Nucleic acid metabolism and genetic information transfer

15 Jul 2020

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Nucleic acid metabolism and genetic information transfer

  1. Nucleic acid metabolism and Genetic information transfer DEVIPRIYA P V M PHARM
  2. CONTENTS  Biosynthesis of purine and pyrimidine nucleotides  Catabolism of purine nucleotides and Hyperuricemia and Gout disease  Organization of mammalian genome  Structure of DNA and RNA and their functions  DNA replication (semi conservative model)  Transcription or RNA synthesis  Genetic code  Translation or Protein synthesis and inhibitors
  3. Biosynthesis of purine nucleotides  Ribose 5-phosphate is the starting material for purine nucleotide synthesis.  lt reacts with ATP to form phosphoribosyl pyrophosphate (PRPP).  Glutamine transfers its amide nitrogen to PRPP and produce 5- phosphoribosyl amine by the action of enzyme PRPP glutamyl amidotransferase.  Phosphoribosylamine reacts with glycine in the presence of ATP to form glycinamide ribosyl 5-phosphate (GAR).  N10-Formyl tetrahydrofolate donates the formyl group and the product formed is formylglycinamide ribosyl 5-phosphate.  Glutamine transfers the second amido amino group to produce formyl glycinamidine ribosyl 5-phosphate.
  4.  5-aminoimidazole ribosyl 5-phosphate is formed in an ATP dependent reaction.  Addition of CO2 occurs to yield aminoimidazole carboxylate ribosyl 5-phosphate.  Aspartate condenses with aminoimidazole carboxylate ribosyl 5- phosphate to form aminoimidazole 4-succinyl carboxamide ribosyl 5-phosphate.  Adenosuccinate lyase cleaves off fumarate to form aminoimidazole 4-carboxamide ribosyl 5-phosphate.  N1O-Formyl tetrahydrofolate donates a one-carbon moiety to produce formaminoimidazole 4-carboxamide ribosyl 5-phosphate.  The final reaction catalysed by cyclohydrolase leads to ring closure with an elimination of water molecule.  The product obtained is inosine monophosphate( lMP), the parent purine nucleotide from which other purine nucleotides can be synthesized.
  5.  The purines can be directly converted to the corresponding nucleotides, and this process is known as salvage pathway.  Adenine phosphoribosyl transferase catalyses the formation of AMP from adenine.  Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) converts guanine and hypoxanthine, respectively, to GMP and IMP.
  6. Degradation of purine nucleotides  The end product of purine metabolism in humans is uric acid.  The nucleotide monophosphates (AMP, IMP and GMP) are converted to their respective nucleoside forms (adenosine, inosine and guanosine) by the action of nucleotidase.  The amino group, either from AMP or adenosine, can be removed to produce IMP or inosine, respectively.  Inosine and guanosine are respectively, converted to hypoxanthine and guanine by purine nucleoside phosphorylase.  Adenosine is not degraded by this enzyme, hence it has to be converted to inosine.  Guanine undergoes deamination by guanase to form xanthine.  Xanthine oxidase converts hypoxanthine to xanthine, and xanthine to uric acid.
  7. Disorders of purine metabolism Hyperuricemia and gout:  Hyperuricemia refers to an elevation in the serum uric acid concentration.  This is sometimes associated with increased uric acid excretion (uricosuria).  Gout is a metabolic disease associated with overproduction of uric acid.  ln severe hyperuricemia, crystals of sodium urate get deposited in the soft tissues, particularly in the joints.  Such deposits are commonly known as tophi.  This causes inflammation in the joints resulting in a painful gouty arthritis.  Sodium urate and/or uric acid may also precipitate in kidneys and ureters that results in renal damage and stone formation
  8.  Gout was found to be often associated with high living, over-eating and alcohol consumption.  Gout is of two types-primary and secondary.  Primary gout is an inborn error of metabolism due to overproduction of uric acid. This is mostly related to increased synthesis of purine nucleotide.  Secondary gout is due to various diseases causing increased synthesis or decreased excretion of uric acid.  The drug of choice for the treatment of primary gout is allopurinol.  Besides the drug therapy, restriction in dietary intake of purines and alcohol is advised.  Consumption of plenty of water will also be useful.  The anti-inflammatory drug colchicine is used for the treatment of gouty arthritis.  Other antiinflammatory drugs-such as phenylbutazone, indomethacin , oxyphenbutazone, corticosteroids are also useful.
