Lecture 11: Molecular Biology I, BIO316 PDF

Molecular Biology I BIO316 Lecture 11 Major principles of Translation Prepared by Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics   Overview on translation...

Molecular Biology I BIO316 Lecture 11 Major principles of Translation Prepared by Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics   Overview on translation Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Translation is the process in which the sequence of codons within mRNA provides the information to synthesize the sequence of amino acids that constitute a polypeptide.  One or more polypeptides then fold and assemble to create a functional protein.  Proteins are critically important as active participants in cell structure and function.  The primary role of DNA is to store the information needed for the synthesis of all the proteins that an organism makes.  Genes that encode an amino acid sequence are known as protein-encoding genes = Structural genes.  The RNA transcribed from protein-encoding genes is called messenger RNA (mRNA).  Dr. Rami Elshazli  The Genetic Basis For Protein Synthesis Associate Professor of Biochemistry and Molecular Genetics  The main function of the genetic material is to encode  When a person inherits two defective the production of cellular proteins in the correct cell, at copies of the gene that encodes the proper time, and in suitable amounts. homogentisic acid oxidase, he or she  This is an extremely complicated task because living cannot convert homogentisic acid into cells make thousands of different proteins. maleylacetoacetic acid.  Such a person accumulates large  The metabolic pathways consist of a series of metabolic amounts of homogentisic acid in the conversions of one molecule to another, each step body and has symptoms of the disease catalyzed by a specific enzyme. known as alkaptonuria.  Each enzyme is a distinctly different protein that catalyzes a particular chemical reaction.  Similarly, if a person has two mutant  Enzymes are only one category of proteins. (defective) alleles of the gene encoding  All proteins are encoded by genes, and many of them do phenylalanine hydroxylase, he or she is not function as enzymes. unable to synthesize the enzyme  Some proteins are composed of two or more different phenylalanine hydroxylase and has the polypeptides. disease called phenylketonuria (PKU).  One gene can encode two or more polypeptides due to alternative splicing.  The Genetic Basis For Protein Synthesis  The sequence of a protein-encoding gene provides a template for the synthesis of mRNA, which contains the information to synthesize a polypeptide.  During Translation, the Codons in mRNA Provide the Information to Make a Polypeptide with a Specific Amino Acid Sequence.  Translation involves an interpretation of the language of mRNA (nucleotide sequence) into the language of proteins (amino acid sequence).  The ability of mRNA to be translated into a specific sequence of amino acids relies on the genetic code.  The sequence of bases within an mRNA molecule provides coded information that is read in groups of three nucleotides known as codons. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli  The Genetic Basis For Protein Synthesis Associate Professor of Biochemistry and Molecular Genetics  The sequence of three bases in most codons specifies a particular amino acid.  For example, the codon AGC specifies the amino acid serine, whereas the codon GGG encodes the amino acid glycine.  The codon AUG, which specifies methionine, is used as a start codon; it is usually the first codon that begins a polypeptide sequence.  The AUG codon is also used to specify additional  The codons in mRNA are recognized methionine within the coding sequence.  mRNA molecule also has regions by the anticodons in transfer RNA that precede the start codon and (tRNA) molecules.  Three codons —UAA, UAG, and UGA—are used to end follow the stop codon.  Anticodons are three-nucleotide the process of translation and are known as stop  Because these regions do not sequences that are complementary codons. encode a polypeptide, they are to codons in mRNA.  They are also called termination codons, or nonsense called the 5′-untranslated region  The tRNA molecules carry the amino codons. and 3′-untranslated region. acids that correspond to the codons in the mRNA.  Dr. Rami Elshazli  The Genetic Basis For Protein Synthesis Associate Professor of Biochemistry and Molecular Genetics  The genetic code is composed of 64 different codons.  Because polypeptides are composed of 20 different kinds of amino acids, a minimum of 20 codons is needed to specify all the amino acids.  