Free Molecular biology lecture notes pdf BIOCHEMISTRY

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DNA REPLICATION
TRANSCRIPTION
TRANSLATION

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Free Molecular biology lecture notes pdf BIOCHEMISTRY

  1. 1. LECTURE NOTES ON MOLECULAR BIOLOGY  Lecture notes on DNA replication, Transcription and Translation Vijay Marakala
  2. 2. 1 Definition Prokaryotic replication Semiconservative mechanism Basic requirements - Substrates - Template - Enzymes and proteins - Primers Stages of replication - Initiation - Elongation - Termination Differences between prokaryotic and eukaryotic replication Inhibitors of DNA replication The duplication or synthesis of DNA is called replication Definition: DNA REPLICATION PROKARYOTIC REPLICATION Basic mechanism of replication is same both in prokaryotic and eukaryotic DNA but replication in prokaryotes is simpler and well understood. SEMICONCERVATIVE MECHANISM Each replicated duplex daughter DNA molecule contains one parent strand and one newly sythesised strand. Dr Vijay Marakala, MBBS. M.D. Assistant professor, Department of Biochemistry, SIMS & RC , MUKKA - SURATHKAL, MANGALORE. vkunder637@gmail.com LECTURE NOTES ON DNA REPLICATION Please refer following textbooks Biochemistry by Pankaja Naik Textbook of Biochemistry by DM Vasudevan Harper’s Illustrated Biochemistry
  3. 3. 2 Protein or Enzyme Function DNA A protein Opens duplex at origin of replication DNA B protein (Helicase) Unwinds DNA Primase Synthesises RNA primer SSB(Single strand binding protein) Binds separated single stranded DNA and stabilizes it DNA topoisomerase I Relieves torsional strain by cutting and joining single strand DNA topoisomerase II Relieves torsional strain by cutting and joining both strands DNA polymerase DNA chain elongation DNA ligase Joins Okazaki fragments Ter binding protein Prevents the helicase from further unwinding and facilitates termination BASIC REQUIREMENTS Substrates: dATP, dGTP, dCTP, dTTP Template: Separated DNA strands are the template for synthesis of new daughter strands. Enzymes and proteins involved in replication DNA polymerase I – Removal of RNA primer and replacing with deoxyribonucleotides DNA polymerase II – Proofreading and DNA repair DNA polymerase III - DNA chain elongation There are 3 types of DNA polymerases in prokaryotes
  4. 4. 3 Enzymes and proteins involved in replication Primer Primase synthesises RNA primer (in a 5’ to 3’ direction) using DNA as a template. DNA polymerase initially adds a deoxyribonucleotide to the 3’-OH group of the primer and then continues to add deoxyribonucleotides to the 3’- end of the growing strand STAGES OF REPLICATION 1. INITIATION 2. ELONGATION 3. TERMINATION Initiation  DNA A protein recognizes and binds to “ORI” and unwinds or separates the DNA  DNA B protein (helicase) binds to this region and further unwinds the DNA and formation of replication bubble  Stress produced by unwinding is released by topoisomerases  SSB proteins stabilizes the separated strands and prevents reassociation.  Binding of primase results in synthesis of RNA primer(5’ to 3’ direction) which are complememtary to bases of DNA “ORI” means origin of replication i.e specific sequence of DNA DNA replication is bidirectional DNA replication always starts from 5’ to 3’ direction Replication bubble
  5. 5. 4 Elongation For animation refer DNA replication ppt Because of antiparallel nature of two strands, the synthesis of DNA along the two strands is different.  One strand of DNA is synthesized continuously and it is known as leading strand.  Another strand is synthesized discontinuously in the form of okazaki fragments. It is known as lagging strand. The length of these segments ranges from 1000 to 2000 bases. They are also synthesized in 5' to 3' direction only.  The gaps between okazaki fragments are filled by DNA polymerase I. RNA primers are removed by 5' → 3' exonuclease activity of DNA polymerase I.  Finally DNA ligase joins the ends of okazaki fragments.
