DNA Translation

Learning Objectives Student will be able to Discuss DNA Translation Explain Post Translational Modifications Solve clinical problem

Translation The process of Protein synthesis is called translation In this process the language of nucleotide sequence on the mRNA is translated into the language of an amino acid sequence. Translation requires a genetic code through which the information contained in the nucleic acid sequence is expressed to produce a specific sequence of amino acids.

The Genetic code Is a dictionary that identify 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 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. Therefore, 64 different combinations of bases, taken three at a time (a triplet code) as shown in Figure 31.2.

This table (or “dictionary”) can be used to translate any codon and, thus, to determine which amino acids are coded for by an mRNA sequence. For example, the codon 5'-AUG-3' codes for methionine the [ AUG is the initiation (start) codon for translation.] Sixty-one of the 64 codons code for the 20 common amino acids. Termination (“stop” or “nonsense”) codons: 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, synthesis of the polypeptide will stops.

DNA template strand 5 DNA 3 molecule Gene 1 5 3 TRANSCRIPTION Figure 17.4 DNA template strand 5 DNA 3 A C C A A A C C G A G T molecule T G G T T T G G C T C A Gene 1 5 3 TRANSCRIPTION Gene 2 U G G U U U G G C U C A mRNA 5 3 Codon TRANSLATION Figure 17.4 The triplet code. Protein Trp Phe Gly Ser Gene 3 Amino acid

Translation Translation is the RNA-directed synthesis of a polypeptide TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide Amino acids tRNA with amino acid attached tRNA Anticodon Trp Phe Gly A G C U Codons 5 3 Translation is the RNA-directed synthesis of a polypeptide Translation involves mRNA Ribosomes - Ribosomal RNA Transfer RNA Genetic coding - codons

Transfer RNA Consists of a single RNA strand that is only about 80 nucleotides long Each carries a specific amino acid on one end and has an anticodon on the other end A special group of enzymes pairs up the proper tRNA molecules with their corresponding amino acids. tRNA brings the amino acids to the ribosomes, (a) 3 C A G U * 5 Amino acid attachment site Hydrogen bonds Anticodon The “anticodon” is the 3 RNA bases that matches the 3 bases of the codon on the mRNA molecule

Transfer RNA 3 dimensional tRNA molecule is roughly “L” shaped 5 3 A (b) Three-dimensional structure Symbol used in the book Amino acid attachment site Hydrogen bonds Anticodon A G 5 3 (c)

Ribosomes Ribosomes facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis The 2 ribosomal subunits are constructed of proteins and RNA molecules named ribosomal RNA or rRNA TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide Exit tunnel Growing polypeptide tRNA molecules E P A Large subunit Small Computer model of functioning ribosome. This is a model of a bacterial ribosome, showing its overall shape. The eukaryotic ribosome is roughly similar. A ribosomal subunit is an aggregate of ribosomal RNA molecules and proteins. (a) 5 3

Ribosome The ribosome has three binding sites for tRNA The P site The A site The E site E P A P site (Peptidyl-tRNA binding site) E site (Exit site) mRNA binding site A site (Aminoacyl- tRNA binding site) Large subunit Small Schematic model showing binding sites. A ribosome has an mRNA binding site and three tRNA binding sites, known as the A, P, and E sites. This schematic ribosome will appear in later diagrams. (b)

Building a Polypeptide Amino end Growing polypeptide Next amino acid to be added to polypeptide chain tRNA mRNA Codons 3 5 Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its anticodon base-pairs with an mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A site holds the tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site. (c)

Building a Molecule of tRNA A specific enzyme called an aminoacyl-tRNA synthetase joins each amino acid to the correct tRNA Amino acid Aminoacyl-tRNA synthetase (enzyme) Active site binds the amino acid and ATP. 1 P P P Adenosine ATP ATP loses two P groups and joins amino acid as AMP. 2 P Adenosine Pyrophosphate P Pi Pi Phosphates Pi tRNA 3 Appropriate tRNA covalently Bonds to amino Acid, displacing AMP. P Adenosine AMP Activated amino acid is released by the enzyme. 4 Aminoacyl tRNA (an “activated amino acid”) Figure 17.15

Building a Polypeptide We can divide translation into three stages Initiation Elongation Termination The AUG start codon is recognized by methionyl-tRNA or Met Once the start codon has been identified, the ribosome incorporates amino acids into a polypeptide chain RNA is decoded by tRNA (transfer RNA) molecules, which each transport specific amino acids to the growing chain Translation ends when a stop codon (UAA, UAG, UGA) is reached

Translation: Initiation mRNA binds to a ribosome, and the transfer RNA corresponding to the START codon binds to this complex. Ribosomes are composed of 2 subunits (large and small), which come together when the messenger RNA attaches during the initiation process.

Translation: Elongation Elongation: the ribosome moves down the messenger RNA, adding new amino acids to the growing polypeptide chain. The ribosome has 2 sites for binding transfer RNA. The first RNA with its attached amino acid binds to the first site, and then the transfer RNA corresponding to the second codon bind to the second site.

Translation: Elongation The ribosome then removes the amino acid from the first transfer RNA and attaches it to the second amino acid. At this point, the first transfer RNA is empty: no attached amino acid, and the second transfer RNA has a chain of 2 amino acids attached to it.

Translation: Termination The elongation cycle repeats as the ribosome moves down the messenger RNA, translating it one codon and one amino acid at a time. The process repeats until a STOP codon is reached.

Post Translational Modifications of Polypeptide Chain Many polypeptide chains are covalently modified either while they are attached to the ribosome or after their synthesis.