With four types of bases in mRNA (A, U, G, and C), a genetic code containing two bases in a codon would not be sufficient because it would specify only 42 = 16 possible types of amino acids.  By comparison, a three-base codon system could form 43 = 64 different codons.  Because the number of possible codons exceeds 20, the genetic code is said to contain degeneracy.  Degeneracy means that more than one codon can specify the same amino acid.  For example, the codons GGU, GGC, GGA, and GGG all specify the amino acid glycine.  Such codons are termed synonymous codons.  Dr. Rami Elshazli  The Genetic Basis For Protein Synthesis Associate Professor of Biochemistry and Molecular Genetics  In most instances of synonymous codons, the third base in the codon is the base that varies.  The third base is referred to as the wobble base = ‫ﻗﺎﻋدة‬ ‫ﻣﺗذﺑذة‬  The start codon (AUG) defines the starting of reading frame of an mRNA.  Selenocysteine (Sec) and pyrrolysine (Pyl) are called the twenty-first and twenty-second amino acids in polypeptides.  Selenocysteine is found in several enzymes involved in oxidation-reduction reactions in bacteria, archaea, and eukaryotes.  Pyrrolysine is found in a few enzymes of methane- producing archaea.  Selenocysteine and Pyrrolysine are encoded by the codons UGA and UAG, which usually function as stop codons.   The polypeptide chain  Polypeptide synthesis has a directionality that parallels the order of codons in the mRNA.  As a polypeptide is made, a peptide bond is formed between the carboxyl group in the last amino acid of the polypeptide and the amino group in the amino acid being added.  This occurs via a condensation reaction that releases a water molecule.  The newest amino acid added to a growing polypeptide always has a free carboxyl group.  The first amino acid is said to be at the amino-terminal end = N-terminus of the polypeptide.  An amino group (NH3+) is found at this site.  The first amino acid is specified by a codon that is near the 5′ end of the mRNA. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli  The polypeptide chain Associate Professor of Biochemistry and Molecular Genetics  The last amino acid in a completed polypeptide is located at the carboxyl-terminal end = C-terminus.  A carboxyl group (COO–) is always found at this site.  This last amino acid is specified by a codon that is closer to the 3′ end of the mRNA.  The first amino acid in a polypeptide (usually methionine) is located at the amino-terminal end, and the last amino acid is at the carboxyl-terminal end.  Therefore, the directionality of amino acids in a polypeptide is from the amino-terminal end to the carboxyl-terminal end, which corresponds to the 5′ to 3′ orientation of codons in mRNA.  Dr. Rami Elshazli  The polypeptide chain Associate Professor of Biochemistry and Molecular Genetics  Each amino acid contains a unique side chain (R group) that has its own particular chemical properties.  For example, aliphatic and aromatic amino acids are relatively nonpolar, which means they are less likely to associate with water.  These hydrophobic (“water-fearing”) amino acids are often buried within the interior of a folded protein.  In contrast, the polar amino acids are hydrophilic (“water-loving”) and are more likely to be on the surface of a protein, where they can favorably interact with the surrounding water of cells or extracellular fluids.  The chemical properties of the amino acids and their sequences in a polypeptide are critical factors that determine the unique structure of the polypeptide.  Dr. Rami Elshazli  Hierarchal Representation of Proteins Associate Professor of Biochemistry and Molecular Genetics  Primary Structure:  It is a polypeptide with a defined amino acid sequence.  This sequence is the primary structure of a polypeptide.  The primary structure of a typical polypeptide may be a few hundred or a couple of thousand amino acids in length.  To become a functional unit, most polypeptides quickly adopt a three-dimensional structure.  The folding process begins while the polypeptide is still being translated.  The chemical properties of the amino acid side chains play a central role in determining the folding pattern of a protein.  In addition, the folding of some polypeptides is aided by chaperones—proteins that bind to polypeptides and facilitate their proper folding.  Dr. Rami Elshazli  The polypeptide chain Associate Professor of Biochemistry and Molecular Genetics  Secondary Structure:  The folding of polypeptides is governed by their primary structure and occurs in multiple stages.  