• +919785333312/14

#### LET'S TALK LIFE SCIENCE MOLECULAR BIOLOGY

Suraj Prakash Sharma | Ekta Chotia

GENETIC CODE
432

6.1.         Characteristics of a genetic code :

• It is a triplet code. Each three-nucleotide codon in the mRNA specifies one amino acid
• It is comma free (i.e. no punctuation mark a used in between codons.) mRNA is read three bases at a time without skipping any bases.
• It is non-overlapping/non-ambiguous. Each nucleotide is part of only one codon and is read only once during translation. Genetic code is non-ambiguous i.e. a particular codon is always specific for amino acid. Much of the degeneracy is in the third base position. In many cases such as the codons for glycine, the third base can be any of the four. Thus GGA, GGC, GGG and GGU are all glycine codons. This is often written as GCX or GCN where X or N can be any base.
• It is almost universal. Genetic code is nearly  universal that means a particular codon codes for the same aminoacid in nearly all organisms. Exception is the mitochondrial DNA and protozoa.
• It is degenerate. Genetic code is degenerate. Since there are 61 codons for twenty amino acid so more than one codon can code for a single amino acid. Out Of twenty amino acids, eighteen are encoded by two or more codons.

6.2.         Significance of degeneracy :

1. The effect of mutation is reduced.
2. The number of tRNA required is reduced.
• Polarity. The genetic code has polarity, that is, the code is always read in a fixed direction, i.e., in the 5′ $\rightarrow$ 3′direction.
• The code has start and stop signals. AUG is the usual start signal and defines the open reading frame (cistron). Stop signals are codons with no corresponding tRNA. The nonsense or chain-terminating codons. Generally there are three stop codons: UAG, UAA, and UGA.

Before the genetic code discovered, it was a mystery as how four nucleotides could encode for 20 amino acids. The group of nucleotides that specifies one amino acid is a code word or codon.

6.3.         Singlet code hypothesis

The simplest possible code is a singlet code (a code of single letter) in which one nucleotide codes for one amino acid. A one-letter code could specify four amino acids.

6.4.         Doublet code hypothesis

A code of two letter) in which combination of two nucleotide codes for one amino acid. A two letter code could specify 16 (42 = 16)

6.5.         Triplet code hypothesis

In a triplet code of 64 codons, there is an excess of (64 – 20) 44 codons and, therefore, more than one codons are present for the same amino acid. This excess will be still greater if more than three-letter words are used. In a quadruplet code there will be (4 X 4 X 4 X 4)  256 possible codons.

Triplet code (a code of three letters) could specify sixty four (4 X 4 X 4) amino acids. Therefore, it is likely that there may be 64 triplet codes for 20 amino acids.

One or two nucleotides representing each amino acid would not sufficient.

Scientist conclude that

A one-letter code could specify four amino acids

A two letter code could specify 16 (42 = 16)

To accommodate 20, at least three letters are needed (43 = 64)

Therefore 3 letters to a codon was the most accusative.

The first genetic code ever to be established was the codon (UUU) for phenylalanine (Phe). AUG and GUG encode methionine, which initiates most polypeptide chains. All other amino acids, except tryptophan (which is encoded only by UGG).

Terminating/ Nonsense/Stop Codon:

Three codons namely UAA(ochre), UAG(amber), UGA(opal)  do not code for any aminoacid and hence bring about the termination of protein synthesis. They are recognised by release factors and release the signal of polypeptide from the ribosome.

6.6.         Codon assignment

6.7.        Wobble Base hypothesis :

It was given by Francis Crick. Wobble base pair is the 3rd base of the codon(3’end) and the 1st base of the anticodon( 5’end). Wobble base done not follow Watson-crick pairing. The change in the wobble base does not change the specified amino acid.  So the requirement of tRNA gets minimised and reduced to 32 tRNAs. The hydrogen bonding among the wobble basepair is very weak compared to the 1st two bases.

The four main wobble base pairs are : Guanine-Uracil, Inosine-Hypoxathine-Adenine, Inosine-Hypoxathine-Uracil, Inosine-Hypoxathine-cytosine.

6.8.         Post Translational Modification

Post translational modifications are key mechanisms to increase the complexity in the genome and hence increasing their diversity. Approximately from the 25000 genes, millions of proteins can be formed which constitute the Proteome.