  9. Biosynthesis of pyrimidine nucleotides
  10.  Glutamine transfers its amido nitrogen to CO2 catalysed by carbamoyl phosphate synthetase II to produce carbamoyl phosphate.  Carbamoyl phosphate condenses with aspartate to form carbamoyl aspartate. This reaction is catalysed by aspartate transcarbamoylase.  Dihydroorotase catalyses the pyrimidine ring closure with a loss of H2O.  Orotate is formed by NAD+ dependent dehydrogenation.  Ribose-5-phosphate is added to orotate to produce orotidine monophosphate (OMP). This reaction is catalysed by orotate phosphoribosyltransferase.  OMP undergoes decarboxylation to uridine mono-phosphate (UMP)  By an ATP-dependent kinase reaction, UMP is converted to UDP which serves as a precursor for the synthesis of dUMP, dTMP, UTP and CTP..
  11. Organization of mammalian genome  The total DNA (genetic information) contained in an organism or a cell is regarded as the genome.  The genome is the storehouse of biological information.  lt includes the chromosomes in the nucleus and the DNA in mitochondria, and chloroplasts.
  12. Structure of DNA  DNA is a polymer of deoxyribonucleotides.  It is composed of monomeric units namely deoxyadenylate (dAMP), deoxyguanylate (dGMP), deoxycytidylate (dCMP) and deoxythymidylate (dTMP).  The monomeric deoxvnucleotides in DNA are held together by 3',5'-phosphodiester bridges.  Chargaff's rule of DNA composltion:  DNA had equal numbers of adenine and thymine residues (A = T) and equal numbers of guanine and cytosine residues (G = C).  The double helical structure of DNA was proposed by James Watson and Francis Crick.
  13. 1. The DNA is a right handed double helix. lt consists of two polydeoxyribo nucleotide chains twisted around each other on a common axis. 2. The two strands are antiparallel, i.e., one strand runs in the 5' to 3' direction while the other in 3' to 5‘ direction. 3. The width (or diameter) of a double helix is 20 A0(2 nm). 4. Each turn of the helix is 34 A0 (3.4 nm) with 10 pairs of nucleotidese, ach pair placed at a distance of about 3.4 A0. 5. Each strand of DNA has a hydrophilic 3'-5' phosphodiester bonds on the outside of the molecule while the hydrophobic bases are stacked inside. 6. The two polynucleotide chains are not identical but complementary to each other due to base pairing. 7. The two strands are held together by H-bonds formed by complementary base pairs . ( A-T pair has 2 and G-C pair has 3 hydrogen bonds). 8. The hydrogen bonds are formed between a purine and a pyrimidine only. 9. The complementary base pairing in DNA helix proves Chargaffs rule. 10. The genetic information resides on one of the two strands known as template strand or sense strand. The opposite strand is antisense
  14. Structure of RNA  RNA is a polymer of ribonucleotides held together by 3',5'- phosphodiester bridges.  Pentose: The sugar in RNA is ribose (deoxyribose in DNA).  Pyrimidine : RNA contains the pyrimidine uracil (thymine in DNA).  Single strand: RNA is usually a single stranded polynucleotide.  Chargaff's rule-not obeyed.  Susceptibility to alkali hydrolysis : Alkali can hydrolyseRNA to 2',3'-cyclic diesters (presence of a hydroxyl group at 2' position).  Types of RNA: 1. Messenger RNA (mRNA): 2. Transfer RNA (tRNA): 3. Ribosomal RNA (rRNA: ):
  15.  The mRNA is synthesized in the nucleus as heterogeneous nuclear RNA (hnRNA).  mRNA is capped at the 5'-terminal end by 7-methylguanosine triphosphate.  The 3'-terminal end of mRNA contains a polymer of adenylate residues (20-250 nucleotides) which is known as poly (A) tail.  The structure of tRNA resembles a clove leaf and contain acceptor arm, the anticodon arm, The D arm, the TYC arm, the variable arm.