The first stage involves the formation of a regular, repeating shape known as a secondary structure.  The two main types of secondary structures are the α helix and the β sheet.  A single polypeptide may have some regions that fold into an α helix and other regions that fold into a β sheet.  Certain amino acids are good candidates to form an α helix, whereas others are found in a β-sheet conformation.  Secondary structures within polypeptides are primarily stabilized by the formation of hydrogen bonds between atoms that are located in the polypeptide backbone.  Dr. Rami Elshazli  The polypeptide chain Associate Professor of Biochemistry and Molecular Genetics  Tertiary Structure:  The short regions of secondary structure within a polypeptide are folded relative to each other to make the tertiary structure of the polypeptide.  α-helical regions and β-sheet regions are connected by irregularly shaped segments to determine this tertiary structure.  This structure is determined by various interactions including:  The tendency of hydrophobic amino acids to avoid water = Hydrophobic interactions  Ionic interactions among charged amino acids.  Hydrogen bonding among amino acids in the folded polypeptide.  Weak bonding known as van der Waals interactions.  Disulfide bonding = covalent bond.  Dr. Rami Elshazli  The polypeptide chain Associate Professor of Biochemistry and Molecular Genetics  Quaternary Structure:  A protein is a functional unit that can be composed of one or more polypeptides.  Some proteins are composed of a single polypeptide.  Many proteins are composed of two or more polypeptides that associate with each other to form a quaternary structure.  The individual polypeptides are called subunits of the functional protein, and each of them has its own tertiary structure.  During translation, the codons in mRNA are recognized by tRNA molecules, which act as intermediates in the production of a polypeptide with a specific amino acid sequence.  The genetic code refers to the relationship between three base codons in the mRNA and the amino acids that are incorporated into a polypeptide.  Dr. Rami Elshazli  Structure and Function of tRNA Associate Professor of Biochemistry and Molecular Genetics  During translation, a tRNA molecule has two functions:  It recognizes a three-base codon sequence in mRNA.  It carries an amino acid specific for that codon.  tRNA molecules recognize the codons within mRNA and carry the correct amino acids to the site of polypeptide synthesis.  During mRNA-tRNA recognition, the anticodon in a tRNA molecule binds to a codon in mRNA.  The anticodon in the tRNA is complementary to the codon for the amino acid that it carries.  For example, if the anticodon in the tRNA is 3′–AAG– 5′, it is complementary to a 5′–UUC–3′ codon.  The UUC codon specifies phenylalanine.  Therefore, the tRNA with a 3′–AAG–5′ anticodon must carry a phenylalanine.  This hypothesis is true for 3’-CCG-5’ codon of proline.  Dr. Rami Elshazli  Structure and Function of tRNA Associate Professor of Biochemistry and Molecular Genetics  Recall that the genetic code has 64 codons.  Of these, 61 are sense codons that specify the 20 amino acids.  A cell must produce different tRNA molecules having specific anticodon sequences.  The anticodon in a tRNA specifies the type of amino acid that it carries.  The tRNA molecules are named according to the amino acid they carry.  For example, a tRNA that carries phenylalanine is called tRNAPhe, whereas a tRNA that carries proline is tRNAPro.  The secondary structure of a tRNA exhibits a cloverleaf pattern with three stem-loops and a few variable sites (locations with additional nucleotides not found in all tRNA molecules).  Dr. Rami Elshazli  Structure and Function of tRNA Associate Professor of Biochemistry and Molecular Genetics  A tRNA also has an acceptor stem with a 3′ single-  A conventional numbering system for the stranded region. nucleotides within a tRNA molecule begins at the  The acceptor stem is where an amino acid 5′ end and proceeds toward the 3′ end. becomes attached. (3’ end of tRNA molecule)  The anticodon is located in the second loop region.  In all tRNAs, the nucleotides at the 3′ end contain the sequence CCA.  Certain locations can have additional nucleotides not found in all tRNA molecules.  The modified bases are as follows:  I = inosine.  mI = methylinosine.  T = ribothymidine.  UH2 = dihydrouridine.  m2G = dimethylguanosine.  P = pseudouridine.   Aminoacyl-tRNA Synthetases  In the first step of the reaction, a  Each type of tRNA have the appropriate amino acid synthetase recognizes a specific attached to its 3′ end. amino acid and ATP molecule.  