Gene Expression 2: RNA Translation and Genetic Code PDF

17 GENE EXPRESSION 2. RNA TRANSLATION AND GENETIC CODE ILOs By the end of this lecture, students will be able to 1. Discuss the rules of genetic code 2. Correlate the function of different RNAs to the process of translation 3. Describe the process of translation 4. Interpret r...

17 GENE EXPRESSION 2. RNA TRANSLATION AND GENETIC CODE ILOs By the end of this lecture, students will be able to 1. Discuss the rules of genetic code 2. Correlate the function of different RNAs to the process of translation 3. Describe the process of translation 4. Interpret role of translation and post translational modification in health and disease What is translation? It is the translation of the nucleotide sequence of a mRNA (Codons) into an amino acid sequence of a protein, in order to synthesize proteins. Each codon consists of a sequence of 3 nucleotides i.e. it is a triplet code. Collection of these codons makes up the genetic code. Protein biosynthesis is called translation because it involves translation of information from the 4- letter language and structure of nucleic acid into the 20-letter language and structure of proteins Requirements of translational process m RNA as a carrier of genetic information. tRNA as an adapter molecule, which recognizes an amino acid on one end and its corresponding codon on the other end. At least one specific type of tRNA is required for each amino acid. Ribosomes as the molecular machine coordinating the interaction between mRNA, tRNA, the enzymes and the protein factors required for protein synthesis. Genetic code (Figure 1) It is the relationship between nucleotide sequence in DNA or mRNA AND amino acids in a polypeptide chain. Each amino acid can be specified by more than one codon. There is one start codon: AUG (METHIONINE) There are 3 STOP codons (UAA, UAG, UGA) The genetic information along mRNA is read from 5’ to 3’ direction. Figure 1.genetic code 1 Characteristics of genetic code 1- Genetic code is degenerate i.e. multiple codons can code for the same amino acid except tryptophan and methionine.(both are coded by only one codon) Wobble theory: The 3rd nucleotide in a codon is less important than the other two in determining the specific amino acid to be incorporated. (i.e if an amino acid has several codons, all such cosons will usually have the first 2 letters in common but the third is different, which means the 3 rd is of less importance) 2- Genetic code is unambiguous i.e. each codon specifies no more than one amino acid. 3- Genetic code is non-overlapping and Commaless meaning that the code is read from a fixed starting point as a continuous sequence of bases, taken three at a time without any punctuation between codons. For example, AGCUGGAUACAU is read as AGC UGG AUA CAU. 4-Genetic code is universal i.e. the same code words are used in all organisms (pro- and eukaryotes) Protein Biosynthesis stages 1- Initiation 2- Elongation 3- Termination Stage 1: Initiation For initiation of protein biosynthesis, there must be:- - tRNA - rRNA - mRNA - Eukaryotic initiation factors (eIFs). - GTP, ATP and different amino acids. In this stage, The 80 S eukaryotic ribosome is dissociated into 40 S and 60 S subunits. eIF – 3 and eIF-1 bind to 40 S subunit thus preventing re-association between the 2 subunits. GTP and eIF-2 bind, in addition to binding of mRNA (accompanied by hydrolysis of ATP to ADP+Pi)and Methionine– tRNA (a tRNA specifically involved in binding to the initiation codon AUG). This This is followed by re-association of both ribosomal units, with dissociation of initiation factors and hydrolysis of GTP. This is termed the initiation complex. (Can you enumerate its components?) (Figure2) Figure 2. Initiation of translation 2  N.B: t RNA charging (Figure 3) It means recognition and attachment of the specific amino acid to the 3` hydroxyl adenosine terminus (to the sugar) of tRNA in an ester linkage. Figure 3.tRNA charging Stage 2: Elongation It is a cyclic process involving 3 steps I. Binding of aminoacyl – tRNA to the A site The ribosome has three binding sites for tRNA molecules: the A, P, and E sites.