It includes:

1. Protein folding
2. Chemical modification
3. Proteolytic cleavage
4. Intein splicing

1.            Protein folding

Chaperons are a group or class of proteins that assist in proteinfolding. There are 2 classes: Heat shock proteins and Chaperonins. These chaperonis provide a suitable environment for the protein to undergo folding.

2.            Chemical modification:

PTM can be done at any stage of a life cycle of a protein. It can be done immediately to ensure the proper folding and for stability or to direct them into their respective organelles. Other modifications can be done after folding and localisation which leads to activate or inactivate the catalytic/ biological activity of the protein.

Chemical group                Amino acid

Acetylation                         Lysine

Methylation                        Lysine

Hydroxylation                     Lysine, Proline

Phosphorylation                Serine, Threonine, Tyrosine, Histidine

Acylation                            Serine, Threonine, Cysteine

Formylation                        Glycine

Biotinylation                       Lysine

Ubiquitination                     Lysine

6.8.1.     Acetylation :

Addition of acetyl group  at the lysine residue at the histone tails causes the activation of gene. The enzymes employes is HAT(Histone Acetyl Transferase) where as HDAC (histone deacetylase) shuts off the gene.

6.8.2.     Methylation :

Adding of methyl groups renders the gene inactive and is brought about by HMT (Histone methyl transferase).

6.8.3.     Phosphorylation:

Addition of phosphate group is known as phosphorylation. Kinases are enzymes that transfers the phosphate group and phosphate removes the group. Mostly the donor is ATP. Phosphorylation has several biological functions which includes:

1. Phosphorylation of Na+/K+ pump which leads to its conformational change and thus very impotent for osmoregulation
2. In signalling pathway like proteins to be transpoted to Lysosome requires the tagging of Mannose-6-phosphate
3. In regulation the biological activity of some proteins. Phosphorylation of some proteins can make them switched off or on.
4. Helps in the interaction between the proteins.

6.8.4.     Glycolsylation :

Glycolsylation is the addition of carbohydrate moiety to the protein. N-linked glycosylation occurs at the Aspargine residue of a protein involved in a secretory pathway.  All the proteins trafficked through secretory pathway are glycosylated and is very crucial for proper folding. It occurs in the endoplasmic reticulum. O-linked glycosylation occurs at the serine, threonine residue which are to be transported further. That occurs in Golgi body.

6.8.5.     Ubiquitination:

The proteins which are to be degraded are tagged with ubiquitin (76AA) at the lysine residue of misfolded protein with the Isopeptide bond.

6.8.6.     Proteolytic Cleavage:

Generally some sequences at the N terminus function as signal peptides and undergo cleavage during post translational modification. Examples: a) post translational processing of Pre-POMC(Pro ophio melanocrtin) in the pituitary. b) A signal peptide for approx 24 aminoacids at the N terminus of Pro-Insulin. Active insulin is produced through cleavage of two peptide chains linked together through disulphide bonds. c) Zymogens are also activated by the proteolytic cleavage.

6.8.7.     Intein splicing:

The non functional sequences or portions in a protein are called as Inteins and they are removed during PTM. They are protein introns. Cysteine, threonine or serine are present at the splicing junction. This is conserved throughout the evolution. They have OH and SH  group respectively which act as a Nucleophile and bring about the transesterification reaction which joins the two exteins. This is a self catalysed reaction.  The spliced out intein acts as a potential double standed DNA cutter and contain LAGLIDADG motif. So if this process occurs in the prokaryotic system, this intein will cut the dsDNA and will produce double stranded break which cannot be repaired and eventually lead to the death of the cell. Thats why the Inteins are considered to be selfish. But if this is occurring in the eukaryote, no harm will be there as they have the double copies of the same gene and thus the double stranded produced by the intein can be overcome easily with the double standed break repair.

DnaE is the catalytic subunit of DNA polymerase III present in cynobacterium Synechocystis sp. PCC6803, is a split gene product containing Inteins which undergo Trans-splicing to produce intact DnaE protein.

6.9.         Antibiotics effect translation

Earlier stages

Rifampicin inhibits DNA dependent RNA polymerase by binding its beta-subunit in turn inhibit prokaryotic DNA transcription. It has lipophilic nature makes it a good candidate in the treatment meningitis form of tuberculosis, which requires distribution to the central nervous system  and penetration through the blood-brain barrier.