Enzymes in the cell known as aminoacyl-tRNA  The ATP is hydrolyzed, resulting in synthetases catalyze the attachment of amino acids to the attachment of AMP to the tRNA molecules. amino acid and the release of pyrophosphate.  Cells produce 20 different aminoacyl-tRNA synthetase enzymes, one for each of the 20 distinct amino acids.  The correct tRNA then binds to  Each aminoacyl-tRNA synthetase is named for the the synthetase. specific amino acid it attaches to tRNA.  The amino acid becomes  For example, alanyl-tRNA synthetase recognizes a covalently attached to the 3′ end tRNA with an alanine anticodon tRNAAla. of the tRNA molecule at the acceptor stem and AMP is  Aminoacyl-tRNA synthetases catalyze a chemical released. reaction involving three different molecules:  Finally, the tRNA with its attached  An amino acid. amino acid is released from the  tRNA molecule. enzyme.  ATP. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics   Aminoacyl-tRNA Synthetases  At this stage, the tRNA is called a charged tRNA = aminoacyl-tRNA.  In a charged tRNA molecule, the amino acid is attached to the 3′ end of the tRNA by a covalent bond.  The ability of aminoacyl-tRNA synthetases to  Valine is specified by GUU, recognize tRNAs has been called the “second GUC, GUA, and GUG. genetic code”.  The first two bases are G  The Wobble Rules and U.  According to the wobble  The ability of aminoacyl-tRNA synthetases to rules, the third base can be recognize tRNAs has been called the “second U, C, A, or G. genetic code”.  The genetic code contains degeneracy, which  E. coli cells make a population means that more than one codon can specify of tRNA molecules that have the same amino acid. just 40 different anticodon sequences.  Degeneracy usually occurs at the third position Dr. Rami Elshazli in a codon. Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli  Ribosome structure and assembly Associate Professor of Biochemistry and Molecular Genetics  To begin translation, the bond between the 3′ end of the tRNA and the amino acid must be broken, and a peptide bond must be formed between the adjacent amino acids.  Translation occurs on the surface of a macromolecular complex known as the ribosome.  The ribosome is defined as the macromolecular arena where translation takes place.  Bacterial cells have one type of ribosome that is found within the cytoplasm.  Eukaryotic cells contain biochemically distinct ribosomes in different cellular locations.  The term eukaryotic ribosome refers to ribosomes in the cytosol,  The most abundant type of ribosome functions in the not to those found within organelles. cytosol.  Besides the cytosolic ribosomes, all eukaryotic cells have ribosomes within the mitochondria.  Dr. Rami Elshazli  Ribosome structure and assembly Associate Professor of Biochemistry and Molecular Genetics  Each ribosome is composed of structures called the large and small subunits.  Each ribosomal subunit itself is formed from the assembly of many different proteins and RNA molecules called ribosomal RNA, or rRNA.  In bacterial ribosomes, the 30S subunit is formed from the assembly of 21 different ribosomal proteins and a 16S rRNA molecule.  The 50S subunit contains 34 different proteins and 5S and 23S rRNA molecules. Composition of ribosomes in prokaryotes and eukaryotic cells Cell type Ribosomal size Subunit rRNA component Proteins Large 50S 23S + 5S 34 Prokaryotic 70S Small 30S 16S 21 Large 60S 28S + 5.8S + 5S 49 Eukaryotic 80S Small 40S 18S 33  Dr. Rami Elshazli  Ribosome structure and assembly Associate Professor of Biochemistry and Molecular Genetics  The 30S and 50S refer to the rate at which these subunits sediment when subjected to a centrifugal force.  The centrifugation rate is described as a sedimentation coefficient in Svedberg units (S) in honor of Theodor Svedberg who invented the ultracentrifuge.  Together, the 30S and 50S subunits form a 70S ribosome.  In bacteria, the ribosomal proteins and rRNA molecules are synthesized in the cytoplasm, and the ribosomal subunits are assembled there.  The synthesis of eukaryotic rRNA occurs within the nucleus, and the ribosomal proteins are made in the cytosol, where translation takes place.  