Translation Mechanisms

Transcription

1 Translation Mechanisms Biology I Hayder A. Giha

2 Translation The translation is the process of protein synthesis, where information in nucleotides sequences of a mrna is translated into amino acids sequence of protein The process takes place in ribosomes (in cytoplasm) The shared language between the DNA nucleotides and protein amino acids is the genetic code. Changes in the nucleotides sequence (known as mutation) in specific gene can lead to alteration of the amino acids sequence of the produced protein, which might cause disease or death but may not have effect at all. Many proteins are modified covalently after translation (post-translation modification) before it becomes active.

3 The Genetic code It is a coding system that explains translation of the nucleotide sequence into amino acid sequence It is made up of 64 codons, each 3 consecutive nucleotides (known as triplet codon) code for a single amino acid. The different combinations of the 4 bases generates 64 codons (each 3-letters) of the genetic code (4 3 ). - Codons: The codon is a triplet (means 3) of nucleotides (A, U, G & C) in the mrna, that read by the trna as one amino acid. The nucleotide sequence of the codon always read from the 5 to 3 end (left to right).

4 The Genetic code 1. The genetic code table shows the possible combination any 3 of the 4 bases and the corresponding amino acid. - Out of the 64 codons, 61 codons code for the standard 20 amino acids - Three codons; UAA, UAG & UGA don t code for any of the amino acids, known as termination (or stop or nonsense) codons. - Any one of stop codons can terminate the reading of the mrna i.e. signal termination of translation.

5 Genetic code Table

6 Characteristics of the genetic code The genetic code is valid for all living organisms. The genetic code have 5 characteristics: 1. Unambiguous (specific): A codon code for only one particular amino acid 2. Degeneracy (redundant): One amino acid can be coded by more than one codon e.g. arginine is encoded by 6 different codons 3. Non-overlapping: the mrna is read from a fix starting point and reads each 3 nucleotides at a time, followed by the next 3 bases and so on, no base shared between 2 codons 4. No punctuation (comma-less): The trnas reads the mrna in a continuous sequence, does not skip any base after start reading, till it end at stop codon.

7 Characteristics of the genetic code: 5. Universality: The genetic code is universal in the sense that a codon read the same across all living organisms (e.g. animals, plants, viruses & etc.). - However they are few exceptions.two codons in the mitochondrial genome reads different e.g. UGA is stop codon in the mitochondrial genome reads as tryptophan (amino aid) even in the same cell, for that reason some describe the genetic code as semi-universal.

8 Components required for translation For protein synthesis (translation), the following are needed: - All amino acids; the building blocks of protein - mrna; as template to be translated. - trnas; as adaptor molecule. - Enzymes & protein factors; needed for initiation, elongation and termination. - Source of energy (ATP & GTP) - Functional ribosomes (containing rrna and proteins) as a machinery or site in which all above components interact to construct a protein

9 a. Amino acids All amino acids that appear in the final product should be available as free amino acids. The essential amino acids should be provided in the diet. The standard amino acids are 20, these are the amino acids that have codon in the genetic code. Synthesis of proteins stops at the codon of the missed amino acid.

10 b. Transfer RNAs (trna) Structurally the trna are clover-like in shape with 4 distinct sites or arms trnas are adaptor molecules since each can bind an amino acid by one arm and recognized and bind the codon of that amino acid in mrna by another arm. In human there are at least 50 species of trnas for the 20 amino acids. Each trna is specific for only one amino acid, but one amino acid can be carried by more than one trna, especially for amino acids with several codons.

11 Transfer RNAs (trna) 1.Amino acid attachment site: Each trna have a site at the 3 end for amino acids attachment. - The carboxyl (COOH) group of the amino acid bind with ester bond the 3 hydroxyl (OH) group of the ribose of the adenosine (A) nucleotide in the CCA sequence at the trna 3 end. - The trna with attached amino acid is described as charged trna, and without amino acid is uncharged, while the bound amino acid is activated amino acid. 2.Anti-codon loop: Each trna contains a 3 nucleotides sequence known as anti-codon at the anti-codon region. It is complementary to the codon of the carried amino acid found in mrna.