  16. FUNCTIONS OF DNA FUNCTIONS OF RNA  DNA is the chemical basis of heredity and regarded as the reserve bank of genetic information  DNA is exclusively responsible for maintaining the identity of different species of organisms.  Every aspects of cellular information is under the control of DNA.  The DNA is organized into genes, the fundamental units of genetic information.  The genes control the protein synthesis through the mediation of RNA.  mRNA transfers genetic information from genes to ribosomes to synthesize proteins.  rRNA provides structural framework for ribosomes.  tRNA transfers amino acid to mRNA for protein synthesis.  hnRNA serves as precursor for mRNA and other RNAs.
  17. DNA replication (semi conservative model)  Replication is a process in which DNA copies itself to produce identical daughter molecules of DNA.  Replication is semi conservative.  The parent DNA has two strands complementary to each other.  Both the strands undergo simultaneous replication to produce two daughter molecules.  Each one of the newly synthesized DNA has one-half of the parental DNA (one strand from original) and one-half of new DNA.  This type of replication is known as semiconservative since half of the original DNA is conserved in the daughter DNA  Each strand serves as a template, over which a new complementary strand is synthesized
  18.  A specific protein called dna A binds at the origin for replication causes the double-stranded DNA to separate.  The two complementary strands of DNA separate at the site of replication to form a bubble.  For the synthesis of new DNA, a short fragment of RNA is required as a primer.  The replication of DNA occurs in 5' to 3‘ direction, simultaneously , on both the strands of DNA.  DNA synthesis is continuous in the leading strand and discontinuous in the lagging strand.  The separation of the two strands of parent DNA results in the formation of a replication fork and the active synthesis of DNA occurs in this region.  DNA helicases bind to both the DNA strands at the replication fork and separate the strands.  Single-stranded DNA binding (SSB) proteins / DNA helix- destabilizing proteins provide the template for new DNA synthesis
  19.  The synthesis of a new DNA strand in 5‘ to 3' is catalyzed by DNA polymerase III.  The DNA strand (leading strand) with its 3'-end (3'-OH) oriented towards the fork can be elongated by sequential addition of new nucleotide.  Okazaki pieces are the small fragments of the discontinuously synthesized DNA.  These are produced on the lagging strand of the parent DNA.  Okazaki pieces are joined to form a continuous strand of DNA with the help of DNA polymerase I and DNA ligase.
  20. Transcription or RNA synthesis  Transcription is a process in which RNA is synthesized from DNA.  one of the two strands of DNA serves as a template( non-coding strand or sense strand) and produces working copies of RNA molecules.  The other DNA strand which does not participate in transcription is known as coding strand or antisense strand.  The product formed in transcription is referred to as primary transcript (inactive).  They undergo certain post-transcriptional modifications to produce functionally active RNA molecules  Transcription involves three different stages- initiation,
  21. Initiation:  Sigma factor of RNA polymerase bind to the promoter region in DNA  There are two base sequences on the coding DNA strand which the sigma factor of RNA polymerase can recognize for initiation of transcription.  There are two base sequences o n the coding DNA strand 1. Pribnowbox (TATA box) consists of 6 nucleotide bases( TATAAT) located on the left side about 10 bases away from the starting point of transcription. 2. The '-35' sequence contains a base sequence TTCACA, which is located about 35 bases away on the left side from the site of transcription start. Elongation:  The sigma factor is released and transcription proceeds.  RNA is synthesized from 5' end to 3' end antiparallel to the DNA template.  RNA polymerase utilizes ribonucleotidte triphosphates (ATP, CTP, CTP and UTP) for the formation of RNA
  22. Termination :  The process of transcription stops by termination signals.  Two types of termination are identified. Rho( ρ) dependent termination:  A specific protein (ρ factor) binds to the growing RNA or weakly to DNA, and in the bound state it acts as ATPase and terminates tanscription and release RNA. Rho( ρ) independent termination:  Termination occurs due to the formation of hairpins of newly synthesized RNA.  This occurs due to the presence of palindromes
  23. Genetic code  The 3 nucleotide (triplet) base sequences in mRNA that act as code words for amino acids in protein constitute the genetic code or simply codons.  The codons are composed of the four nucleotide bases, namely the purines-adenine (A) and guanine (C), and the pyrimidines-cytosine (C) and uracil (U).  These 4 bases produce 64 different combinations of 3 base codons.  The nucleotide sequence of the codon on mRNA is written from the 5'-end to 3' end.  61 codons code for the 20 amino acids found in protein.  The 3 codons UAA, UAG and UCA do not code for amino acids. They act as stop signals in protein synthesis. These 3 codons are known as termination codons or non-sensec odons
  24.  The codons AUG and GUG are the chain initiating codons.  Universality : The same codons are used to code for the same amino acids in all the living organisms.  Specificity : A particular codon always codes for the same amino acid, hence the genetic code is highly specific or unambiguous.  Eg: UGG is the codon for tryptophan.  It is non-overlapping, commaless and without any punctuations  The codes are consecutive and are read one after another in a continuous manner, e.g. AUG, CAU, GAU, GCA, etc.  Degenerate: The codon is degenerate or redundant, since there are 6l codons available to code for only 20 amino acids (ie, one amino acid has more than one codon.)  Eg: glycine has four codons.  Wobble hypothesis explains the degeneracy of the genetic code, i.e. existence of multiple codons for a single amino acid.