Dr. Rami Elshazli  Ribosome structure and assembly Associate Professor of Biochemistry and Molecular Genetics A Comparison of Ribosome Composition in Bacteria and Eukaryotes  The 40S subunit is composed of 33 proteins and an 18S rRNA. Small Subunit Large Subunit Assembled Ribosome  The 60S subunit is made of 49 proteins and 5S, Prokaryotic 5.8S, and 28S rRNAs. Sedimentation coefficient 30S 50S 70S  The assembly of the rRNAs and ribosomal Number of proteins 21 34 55 proteins to make the 40S and 60S subunits occurs rRNA molecules 16S rRNA 5S + 23S rRNA 16S + 5S + 23S rRNA within the nucleolus. Eukaryotic  The 40S and 60S subunits are then exported into Sedimentation coefficient 40S 60S 80S the cytosol, where they associate to form an 80S ribosome during translation. Number of proteins 33 49 82 rRNA molecules 18S rRNA 5S + 5.8S + 28S 18S + 5S + 5.8S + 28S  The term polyribosome = polysome is used to describe an mRNA transcript that has many bound ribosomes in the act of translation.   Ribosome structure and assembly  The overall shape of each subunit is determined by the structure of the rRNAs which constitute most of the mass of the ribosome.  During bacterial translation, the mRNA lies on the surface of the 30S subunit within a space between the 30S and 50S subunits.  As the polypeptide is being synthesized, it exits through a channel within the 50S subunit.  Ribosomes contain discrete sites where tRNAs bind and the polypeptide is synthesized.  These three sites are:  The peptidyl site (P site)  The aminoacyl site (A site).  The exit site (E site). Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli  Stages of translation Associate Professor of Biochemistry and Molecular Genetics  A ribosome contains A (aminoacyl), P (peptidyl), and E (exit) sites, which are occupied by tRNA molecules.  The process of translation can be occurred in three stages: initiation, elongation, and termination.  During initiation, the ribosomal subunits, mRNA, and the first tRNA assemble to form a complex.  After the initiation complex is formed, the ribosome slides along the mRNA in the 5′ to 3′ direction, moving over the codons.  As the ribosome moves, tRNA molecules sequentially bind to the mRNA in the ribosome, bringing with them the appropriate amino acids.  Finally, a stop codon is reached, signaling the termination of translation.  At this point, disassembly occurs, and the newly made polypeptide is released.   The Initiation Stage  During initiation, an mRNA and the first tRNA bind to the ribosomal subunits.  A specific tRNA functions as the initiator tRNA, which recognizes the start codon in the mRNA.  In bacteria, the initiator tRNA, which is designated tRNAfMet, carries a methionine that has been covalently modified to N-formylmethionine.  First, IF1 and IF3 bind to the 30S  A formyl group (—CHO) is attached to the nitrogen subunit. atom in methionine after the methionine has been  IF1 and IF3 prevent the attached to the tRNA. association of the 50S subunit.  Next, the mRNA binds to the 30S  The mRNA, tRNAfMet, and ribosomal subunits associate subunit. with each other to form an initiation complex.  This binding is facilitated by a  The formation of this complex requires the nine-nucleotide sequence within participation of three initiation factors: IF1, IF2, and the bacterial mRNA called the IF3. Shine-Dalgarno sequence. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli  The Initiation Stage Associate Professor of Biochemistry and Molecular Genetics  How does the Shine-Dalgarno sequence facilitate the binding of mRNA to the ribosome?  The Shine-Dalgarno sequence is complementary to a short sequence within the 16S rRNA, which promotes the hydrogen bonding of the mRNA to the 30S subunit.   During translation of the  The Initiation Stage entire polypeptide, the formyl group or the  A tRNAfMet binds to the mRNA that is already attached to the entire formylmethionine 30S subunit. may be removed.  This step requires the function of IF2, which uses GTP.  The tRNAfMet binds to the start codon, which is typically a few  Thus, some polypeptides may nucleotides downstream from the Shine-Dalgarno sequence. not have formylmethionine or methionine as their first amino acid.  After the mRNA and tRNAfMet have become bound to the 30S subunit.  IF1 and IF3 are released.  Then, IF2 hydrolyzes its GTP and is also released.  The start codon is usually AUG, and the anticodon is UAC.  The first amino acid in the polypeptide is a formylmethionine  This allows the 50S because only a tRNAfMet can initiate translation. ribosomal subunit to associate with the 30S Dr. Rami Elshazli subunit. Associate Professor of Biochemistry and Molecular Genetics  A Simplified Comparison of Translational Protein Factors in Bacteria and  The Initiation Stage Eukaryotes Bacterial Factors Eukaryotic Factors Function  In eukaryotes, the assembly of the initiation complex has Initiation Factors similarities to what occurs in bacteria. IF1, IF3 eIF1, eIF3, eIF6 Prevent the association between the  However, additional factors are required for the initiation small and large ribosomal subunits and favor their dissociation process. IF2 eIF2 Promote the binding of the initiator tRNA to the small ribosomal subunit.  