12 Transfer RNAs (trna) & mrna c. Aminoacyl-tRNA synthetases: A family of enzymes catalyze the attachment of amino acids to their corresponding trnas. Each enzyme recognize specific amino acid and its trna. - Each aminoacyl-trna synthetase catalyzes 2-step reaction, consuming 2 high energy bonds of a single ATP molecule, to form a covalent bond between the amino acid and the A nucleotide at the 3 end of trna. - Also the enzymes have a proofreading (editing) activity as it correct wrong binding between trna and amino acids. d. Messenger RNA: Specific mrna required as a template that depict the amino acids and their sequences in the translated polypeptide chain.

13 e. Functionally competent ribosome Ribosomes are large complex of proteins and rrnas, consists of 2 subunits, one large (60S) and one small (40S), together form the 80S full ribosome in eukaryotes. - The S stand for Svedberg unit, measure sedimentation coefficients (molecular mass & shape). - The 60S catalyze the formation of the peptide bonds that link amino acids together in proteins. - The 40S binds mrna and ensure base pairing between codons and anti-codons.

14 Functionally competent ribosomes 1. Ribosomal RNA: Eukaryotic ribosomes contain 4 rrna. The 60S contain 5S RNA, 5.8S RNA & 28S RNA, while the 40S contain 18S RNA. 2. Ribosomal proteins: The 60S subunit contains about 50 proteins and the 40S about 30 proteins, all play a number of roles in the structure and function of the ribosomes. 3. A, P, and E sites on the ribosomes: The ribosomes has 3 binding sites for trna; the A, P, & E, each extends over both subunits.

15 Functionally competent ribosome - During translation the A site binds the incoming new aminoacyl-trna as directed by the mrna codon in the A site to add a new amino acid to the polypeptide chain. - The P site occupied by peptidyltrna which carries the peptide chain already being synthesized. - The E site occupied by the uncharged trna as its about to exit after delivering the amino acid.

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17 Functionally competent ribosomes 4. Cellular location of the ribosomes: the ribosomes are either free in the cytosol or attached to the endoplasmic reticulum - ER (make ER as rough ER). - Proteins synthesized in ribosomes attached to ER are for export outside the cell or for integration in the plasma membrane, ER or Golgi membranes or incorporated in the lysosomes. - While proteins synthesized in free cytosolic ribosomes are required for use in the cytosol itself or destined for nucleus, mitochondria and peroxisomes. - The mitochondria in addition synthesize its own proteins by its own ribosomes using its own DNA.

18 Components required for translation f. Protein factors: Initiation, elongation and termination (releasing) factors are needed for peptide synthesis (have catalytic function or stabilize the ribosome) g. ATP and GTP: are required as source of energy. - Four high energy bonds are consumed for addition of one amino acid to the peptide chain: - Two high energy bonds from one ATP (ATP to AMP) used in a aminoacyl-tran synthetase reaction - Two GTPs, one for binding of aminoacyl-trna to the A site and one for the translocation step. - Additional ATP and GTP needed for initiation and another GTP for termination.

19 Codons and anti-codons Codon recognition by trna: - The correct-fullness of base-pairing between the mrna codon and the trna anti-codon is essential. - Sometimes one trna can recognized more than one codon, but all recognized codons codes for the same amino acid. A. Anti-parallel binding between codon and anti-codon: - The mrna codon is read from 5' to 3' by trna anti-codon pairing in flipped orientation, 3' to 5'. - So the codon and anti-codon are complementary (base pairing A-U and G-C) and anti-parallel (5' to 3' and 3' to 5' orientation).