Initiation

• Linezolid inhibit the formation of the initiation complex, though the mechanism is not fully understand.
• Aminoacyl tRNA entry
• Tetracyclines and Tigecycline (a glycylcycline related to tetracyclines) inhibting the binding of aminoacyl tRNAs by block the A site on the ribosome.
• Aminoglycosides, causing amplified rate of error in production of polypeptide chain by premature termination because causing hamper within the proofreading process also inhibit  translocation, disrupt the integrity of bacterial cell membrane and binds with 30S ribosomal subunit.
• Elongation
• Kirromycin: Function of EF-Tu inhibited by an antibiotic name kirromycin. When EF-Tu is bound by kirromycin, then EF-Tu-GDP complex cannot be released from the ribosome and prevent formation of the peptide bond between the peptidyl-tRNA and the aminoacyl-tRNA. Thus arrest of translation occurs.
• Peptidyl transfer
• Chloramphenicol in both bacteria and mitochondri blocks the peptidyl transfer reaction in 50 S subunit of ribosome.
• Macrolides inhibiting peptidyl transfer as well as ribosomal translocation in 50 S subunit.
• Syneragistic act reported in case of Quinupristin/dalfopristin, with dalfopristin enhancing the binding of quinupristin to 50 S subunit and inhibiting peptidyl transfer, Quinupristin binds to a close site on the 50S ribosomal subunit and inhibiting elongation of the polypeptide simintaniouly causing incomplete chains to be released.
• Cycloheximide: It binds to 80 S ribosome and inhibit the peptidyl transferase reaction.
• Ribosomal translocation
• Clindamycin inhibition of ribosomal translocation.
• Aminoglycosides and macrolides have evidence of inhibition of ribosomal translocation.
• Fusidic acid block elongation by preventing the turnover of elongation factor G (EF-G) from the ribosome.
• Ricin: Ricin is a toxic protein extrac from the castor bean (Ricinus communis) cause depurination of a specific adenosine in 28 S RNA of 60 S subunit. Ricin is classified as a type 2 ribosome inactivating protein (RIP) which is also known as holotoxins, are heterodimeric glycoproteins Neomycin. Whereas single enzymatic protein chain present in Type 1 RIPs.
• Neomycin: It is an aminoglycoside antibiotic, which is present in various topical medications such as creams, ointments, and eyedrops and other medicinally important drugs.

Termination

• Puromycin: It has a structure similar to the tyrosinyl aminoacyl- tRNA or we can say it has structure analogue 3’ end of amino acytRNA. It binds to the ribosomal A site and participates in peptide bond formation, producing peptidyl-puromycin and its amide bond not cleave by peptidal transferase thus not engage in translocation and quickly dissociates from the ribosome causing a premature termination of polypeptide synthesis.
• Macrolides and clindamycin (both also having other potential mechanisms) responsible for premature dissociation of the peptidyl-tRNA from the ribosome.
• Streptogramins: It also responsible for premature release of the peptide chain.

Binding site

The following antibiotics bind to the 50S ribosomal subunit:

Chloramphenicol: It bacteriostatic (that is, it stops bacterial growth) inhibit peptide bond formation because bind to A2451 and A2452 residues in the 23S rRNA of 50 S subunit and inhibit peptidyl transferase reaction. It directly interferes with substrate binding.

Erythromycin: It inhibits peptide chain formation in elongation by bind to 50 S subunit.

Linezolid: Linezolid binds to the 23S rRNA present in 50S subunit (peptidyl transferase activity) and inhibit protein synthesis. It site close to the binding sites of chloramphenicol,  lincomycin, and other antibiotics. Due to this unique mechanism of action, cross-resistance between linezolid and other protein synthesis inhibitors is highly uncommon or absent.

Tetracycline: It binds to the 16S rRNA present in 30S ribosomal subunit and prevents the amino-acyl tRNA from binding to the A site of the ribosome and thus inhibiting cell growth. It has reversible nature of binding.

Streptomycin: It is a aminoglycosides and trisaccharide, responsible for misreading of mRNA in relatively low concentration by binding through 30 S subunit. paromomycin, kanamycin and streptomycin are aminoglycosides are known for their ability to bind   to duplex RNA with high affinity.

Diptheria toxin: It is an exotoxin of Cornephage β (lysogenic phage) infected cornybecterium diptheriae, which inactivate eEF2 (E.K.) by ADP-ribosylation, A fragment of toxin responsible for ribosylation.

Page no. 432