The initiation factors are designated eIF (eukaryotic Initiation eIF4 Involved with the recognition of the Factor) to distinguish them from bacterial initiation factors. 7-methylguanosine cap and the  The initiator tRNA in eukaryotes carries methionine rather than binding of the mRNA to the small ribosomal subunit. formylmethionine, as in bacteria. eIF5 Helps to dissociate the other  Eukaryotic initiation factor (eIF2) binds directly to tRNAMet to elongation factors, which allows the large ribosomal subunit to bind. recruit it to the 40S subunit.  Eukaryotic mRNAs do not have a Shine-Dalgarno sequence.  How are eukaryotic mRNAs recognized by the ribosome?  The mRNA is recognized by eIF4, which is a multiprotein complex that recognizes the 7-methylguanosine cap and facilitates the binding of the mRNA to the 40S subunit. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli  The Initiation Stage Associate Professor of Biochemistry and Molecular Genetics  After the initial binding of mRNA to the ribosome, the next step is locating an AUG start codon that is downstream from the 5′ cap.  The ribosome uses the first AUG codon that it encounters as a start codon.  When a start codon is identified, the 60S subunit assembles onto the 40S subunit with the aid of eIF5, forming the initiation complex.  The Kozak sequence is a protein translation initiation site in eukaryotic mRNA.  Dr. Rami Elshazli  The Initiation Stage Associate Professor of Biochemistry and Molecular Genetics  The Kozak sequence is a specific nucleotide sequence that plays a critical role in the initiation of translation in eukaryotic messenger RNA (mRNA).  It surrounds the start codon (AUG) and enhances the efficiency of ribosome binding and translation initiation.  The consensus Kozak sequence is 5'-gccRccAUGG-3’.  AUG: The start codon where translation begins.  R: A purine (adenine A or guanine G) at the -3 position relative to the start codon.  These positions immediately upstream and downstream of the start codon are critical for the sequence's functionality.  -3 position (R): A purine is strongly favored at this position.  +4 position (G): A guanine is preferred immediately after the start codon.  The Kozak Sequence helps the ribosome to identify the correct start codon.   The Elongation Stage  This transfer is accompanied by the formation of a peptide  During the elongation stage of translation, amino acids bond between the amino acid are added - one at a time - to a growing polypeptide. at the A site and the  A short polypeptide is already attached to the tRNA at polypeptide, lengthening the the P site of the ribosome. polypeptide by one amino  A charged tRNA carrying a single amino acid binds to acid. the A site.  The peptidyl transfer reaction  This binding occurs because the anticodon in the tRNA is catalyzed by a component is complementary to the codon in the mRNA. of the 50S subunit known as peptidyl transferase, which is  The hydrolysis of GTP by the elongation factor EF-Tu composed of several proteins provides energy for the binding of a tRNA to the A site. and rRNA.  The 16S rRNA detect an incorrect tRNA when it is bound at the A site and prevents elongation.  The 23S rRNA catalyzes the bond formation between  The next step of elongation is a reaction called peptidyl adjacent amino acids. transfer—the polypeptide is removed from the tRNA in  In other words, the ribosome the P site and transferred to the amino acid at the A site. is a ribozyme! Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  A Simplified Comparison of Translational Protein Factors in Bacteria and  The Elongation Stage Eukaryotes Bacterial Factors Eukaryotic Factors Function  After the peptidyl transfer reaction is complete, the ribosome Elongation Factors translocases to the next codon in the mRNA. EF-Tu eEF1α Involved in the binding of tRNAs to  This moves the tRNAs at the P and A sites to the E and P sites, the A site. respectively. EF-Ts eEF1βγ Nucleotide exchange factors required for the functioning of EF-Tu and eEF1α.  Finally, the uncharged tRNA exits from the E site. EF-G eEF2 Required for translocation.  The next codon in the mRNA is now exposed in the unoccupied A site.  At this point, a charged tRNA can enter the empty A site, and the same steps can add the next amino acid to the growing polypeptide.  The A site (charged tRNA) is an aminoacyl-tRNA binding site.  The P site is the peptidyl-tRNA binding site usually contains a tRNA with an attached peptide.  The E site (Exit site) is where the uncharged tRNA exits.  