20 B. Wobble hypothesis - The mechanism by which the anti-codon of specific trna can recognize and bind more than one codon for specific amino acid is described as wobble hypothesis. - In this hypothesis the interaction between the 3 rd nucleotide in the codon with the corresponding nucleotide in the anti-codon, not always follow the base-pairing rule e.g. G can bind C or U

21 Wobble hypothesis - The wobbling of the genetic code at the 3 rd position, permit one trna to recognize more than one codon for specific amino acid. - For that reason the number of trnas (50) is less than the number of the codons in the genetic code (61).

22 Steps in protein translation The mrnas are translated from their 5' end to the 3' end, using a set of 50 trnas charged with 20 amino acids. The product proteins are synthesized from the amino terminal (NH2) to the carboxyl terminal (COOH) ends. The process of translation is divided into 3 phases; initiation, elongation and termination (release). The produced protein may be modified in a process known as post-translation modification.

23 a. Initiation of translation A. Initiation: - Include assembly of the components of the translation machine (ribosome), before peptide bonds formation. - The machine components include; 2 ribosomal subunits (60S & 40S), the mrna to be translated, aminoacyl-trna containing the 1 st amino acid, GTP and ATP as source of energy, and initiation factors (in eukaryotes about 10 proteins designated as eukaryotic initiation factors- eifs) that assemble this complex into functioning system.

24 a. Initiation of translation A. Initiation: - The 1 st codon in eukaryotes mrnas is always AUG which is recognized by special initiator trna. - Recognition is facilitated by eif2 plus other eifs. - The charged amino-acyl-trna enter the P site of the full ribosome and one GTP is consumed (gives GDP). The 1 st amino acid is always methionine (Met) - Note: the initiator trna is the only trna recognized by eif2 and only trna that go direct to the P site.

25 Initiation: A binary complex of eukaryotic translation initiation factor 2 (eif2) and GTP binds to methionyl transfer RNA (Met trna i Met ), and the ternary complex associates with the 40S ribosomal subunit. The association of additional factors, such as eif3 and eif1a (1A), with the 40S subunit promotes ternary complex binding and generates a 43S pre-initiation complex. The capbinding complex, which consists of eif4e (4E), eif4g and eif4a (4A), binds to the 7-methyl-GTP (m 7 GTP) cap structure at the 5' end of a messenger RNA (mrna). eif4g also binds to the poly(a)- binding protein (PABP), thereby bridging the 5' and 3' ends of the mrna. This mrna circularization and the ATP-dependent helicase activity of eif4a are thought to promote the binding of the 43S pre-initiation complex to the mrna, which produces a 48S pre-initiation complex. Following scanning of the ribosome to the AUG start codon, GTP is hydrolysed by eif2, which triggers the dissociation of factors from the 48S complex and allows the eif5b- and GTPdependent binding of the large, 60S ribosomal subunit. Although the precise timing and requirements for the release of factors from the pre-initiation complexes are not clear, the 80S product of the pathway is competent for translation elongation and protein synthesis.

26 b. Elongation - Elongation involves addition of an amino acid to the carboxyl end of the growing chain. - During elongation the ribosome moves from 5' end to 3' end of the mrna. - An aminoacyl-trna enters the free A site of the ribosome, its determined by the codon of the mrna that appears in the empty A site, and the entry is facilitated by elongation factors eef-1α and eef-1bg.

27 - Formation of peptide bond between the amino group of the amino acid charged in the aminoacyl-trna and the carboxyl group of the growing peptide chain in the P site.

28 Elongation This bond formation is catalyzed by peptidyl transferase (an intrinsic activity of the 28S rrna so, this rrna is referred to as ribozyme). Thereafter the ribosome moves 3 nucleotides towards the 3' end of the mrna, this process is known as translocation and it needs eef-2 and one GTP. The uncharged trna moves into the E site of the ribosome and released and peptidyl-trna moves into the P site.