  6. 6. 5 Features Prokaryotes Eukaryotes RNA primer length 50 nucleotides 9 nucleotides DNA polymerase I,II,III α,β,γ,δ,ε Number of origins Single Multiple Nucleotide length of Okazaki 1000-2000 nucleotides 200 nucleotides Rate of replication 500 nucleotides/sec 50 nucleotides/sec Termination A specific protein “ter binding protein” binds a specific sequences “ter” sequences and prevents the helicase from further unwinding of DNA and facilitates the termination of replication. Differences between prokaryotic and eukaryotic replication Inhibitors of DNA replication Nalidixic acid Novobiocin Ciproploxacillin Inhibit prokaryotic topoisomerase II. Widely used as antibiotics for treating urinary tract infections and other infections. Adriamycin Etoposide Doxorubicin Inhibit eukaryotic topoisomerase II. Widely used as anticancer drugs
  7. 7. 6 LECTURE NOTES ON TRANSCRIPTION Dr Vijay Marakala, MBBS. M.D. Assistant professor, Department of Biochemistry, SIMS & RC , MUKKA - SURATHKAL, MANGALORE. vkunder637@gmail.com Definition Similarities and differences between replication and transcription Basic requirements for transcription  Template  Substrate  Enzyme Stages of transcription  Initiation  Elongation  Termination Differences between prokaryotic and eukaryotic transcription Post-transcriptional processing  Cleavage of precursor of RNA  Terminal addition of nucleotides  Base modification  Splicing Inhibitors of transcription Please refer following textbooks Biochemistry by Pankaja Naik Textbook of Biochemistry by DM Vasudevan Harper’s Illustrated Biochemistry Definition Synthesis of RNA using DNA as a template Similarities and differences between replication and transcription Similarities  3 stages  Synthesis occurs in the 5’→3’ direction  Follows Watson-Crick base pairing Differences  Ribonucleotides are used in RNA synthesis  Uracil replaces Thymine  Primer is not required  Only a very small portion of genome is transcribed Basic requirements for transcription Template The strand of DNA that is transcribed into RNA is called as template or sense strand whereas other strand is called as coding or anti-sense strand. For more diagram please go through following ppt slides Transcription Post-transcriptional processing
  8. 8. 7 Substrates: ATP, GTP, CTP and UTP Enzyme:  In prokaryotes, DNA directed RNA polymerase is the major enzyme of transcription.  It catalyzes the synthesis of all three types of RNAs like mRNA, tRNA and rRNA.  Holoenzyme consists of five subunits. They are ∝ ∝ ββ′ and σ.  The coenzyme consists of only four subunits αα ββ' Stages of transcription Initiation Elongation Termination Initiation 1. Initiation of RNA synthesis involves binding of RNA polymerase to the template strand. Certain regions of DNA serves as initiation signals. They are known as promoter sites. 2. RNA polymerase identifies promoter by virtue of σ factor. 3. Unwinding of DNA occurs and sets the stage for first phosphodiester linkage formation. Promoters Specific sequence of DNA functions as transcription signals. They are referred as Promoters. Usually they are located away (upstream) from start point (+1) of transcription. Two such promoters are known in prokaryotes. One promoter is located 10 nucleotides away from start site.It is known as –10 region or Pribnow or TATA box. Another promoter is located 35 nucleotides away from start site. It is known as –35 region. Usually the promoters facilitate dissociation of DNA strands so that DNA unwinds to favors transcription by RNA polymerase.
  9. 9. 8 Elongation  Elongation of RNA molecule occurs in 5' → 3' direction as the RNA polymerase polymerizes rNTPs anti-parallel to template strand.  As RNA polymerase progress along the DNA molecule unwinding of DNA takes place ahead of 3' end of nascent RNA. The growing RNA or nascent RNA is base paired to template strand. Termination Prokaryotic termination of transcription occurs by one of the two well characterized mechanisms. Rho-dependent Rho-independent Rho-dependent Termination Rho-dependent termination requires a protein factor called rho whichever recognizes then termination signal that displaces the RNA polymerase from template resulting in termination of RNA synthesis. Rho-independent Termination Rho-independent termination involves a secondary structure (hair- pin loop) formed in the newly synthesized RNA, which dislodges the RNA polymerase from DNA template resulting in the release of transcript.