Under normal cellular conditions, a polypeptide can elongate at a rate of 10 to 20 amino acids per second in bacteria or 2 to 6 amino Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics acids per second in eukaryotes.   The Termination Stage  The completed polypeptide is attached to a tRNA in the P site.  Termination occurs When a stop codon is reached in the  A stop codon is located at the A mRNA. site.  In most species, the three stop codons are UAA, UAG, and UGA.  Release factors can specifically  The stop codons are recognized by proteins known as release bind to a stop codon sequence. factors.  In bacteria, RF1 recognizes UAA and UAG, and RF2 recognizes A Simplified Comparison of Translational Protein Factors in Bacteria and Eukaryotes UGA and UAA. Bacterial Eukaryotic Function  A third release factor (RF3) is Factors Factors also required in termination. Termination or release Factors RF1, RF2 eRF1 Recognize a stop codon and  In eukaryotes, a single release trigger the cleavage of the factor (eRF1) recognizes all three polypeptide from the tRNA. stop codons. RF3 eRF3 GTPases that are also involved in termination.  The eRF3 is also required for termination. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli  The Termination Stage Associate Professor of Biochemistry and Molecular Genetics  In the first step, RF1 or RF2 binds to the stop codon at the A site and RF3 binds at a different location on the ribosome.  After RF1 (or RF2) and RF3 have bound, the bond between the polypeptide and the tRNA is hydrolyzed.  The polypeptide and tRNA are then released from the ribosome.  The final step in translational termination is the disassembly of ribosomal subunits, mRNA, and the release factors.  Bacterial translation can begin before transcription is completed.  Dr. Rami Elshazli  The Termination Stage Associate Professor of Biochemistry and Molecular Genetics Comparison of Bacterial and Eukaryotic Translation  The translation of a bacterial mRNA begins before the mRNA Prokaryotic Eukaryotic transcript is completed.  As soon as an mRNA strand is long enough, a ribosome Ribosome composition 70S ribosomes: 80S ribosomes: 30S subunit  40S subunit  attaches to the 5′ end and begins translation, even before 21 proteins + 16S rRNA 33 proteins + 18S rRNA 50S subunit  60S subunit  RNA polymerase has reached the transcriptional termination 34 proteins + (5S + 23S 49 proteins + (5S + 5.8S +28S site. rRNAs) rRNAs) Initiator tRNA tRNAfMet tRNAMet  This phenomenon in bacterial cells is referred to as coupling Formation of the Requires IF1, IF2, and IF3. Requires more initiation initiation complex factors than in bacterial between transcription and translation. translation  The coupling of these processes does not usually occur in Initial binding of mRNA Requires a Shine-Dalgarno Requires a 7-methylguanosine eukaryotes, because transcription takes place in the nucleus to the ribosome sequence. cap. of eukaryotic cells, whereas translation occurs in the cytosol. Selection of a start codon AUG located downstream According to Kozak’s rules. from the Shine-Dalgarno sequence. Elongation rate 10 to 20 amino acids per 2 to 6 amino acids per second. second. Termination Requires RF1, RF2, and RF3. Requires eRF1 and eRF3. Coupled to transcription Yes No  Translation summary Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli  The Termination Stage Associate Professor of Biochemistry and Molecular Genetics Mechanisms of inhibition of bacterial translation via antibiotics  Many different diseases that affect people are caused by pathogenic bacteria. Antibiotic Description  An antibiotic is any substance produced by a microorganism Chloramphenicol Blocks elongation by acting as competitive inhibitor of peptidyl transferase. that inhibits the growth of other microorganisms, such as Erythromycin Binds to the 23S rRNA and blocks elongation by interfering pathogenic bacteria. with the translocation step.  Most antibiotics are small organic molecules, with masses less Puromycin Binds to the A site and causes premature release of the than 2000 Daltons. peptide. This early termination of translation results in polypeptides that are shorter than normal. Tetracycline Blocks elongation by inhibiting the binding of aminoacyl tRNA to the ribosome. Streptomycin Interferes the normal pairing between aminoacyl tRNAs and codons. This causes misreading, and thereby produces abnormal proteins.  Antibiotics exert their effect because they interfere with bacterial translation.  Therefore, they can be used to treat bacterial infections in humans.  Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics