Introduction to Protein synthesis and genetic code
- Genetic information is stored in the chromosome and transmitted to daughter cells through replication. It is expressed to RNA through transcription.
- From the mRNA genetic information is translated to polypeptides.
- the flow of information from DNA -> RNA -> protein is called central dogma.
- The set of triplet code words in DNA or mRNA coding for amino acids of protein. This is the dictionary that gives correspondence between a sequence of nucleotide bases and a sequence of amino acids in a protein. Each individual word in the code is composed 3 nucleotides. These genetic words are called codons.
- Altogether there are 64 codons of which 61 codons code for 20 amino acids in a protein. 3 codons act as termination signal.
- 61 codons coding for amino acids are called sense codons and the rest( UAG, UGA, UAA) are called nonsense codons.
Characteristics of genetic code
- Non overlapping.
- Non punctuated.
- This means multiple codons code for a single amino acid. There are six codons for serine, four for glycine and one for methionine.
- AUG for methionine.
- GGA, GGC, GGG and GGU for glycine
- One specific codon codes for only one specific amino acid.
- For example -> phenylalanine is coded by both UUU and UUC but UUU will code only for phenylalanine.
- So given a specific codon only a specific amino acid will come although given a specific amino acid, more than one codon may be called.
Non overlapping & non punctuated
- Non overlapping -> codons are consecutive. They are read one after another in a continuous manner.
- Non punctuation -> no comma between two codons.
- Until recently the genetic code was thought to be universal that means one specific codon will code for one specific amino acid in all the organisms.
- Recently it has been shown that four codons behave differently in cytoplasm and mitochondria of the same cell. For example UGA codes for termination codon in cytoplasm and tryptophan in mitochondria
- The degeneracy of genetic code resides mostly in the last base of the codon, suggesting that the base pairing between the last nucleotide of the codon and the corresponding nucleotide in the anticodon is not strict and less strong.
- This phenomenon is called Wobble.
Example of wobble
- The two codons for arginine AGA and AGG can bind to the same anticodon having uracil at the 5’ end.
- Similarly three codons for glycine GGC, GGU, and GGA can form base pair with the same anticodon CCI.
- Permanent and heritable change in the genome which will appear in the daughter DNA , after replication, in the RNA after transcription and in the protein after translation.
- 1) Point mutation
- 2) Frame shift mutation.
- single base alteration in the gene.
2 classifications :-
- transition and transversion.
- silent mutation, nonsense mutation and missense mutation.
Transition & Transversion
- Transition -> when one purine base is replaced by another purine base.
- for example – adenine -> guanine and vice versa.
- Transversion -> purine is replaced by pyrimidine.
- For example — A -> C, A -> T, G -> C, G -> T and vice versa.
- Silent mutation -> The codon containing the changed base may code for the same amino acid. For example -> if serine codon UCA is changed to UCU, it still codes for serine. So there will be no change in the protein product.
- Other examples -> AGG and AGA both code for arginine
Non sense mutation
- The codon containing the changed base may become a termination codon.
- For example if serine codon UCA is given a different 2nd base ( to become UAA) the new codon becomes a termination codon which causes premature termination of protein synthesis at that point.
- Example -> β thalassemia in which β chain of hemoglobin is prematurely terminated.
- The codon containing the changed base may code for a different amino acid, for example if the serine codon UCA is given a different 1st base (to become CCA ), it will code for a different amino acid in this case proline.
- This substitution of an incorrect amino acid is called missense mutation. Example :- sickle cell anemia. GAG -> GUG.
Frame shift mutation
- The deletion or addition of a single nucleotide in the coding strand of a gene results in an altered reading frame in the mRNA.
- When the mRNA is translated to protein the machinery does not recognize that a base is missing or extra since there is no punctuation in the reading frame.
- Thus there will be major alteration in the sequence of amino acids distal to the deletion or addition.
Example of mutation
- Point mutation ( missense) -> sickle cell anemia.
- Point mutation ( nonsense) -> α and β thalassemias.
- Frame shift mutation -> thalassemias
Effects of mutation
- Lethal effect -> if the mutation is incompatible to the life. For example -> β-thalassemia major.
- Carcinogenic effect -> mutation may result in uncontrolled cell division leading to cancer.
- Disease -> sickle cell anemia , thalassemia.
Translation or protein synthesis
Components needed for protein synthesis.
- 1) amino acids.
- 2) tRNA.
- 3) mRNA.
- 4)aminoacyl tRNA synthase.
- 5) ribosome.
- 6) protein factors.
- 7) ATP / GTP.
Translation in pro and eukaryotes are similar with some differences
- 1) The 1st amino acid is methionine in eukaryotes but N formyl methionine in prokaryotes.
- 2) Shine- Delgarno sequence is present at the start site in prokaryotes, Kozak sequence in eukaryotes.
- 3)eukaryotic ribosomes are larger ( 80 S).
- In prokaryotes protein synthesis begins before transcription is complete i.e. transcription and translation go on simultaneously.
- In eukaryotes transcription is nuclear one and translation occurs in cytoplasm. So simultaneous transcription and translation is not possible. Also there is necessity of generation of mRNA from hnRNA.
- Eukaryotes have 9 initiation factors. Prokaryotes have 3.
Codon recognition by the tRNA
- Recognition of a particular codon in an mRNA sequence is done by the anti codon sequence of tRNA.
- Some tRNAs recognize more than one codon for a given amino acid.
- Binding of anti codon and codon follows the rules of complementary and anti parallel binding.
- Codons are read in 5’- 3’ direction by an anti codon pairing in 3’ – 5’ direction.
Steps of protein synthesis
- Activation of amino acid.
- Post translational modification.
- Ribosome serves as machinery for protein synthesis
Activation of amino acid.
- Amino acid + ATP -> (amino acyl tRNA synthatase ) E-AMP- amino acid.
- E- AMP- amino acid + tRNA -> (E ) -> amino acyl tRNA. Enzyme and AMP will be released.
- This is a family of enzymes. Each member of the family recognizes a specific amino acid and the tRNA that corresponds to that amino acid.
Initiation of protein synthesis
- At first the ribosome selects the mRNA and binds to it. The ribosome finds the correct reading frame on the mRNA and translation begins.
- The process involves tRNA, rRNA, mRNA , at least 9 eukaryotic initiation factors ( e .i .f s ) and also GTP, ATP, amino acids.
- Prokaryotes have 3 initiation factors.
A,P and E sites of ribosome.
- The ribosomes have two binding sites and one ejection site for the tRNA molecule.
- During translation the A site binds with the incoming amino acyl tRNA as directed by the codon currently occupying this site.
- the P site is occupied by the tRNA containing the polypeptide chain that has already been synthesized.
Sub steps of initiation
- Ribosomal dissociation.
- Formation of 43 S pre initiation complex.
- Formation 48 s initiation complex.
- Formation of 80 s initiation complex.
Ribosomal dissociation and formation of 43S pre initiation complex
- Two initiation factors eif 3 and 1A bind to 40S subunit. This favors dissociation of the 80S ribosome into 40S and 60S.
- 40S ribosome now binds to GTP, eif 2 and met tRNA to form 43S pre initiation complex.
- Met tRNA is the tRNA involved in binding to the initiation codon AUG.
Formation of 48S and 80S complex
- mRNA binds to the 43S pre initiation complex, and along with it eif 4F, 4A and 4B bind -> 48S initiation complex. Now this complex scans the mRNA for a suitable initiation codon.
- Binding of 60S subunit with 48S will form 80S ribosome with release of initiation factors and GTP.
- A cyclic process, involves several steps catalyzed by proteins called elongation factors (e.e.f ).
- The steps are:
- Binding of amino acyl tRNA at the A site
- Peptide bond formation.
Binding of amino acyl tRNA to the A site
- At the end of initiation 80 S ribosome has A site free. Proper amino acyl tRNA will bind at the A site by proper codon recognition.
- EF1α and GTP allow amino acyl tRNA to enter the A site. GTP is hydrolyzed to GDP and Pi.
Peptide bond formation.