29 Termination of translation - The termination occurs when one of the 3 stop codons (UAA, UAG or UGA) in the mrna appears in the free A site. - In eukaryotes a single release factor, erf (recognize stop codons), facilitate the release of the synthesized protein. - That is followed by detachment of the different components of the translation machinery. - The ribosomal subunits, mrna, trna and protein factors recycle to be used in synthesis of another polypeptide

30 Translation D. Polysomes: Because of the length of most mrnas, more than one ribosome can translate one mrna, all at the same time, such complex is called polysome or polyribosome E. Regulation of translation: Most of the gene expression is regulated at transcription. However, regulation of the rate of translation, accomplished by covalent modification of eif-2 (phosphorylated eif-2 is inactive)

31 Antibiotic (antimicrobial) target bacterial translation Many antibiotics taken by human against bacteria (prokaryotes) affects protein synthesis in bacteria but not in human because the initiation of translation is different between eukaryote and prokaryote The two subunits of the prokaryotes ribosomes are 30S and 50S, which form the full ribosome 70S For the other differences see the table next slide

32 Translation: Comparison between eukaryote & prokaryotes Eukaryotic Translation 1. Ribosomes 80 S 70 S Prokaryotic Translation 2. Sites Transcription occurs in nucleus while translation takes place in cytoplasm. It is a continuous process as both transcription and translation occur in cytoplasm 3 First amino acid Met (methionine). fmet.(formyl methionine) 4 Initiation codon Usually AUG, occasionally GUG or CUG. 5 Velocity Slower process, adds 1 amino acid per second 6 Initiation factors It requires a set of 9 eif 1, 2, 3, elf 4A. 4B, 4C, 4D, Fate of initiating Removed from the polypeptide methionine chain. 8 Release factor in requires single erf 1 the termination 9 mrna stability It is quite stable, has a life of few hours to few days. Usually AUG, occasionally GUG or UUG. Faster process, adds about 20 amino acids per second. It requires 3 initiation factors IFI. IF2. IF3. Only formyl group is removed methionine is retained. It requires two release factors RF1 and RF2 less stable, life is short (some seconds to some minutes)

33 Post-translation modification of proteins Many proteins are covalently modified while still attached to the ribosomes or after their synthesis is complete. These modifications may include, removal of part of the translated protein or covalent addition of one or more of the chemical groups.

34 a. Trimming New proteins to be secreted from the cell are synthesized as large molecules (precursor molecules), which are not active, to be active the proteins are cleaved by endoproteases. Examples of the precursor molecules are the zymogens, they are inactive precursor for enzymes, activated by cleavage when and where are needed, in or outside the cell. Also insulin is synthesized as pre-pro-insulin, the active form is insulin, the signal peptide and C peptide are removed

35 b. Covalent modifications Enzymes and other proteins may be activated or inactivated by addition of chemical groups. Below are some of these modifications: 1. Phosphorylation: Occurs by addition of P to the OH (hydroxyl) group of amino acids (serine, threonine or tyrosine) of the protein. The phosphorylation is catalyzed by protein kinases, and reversed (de-phosphorylation) by phosphatase enzymes. Both phosphorylation or de-phosphorylation may increase or decrease the activity of the protein. 2. Glycosylation: Many proteins destined to be secreted outside the cell, or to become part of the plasma membrane or lysosomes, have carbohydrate molecule attached to certain amino acids (serine or threonine or asparagine) in the protein. The addition of sugar occurs in the endoplasmic reticulum and Golgi Apparatus.

36 Covalent modifications 3. Hydroxylation: The proline and lysine amino acids of collagen are extensively hydroxylated (OH) in the endoplasmic reticulum. 4. Other covalent modification, examples: - Addition of carboxyl groups (carboxylation), a modification essential for many blood clotting factors. - Biotin (vitamin) covalently bound to the amino groups of certain enzymes that catalyze certain reactions. - Many proteins are acetylated (bind acetyl group).