  10. 10. 9 Binds to tightly to DNA and prevent unwinding. Actinomycin D Binds to β subunit of prokaryotic RNA polymerase Rifampicin Inhibits eukaryotic RNA polymerase α-Amanitin Differences between prokaryotic and eukaryotic transcription The basic mechanism of transcription is same in eukaryotes as that of prokaryotes; however eukaryotic transcription differs mainly with respect to RNA polymerase: type I, II and III Promoter site: -25 TATA box(Hogness box) and -75 CAAT box Post-transcriptional processing All three types of RNAs are synthesized in precursor forms in eukaryotes. These precursors are converted to functional RNA molecules by post-trasncriptional modifications. Usually, these modifications takes place in nucleus. Some prokaryotic RNAs also undergo these modifications.(prokaryotic mRNA is not processed post-transcriptionally)  Cleavage of precursor of RNA  Terminal addition of nucleotides  Base modification  Splicing mRNA Processing 1. Capping at the 5’ end 2. Addition of poly-A tail at the 3’ end 3. Splicing to remove intron tRNA Processing 1. Cleavage of 5’ leader sequence 2. Splicing 3. Replacement of 3’ UU by CCA 4. Modifications of several bases rRNA Processing  Ribosomal RNAs are synthesized in large precursor form known as pre rRNA Inhibitors of Transcription
  11. 11. 10 LECTURE NOTES ON TRANSLATION [protein biosynthesis] Dr Vijay Marakala, MBBS. M.D. Assistant professor, Department of Biochemistry, SIMS & RC, MUKKA - SURATHKAL, MANGALORE. vkunder637@gmail.com Definition Basic requirements  mRNA to be translated  tRNAs  Ribosomes  Energy in the form of ATP and GTP  Enzymes and specific factors Stages of translation  Activation of amino acids  Initiation  Elongation  Termination Post-translational modification  Proteolytic cleavage  Modification of amino acids  Subunit aggregation  Protein folding and chaperones Inhibitors of translation Please refer following textbooks Biochemistry by Pankaja Naik Textbook of Biochemistry by DM Vasudevan Harper’s Illustrated Biochemistry Definition Process by which ribosomes convert the information carried by mRNA in the form of genetic code to the synthesis of new protein. Basic requirements  mRNA to be translated  tRNAs  Ribosomes  Energy in the form of ATP and GTP  Enzymes and specific factors Stages of Translation  Activation of amino acids  Initiation  Elongation  Termination Activation of amino acids For protein synthesis, initial activation of amino acid is required. This activation is essential because energy is required for peptide bond formation. Activation involves esterification of amino acid with tRNA.
  12. 12. 11 Initiation Basic requirements for Initiation  Ribosome  mRNA to be translated  The initiating Met-tRNAi met  Initiation factors – Prokaryotes-IF1, IF2 &IF3. Eukaryotes-EIF1 to EIF9 Steps involved in Initiation  Ribosomal dissociation  Formation of 43S pre-initiation complex  Formation of 48S initiation complex  Formation of 80S initiation complex
  13. 13. 12 Elongation Basic requirements for Elongation  Various elongation factors –for Prokaryotes(EF), for Eukaryotes- eEF  80S initiation complex  The next aminoacyl tRNA specified by the next coding triplet in mRNA Steps involved in Elongation  Binding of next aminoacyl tRNA specified by the next coding triplet in mRNA to ‘A’ site  Formation of peptide bond  Translocation Formation of peptide bond Two amino acyl-tRNAs on the two sites of ribosome sets the stage for first peptide bond formation. The peptidyl transferase activity of 60S ribosomal subunit catalyzes the peptide bond formation between two amino acids. This process is also known as trans-peptidation, because the peptide bond formation involves transfer of met from tRNA located in the P site to α-amino group of amino acyl-tRNA in the A site. Nucleophilic attack of α-amino group of incoming amino acyl-tRNA on carboxyl group of met of met tRNA generates peptide bond. As a result, a dipeptide is generated on tRNA of A site leaving empty tRNA f on P site
  14. 14. 13 Termination  The elongation steps are repeated until one of the three (UAA, UAG, UGA) termination or nonsense codons of mRNA appear in the ‘A’ site.  Once the ribosome reaches a termination codon, releasing factors are capable of recognizing the termination signal present in the ‘A’ site.  Prokaryotes have 3 release factors-RF1, RF2 &RF3  Eukaryotes have only one release factor eRF. Post-translational modification In order to achieve native biologically active form of polypeptide, it must undergo processing and folding in proper three dimensional conformation. These alterations are known as post-translational modifications.  Proteolytic cleavage  Modification of amino acids  Subunit aggregation  Protein folding and chaperones
  15. 15. 14 Modification of amino acids SUBUNIT AGGREGATION Protein folding and chaperones Proper folding of protein into its three dimensional confirmation is assisted by chaperones. Improper folding leads to inactive protein or sometimes may cause diseases. Inhibitors of Translation

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