(Figure 4) In the complete 80S ribosome subunit, A site is free (N.B. A=aminoacyl binding site) Binding of aminoacyl t-RNA to A site needs activation of aminoacyl tRNA by binding of eukaryote elongation factor - 1 (e EF-1) and GTP. When aminoacyl tRNA binds to A site, GTP is hydrolysed and e EF-1 is released. Figure 4..Binding sites of tRNA in ribosome N.B: Anticodon: Each tRNA molecule contains a three-base nucleotide sequence, the anticodon, which pairs with a specific codon on the mRNA within the ribosome. This codon specifies which aminoacid will be inserted in the growing peptide sequence. The first codon on mRNA always codes for methionine. II. Peptide bond formation (Figure 5) The alpha amino group of the new amino acid carried by t-RNA in the A site attacks the carboxylic group of the peptidyl-tRNA in the P site. This reaction needs peptidyl transferase enzyme (RIBOZYME) The reaction results in attachment of the growing peptide chain to the tRNA in the A site. III. Translocation (Figure 5) Upon removal of the peptide from t-RNA in the P site, the discharged t-RNA quickly dissociates. eEF-2 and GTP are responsible for translocation of the newly formed peptidy t-RNA from A site to P site 3  The A site is now free to receive a new aminoacyl-RNA Stage 3: Termination (Figure 6) After many cycles of elongation, the non-sense or stop codon of mRNA (UAA, UAG or UGA) appears in the A site. Normally, there is no tRNA with an anticodon capable of recognizing such a termination signal. Releasing factors (eRFS) can recognize the termination signals in the A site Releasing factors(eRFs), GTP and peptidyl transferase promote the hydrolysis of the bond between the peptide chain and t-RNA at P site 80S subunit dissociates and all the factors , tRNA , mRNA, GDP and Pi are released Figure 5. Process of elongation Figure 6.Termination of translation N.B. The formation of one peptide bond requires energy resulting from hydrolysis of 4 high energy phosphate bonds:- Charging of tRNA with amino acyl moiety requires hydrolysis of an ATP to an AMP. (2 high energy bonds) The entry of amino tRNA into the A site requires one GTP hydrolysis to GDP. The translocation of the newly formed peptidyl – tRNA in the A site into the P site results in hydrolysis of one GTP to GDP N.B. Errors in translation will result in faulty proteins, which will be either targeted for degradation in proteasome or will be non- functioning or abnormally functioning. Protein maturation Aim: Activation of protein to a functional form 4  Localization in subcellular compartment Secretion from the cell Protein maturation involves the following: I- Protein folding: Folding to 3D structures, aided by molecular chaperone. (Refer to protein structure lecture). Misfolded proteins are targeted for destruction II- Post – translational processing: a- Proteolysis: It means removal of amino terminal, carboxy terminal or internal sequences. Examples: 1-Removal of amino terminal methionine residues 2-Removal of signal peptides by signal peptidases (signal peptides help translocate the proteins to its final destination and hence are removed after protein transport) (figure 7) b- Modifications of individual amino acids Figure 7.Proteolysis Hydroxylation: of proline and lysine for collagen synthesis Phosphorylation of serine, threonine or tyrosine (Important in cell signaling) γ-carboxylation of glutamic acid in prothrombin and osteocalcin (this helps Ca 2+ binding which is necessary for blood clotting and bone ossification in both proteins respectively) c- Addition of certain groups Glycosylation: Attachment of CHO side chain to form glycoproteins. Glycosylation can help stabilize glycoproteins against degradation or provide proper conformation for protein function. CHO can be either N linked( to asparagine), or O-linked( to serine and threonine) Acylation: Addition of fatty acids to various amino acid side chains. Acylation has several functional effects on proteins, especially to help anchor them to membranes. Clinical implications: I) Many antibiotic work by altering the translation of bacterial DNA and are generally classified as bacterial protein synthesis inhibitors as Tetracyclines and Macrolides like Erythromycin. II) Some toxins can cause death by inhibiting eukaryotic translation: Shiga toxin / ricin (produced by E-coli bacteria and causes bloody diarrhea) inhibits tRNA binding by acting on 60S subunit Diphtheria toxin (produced by bacteria and causes difficult breathing, heart failure, paralysis, and even death) inhibits translocation through binding to Eukaryotic- EF-2 5