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. The cellular site of the cleavage reaction depends on the protein to be modified. Some precursor proteins are cleaved in the endoplasmic reticulum or the Golgi apparatus, others are cleaved in developing secretory vesicles (for example, insulin, 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. For example, the pancreatic zymogen, trypsinogen, becomes activated to trypsin in the small intestine (see Figure 19.5, p. 249). The synthesis of proteases as zymogens protects the cell from being digested by its own products.

Covalent Modification

Covalent Modification Phosphorylation Glycosylation Hydroxylation Carboxylation Biotinylation Acetylation

Covalent Modification Methylation Alkylation Glutamylation Lipoylation Sulfation

Covalent Modification Phosphorylation The addition of a phosphate (PO4) group to a protein or a small molecule. Can occur on Serine, Threonine, Tyrosine.

Covalent Modification Glycosylation The addition of saccharide to a protein or a lipid molecule. N-Linked Glycosylation Amide nitrogen of Asparagine O-Linked Glycosylation Hydroxyl oxygen of Serine and Therionine.

Covalent Modification Hydroxylation The addition of hydroxyl group to proline of protein. Carboxylation The addition of carboxyl group to glutamate.

Covalent Modification Biotinylation The addition of biotin to protein or nucleic acid. Acetylation The addition of an acetyl group, usually at the N-terminus of the protein.

Covalent Modification Methylation The addition of a methyl group, usually at lysine or arginine residues. Alkylation The addition of an alkyl group (e.g. methyl, ethyl).

Covalent Modification Glutamylation Covalent linkage of glutamic acid residues to tubulin and some other Lipoylation The attachment of a lipoate functionality Sulfation The addition of a sulfate group to a tyrosine.

Effect of Different Antibiotics on Translation Sterptomycin binds to the 30S subunit and distort its structure, Interfereing with the initiation of the Protein Synthesis. Tetracyclines interact with 30S subunit, blocking access of the aminoacyl-tRNA to the A-site thereby inhibiting elongation.

Puromycin bears a structural resemblance to aminoacyl tRNA and accepts peptide from the P site, causing inhibition of elongation. Chloramphenicol Inhibits prokaryotic peptidyltransferase. High levels may also inhibit mitochondrial protein synthesis.

Erythromycin binds irreversibly to a site on the 50S subunit and blocks the tunnel by which the peptide leaves the ribosome. Thereby inhibiting translocation. Diptheria toxin Inactivates the eukaryotic elongation factor EF2 hereby preventing translocation.

Practice Questions

Q1. During translation, Catalyzing bonding between adjacent amino acids is through which of the following enzyme: helps in a. peptidyl transferase b. aminoacyl-tRNA synthetase c. Shifting of ribosome from one codon to other on mRNA d. Removal of tRNA after formation of peptide bond

Ans: A a. The peptidyl transferase is an aminoacyltransferase (EC 2.3.2.12) as well as the primary enzymatic function of the ribosome, which forms peptide bonds between adjacent amino acids using tRNAs during the translation process of protein biosynthesis. b. An enzyme called aminoacyl-tRNA synthetase adds the correct amino acid to its tRNA. The correct amino acid is added to its tRNA by a specific enzyme called an aminoacyl-tRNA synthetase. c. Shifting of ribosome from one codon to other on mRNA d. Removal of tRNA after formation of peptide bond

2. In Prokaryotes, first amino acid in polypeptide chain is a 2. In Prokaryotes, first amino acid in polypeptide chain is a. Methionine b. N-formyl methionine c. N-methyl methionine d. Methyl methionine

Ans B Methionine is used as a first amino acid in eukaryotes. The first amino acid in a polypeptide chain of prokaryotes is carried by the initiator tRNA, which always carries formylmethionine.

3. During translation, proteins are synthesized by: a 3. During translation, proteins are synthesized by: a. ribosomes using the information on DNA b. lysosomes using the information on DNA c. ribosomes using the information on mRNA d. lysosomes using the information on mRNA

Ans C Translation is the final step on the way from DNA to protein. It is the synthesis of proteins directed by a mRNA template. The information contained in the nucleotide sequence of the mRNA is read as three letter words (triplets), called codons. Each word stands for one amino acid.

4. Tetracycline blocks protein synthesis by inhibiting a 4. Tetracycline blocks protein synthesis by inhibiting a. binding of aminoacyl tRNA b. DNA-dependent RNA polymerase c. peptidyl transferase d. translocase enzyme

Ans A Tetracycline antibiotics are protein synthesis inhibitors, inhibiting the binding of aminoacyl-tRNA to the mRNA-ribosome complex. They do so mainly by binding to the 30S ribosomal subunit in the mRNA translation complex. Rifamycin inhibits prokaryotic DNA transcription into mRNA by inhibiting DNA-dependent RNA polymerase by binding its beta-subunit Chloramphenicol, Macrolides Quinupristin/dalfopristin and Geneticin are inhibiting peptidyl transferase enzyme. Macrolides, clindamycin and aminoglycosides (with all these three having other potential mechanisms of action as well), have evidence of inhibition of ribosomal translocation.

Which of the following process is involved in post-transcription modification? Acetylation addition of a 5' cap Glycocylation Hair pin loop formation

Key: b Acetylation is the process of post-translation modification The process includes three major steps: addition of a 5' cap, addition of a 3' poly-adenylation tail, and splicing Glycosylation is the process of post-translation modification The hairpin loop forms in an mRNA strand during transcription and causes the RNA polymerase to become dissociated from the DNA template strand.