1. Ribosomal RNA: Eukaryotic ribosomes contain four molecules of rRNA. The rRNAs have extensive regions of secondary structure arising from the base-pairing of the complementary sequences of nucleotides in different portions of the molecule. 2. Ribosomal proteins: Ribosomal proteins play a number of roles in the structure and function of the ribosome and its interactions with other components of the translation system. E. Functionally competent ribosomes 3. A, P, and E sites on the ribosome: The ribosome has three binding sites for tRNA molecules—the A, P, and E sites—each of which extends over both subunits. ⚫ During translation, the A site binds an incoming aminoacyl-tRNA as directed by the codon currently occupying this site. This tRNA holds the tRNA carrying the next amino acid to be added to the chain. ⚫ The P site codon is occupied by peptidyl-tRNA. This tRNA holds the tRNA carrying the growing polypeptide chain. ⚫ The E site (exit site) is occupied by the empty tRNA as it is about to exit the ribosome. E. Functionally competent ribosomes 4. Cellular location of ribosomes: ⚫ In eukaryotic cells, the ribosomes are either “free” in the cytosol or are in close association with the endoplasmic reticulum (which is then known as the “rough” endoplasmic reticulum, or RER). ⚫ The RER-associated ribosomes are responsible for synthesizing proteins that are to be exported from the cell as well as those that are destined to become integrated into plasma, endoplasmic reticulum, or Golgi membranes or incorporated in lysosomes. ⚫ Cytosolic ribosomes synthesize proteins required in the cytosol itself or destined for the nucleus, mitochondria, and peroxisomes. F. Protein factors ⚫ Initiation, elongation, and termination (or release) factors are required for peptide synthesis. ⚫ Some of these protein factors perform a catalytic function, whereas others appear to stabilize the synthetic machinery. G. ATP and GTP are required as sources of energy ⚫ Cleavage of four high-energy bonds is required for the addition of one amino acid to the growing polypeptide chain: two from ATP in the aminoacyl-tRNA synthetase reaction, and two from GTP—one for binding the aminoacyl-tRNA to the A site and one for the translocation step. ⚫ (Note: Additional ATP and GTP molecules are required for initiation in eukaryotes and an additional GTP molecule is required for termination). Steps in protein translation ⚫ The mRNA is translated from its 5′ end to its 3′ end, producing a protein synthesized from its amino-terminal (N-terminus) end to its carboxyl- terminal end (C-terminus). ⚫ The process of translation is divided into three separate steps: initiation, elongation, and termination. ⚫ The polypeptide chains produced may be modified by posttranslational modification. A. Initiation ⚫ The initiation stage of translation brings together mRNA, a tRNA bearing the first amino acid of the polypeptide, and the two subunits of a ribosome. ⚫ First, a small ribosomal subunit binds to both mRNA and a specific initiator tRNA, which carries the amino acid methionine. ⚫ In eukaryotes, the small subunit, with the initiator tRNA already bound, binds to the 5' cap of the mRNA and then moves, or scans, downstream along the mRNA until it reaches the start codon; the initiator tRNA then hydrogen-bonds to the AUG start codon. A. Initiation ⚫ In eukaryotes, the initiating AUG is recognized by a special initiator tRNA. ⚫ The union of mRNA, initiator tRNA, and a small ribosomal subunit is followed by the attachment of a large ribosomal subunit, completing the translation initiation complex. ⚫ The amino acid–charged initiator tRNA enters the ribosomal P site, and GTP is hydrolyzed to GDP. ⚫ Proteins called initiation factors are required to bring all these components together. B. Elongation ⚫ In the elongation stage of translation, amino acids are added one by one to the previous amino acid at the C-terminus of the growing chain. ⚫ Each addition involves the participation of several proteins called elongation factors (eEF-1α and eEF-1βγ) and occurs in a three-step cycle. ⚫ Energy expenditure occurs in the first and third steps. Codon recognition requires hydrolysis of one molecule of GTP, which increases the accuracy and efficiency of this step. One more GTP is hydrolyzed to provide energy for the translocation step. B. Elongation ⚫ The formation of the peptide bonds is catalyzed by peptidyl transferase, an activity intrinsic to the 28S rRNA found in the 60S ribosomal subunit. ⚫ After the peptide bond has been formed, the ribosome advances three nucleotides toward the 3′ end of the mRNA. This process is known as translocation and requires the participation of eEF-2 and GTP hydrolysis ⚫ The empty tRNAs that are released from the E site return to the cytoplasm, where they will be reloaded with the appropriate amino acid. C. Termination ⚫ Elongation continues until a stop codon in the mRNA reaches the A site of the ribosome. ⚫ The nucleotide base triplets UAG, UAA, and UGA do not code for amino acids but instead act as signals to stop translation. ⚫ A release factor (eRF), a protein shaped like an aminoacyl tRNA, binds directly to the stop codon in the A site. The release factor causes the addition of a water molecule instead of an amino acid to the polypeptide chain. Completing and Targeting the Functional Protein ⚫ The process of translation is often not sufficient to make a functional protein. ⚫ To function, the completed polypeptide chain must fold correctly into its three-dimensional conformation, bind any cofactors required, and assemble with its partner protein chains, if any. ⚫ Most proteins probably do not fold correctly during their synthesis and require a special class of proteins called molecular chaperones (chaperonin) to do so (helps the polypeptide fold correctly). Completing and Targeting the Functional Protein ⚫ Many molecular chaperones are called heat-shock proteins (designated hsp), because they are synthesized in dramatically increased amounts after a brief exposure of cells to an elevated temperature (for example, 42°C for cells that normally live at 37°C). ⚫ This reflects the operation of a feedback system that responds to an increase in misfolded proteins (such as those produced by elevated temperatures) by boosting the synthesis of the chaperones that help these proteins refold. Post-translational modifications ⚫ Additional steps—post-translational modifications—may be required before the protein can begin doing its particular job in the cell. ⚫ Indeed, many polypeptide chains are covalently modified, either while they are still attached to the ribosome or after their synthesis has been completed. ⚫ These modifications may include the removal of a part of the translated sequence or the covalent addition of one or more chemical groups required for protein activity. ⚫ Certain amino acids may be chemically modified by the attachment of sugars, lipids, phosphate groups, or other additions. A. Trimming ⚫ Many proteins destined for secretion from the cell are initially made as large, precursor molecules that are not functionally active. ⚫ Portions of the protein chain must be removed by specialized endoproteases, resulting in the release of an active molecule. ⚫ Some precursor proteins are cleaved in the endoplasmic reticulum or the Golgi apparatus, others are cleaved in developing secretory vesicles, and still others, such as collagen, are cleaved after secretion. ⚫ Zymogens are inactive precursors of secreted enzymes (including the proteases required for digestion). They become activated through cleavage when they reach their proper sites of action. B. Phosphorylation ⚫ Phosphorylation occurs on the hydroxyl groups of serine, threonine, or, less frequently, tyrosine residues in a protein. ⚫ This phosphorylation is catalyzed by one of a family of protein kinases and may be reversed by the action of cellular protein phosphatases. ⚫ The phosphorylation may increase or decrease the functional activity of the protein. C. Glycosylation ⚫ Many of the proteins that are destined to become part of a plasma membrane or lysosome, or to be secreted from the cell, have carbohydrate chains attached to serine or threonine hydroxyl groups (O-linked) or the amide nitrogen of asparagine (N-linked). ⚫ The addition of sugars occurs in the endoplasmic reticulum and the Golgi apparatus. ⚫ Sometimes glycosylation is used to target the proteins to specific organelles. For example, enzymes destined to be incorporated into lysosomes are modified by the glycosylation of mannose residues. D. Hydroxylation ⚫ Proline and lysine residues of the α chains of collagen are extensively hydroxylated in the endoplasmic reticulum.