  25. Translation or protein synthesis  The biosynthesis of a protein or a polypeptide in a living cell.  The protein synthesis occurs on the ribosomes .  The mRNA is read in the 5‘ to 3' direction and the protein synthesis proceeds from N-terminal end to C- terminal end.  Steps involved in translation: 1. Activation of amino acid 2. Initiation 3. Elongation 4. Termination 5. Post-translational processing.
  26. Activation of amino acid:  The enzymes aminoacyl tRNA synthetase activate the amino acids.  The amino acid is first attached to the enzyme utilizing ATP to form enzyme-AMP-amino acid complex.  The amino acid is then transferred to the 3' end of the tRNA to form aminoacyl tRNA
  27. Initiation of translation :  It involves use of initiation factors (eIF)  The process of translation initiation can be divided into 4 steps 1. Ribosomal dissociation. 2. Formation of 43S preinitiation complex. 3. Formation of 48S initiation complex. 4. Formation of 80S initiation complex.  The 80S ribosome dissociates to form 40S and 60 S subunits  A ternary complex containing met-tRNA and elF-2 bound to GTP attaches to 40S ribosomal subunit to form 43S preinitiation complex.  The binding of mRNA to 43S preinitiation complex results in the formation of 48S initiation complex through the intermediate 43S initiation complex.  48S initiation complex binds to 60 S ribosomal subunit to form 80S initiation complex.  As the 80S complex is formed, the initiation factors bound to 48S initiation complex are
  28. Elongation of translation:  Ribosomes elongate the polypeptide chain by sequential addition of amino acids.  Elongation process involves use of certain elongation factors (EF). Steps: (1)Binding of aminoacyl tRNA to A-site.  The 80 S initiation complex contains met tRNA in the P-site, and the A-site is free. Another aminoacyl-tRNA is placed in the A-site. (2)Peptide bond formation.  The enzyme peptidyltransferase catalyses the formation of peptide bond (3)Translocation.  As the peptide bond formation occurs, the ribosome moves to the next codon of the mRNA (towards 3'-end).  Translocation involves the movement of growing peptide chain from A-site to P-site.  Translocation requires EF-2 and GTP.
  29. Termination of translation:  After successive addition of amino acids, ribosome reaches the termination codon sequence (UAA, UAG or UGA) on the mRNA.  As the termination codon occupies the ribosomal A- site, the release factor eRF recognizes the stop signal.  The 80S ribosome dissociates to form 40S and 605 subunits which are recycled.  The mRNA is also released.
  30. Post-translational processing:  The proteins synthesized in translation are, as such, not functional. so some changes are needed  These modifications include protein folding , trimming by proteolytic degradation, intein splicing and covalent changes which are collectively known as Post-translational modification.
  31. Inhibitors of protein synthesis  Majority of the antibiotics interfere with the bacterial protein synthesis.  Streptomycin inhibit initiation of protein synthesis. It causes misreading of mRNA and interferes with the normal pairing between codons and anticodons.  Tetracycline inhibits the binding of aminoacyl tRNA to the ribosomal complex.  Puromycin enters into the growing peptide chain and causes its release  Chloramphenicol acts as a competitive inhibitor of peptidyltransferase and thus interferes with elongation of peptide chain.  Erythromycin inhibits translocation by binding with 50S subunit of bacterial ribosome.  Diphtheria toxin prevents translocation in protein synthesis by inactivating elongation factor