- The new amino acid at the A site attacks the carboxyl group of the amino acid at the P site and form peptide bond. This reaction is catalyzed by peptidyl- transferase.
- The peptide bond is formed at the A site and the growing peptide chain is now at the A site.
- Peptidyl transferase is a Ribozyme.
- Upon removal of the peptidyl moiety from the tRNA from the P site, the tRNA quickly dissociates from the P site through E site.
- The newly formed peptidyl tRNA at the A site is now translocated to the empty P site by eef2 and GTP. Now the A site is free for another amino acyl tRNA to bind to the next codon.
Energy required for formation of one peptide bond
- 1) hydrolysis of 2 ATP molecules in the formation of amino acyl tRNA.
- 2) 1 GTP for entry of amino acyl tRNA to the A site and 1 GTP for translocation step.
- So there is need for 4 high energy phosphates for synthesis of one peptide bond.
- After many cycles of elongation , when protein synthesis is complete a nonsense termination codon of mRNA ( UAA, UAG, UGA ) appears at the A site.
- Normally there is no tRNA with an anti codon capable of recognizing such a termination signal.
- Releasing factors (erf ) can recognize the termination signal in the A site.
- The releasing factor in conjugation with GTP and peptidyl transferase promotes hydrolysis of the bond between the peptide and the tRNA occupying the P site.
- Thus a water molecule instead of an amino acid is added. This releases the protein and tRNA from the P site.
- 80S ribosome dissociates into 40S and 60S. mRNA is also released from the ribosome.
- Many ribosomes can translate the same mRNA simultaneously.
- Multiple ribosome on the same mRNA molecule form a poly ribosome or polysome.
Post translational modification
After translation the newly synthesized protein may not be in the the active form. To make it active some modifications are required:
- 1) loss of signal sequence.
- 2) folding and S-S formation.
- 3) proteolytic processing.
- 4) modification of individual amino acid and triple helix formation.
- 5) attachment of carbohydrate side chain.
- 6) acetylation, carboxylation, phosphorylation
Post translational modification
- Loss of signal sequence.
- Some proteins have 15 – 30 amino acid residues at the beginning which direct the protein to its proper destination. This signal sequences are ultimately removed by specific peptidases ( signal peptidase).
- Example :- insulin is synthesized with 23 amino acid signal peptide, which is removed. The signal peptide is also called leader sequence.
Folding and S-S bridge formation and proteolytic processing.
- Insulin is synthesized as a single chain prohormone in the ribosome. Folding and S-S bridge formation occurs in the endoplasmic reticulum.
- In the Golgi apparatus a specific protease clips out the segment that connects two chains to form the functional insulin molecule.
Triple helix formation and modification of amino acids
- Collagen is synthesized as single chain pro-collagen molecule. Three pro-collagen molecules align themselves to form the triple helix.
- Specific enzymes carry out hydroxylation of proline and lysine residues to provide cross linking and greater stability.
Inhibition of protein synthesis
- 1) Gentamycin, Streptomycin, Neomycin, Tetracycline -> bind irreversibly with 30S ribosomal subunit blocking initiation of protein synthesis.
- 2) Erythromycin, Chloramphenicol -> bind to 50S ribosomal subunit inhibit elongation.
- 3) Puromycin -> inhibits peptide bond formation both in pro and eukaryotes.
Inhibition of N.A. synthesis
- 1) Norfloxacin, O- Floxacin, ciprofloxacin, lomefloxacin inhibits topoisomerase (DNA gyrase ) in bacteria.
- 2) Nitrofurantoin, Metronidazol -> causes DNA cleavage in bacteria.
- Rifampicin -> inhibits transcription in bacteria.
Drugs used in cancer chemotherapy
- 1) methotrexate -> inhibits dihydrofolate reductase
- 2) 5 fluorouracil, 5 iodouracil, 6 azauridine, allopurinol -> inhibit Pyrimidine synthesis.
- 3) 6 thioguanine, 6 mercaptopurine -> inhibit purine synthesis.
- Cyta arabine -> inhibits replication.
Other Biochemistry Notes
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