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post translation modifications, translation, transcription, replication

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CENTRAL DOGMA OF MOLECULAR BIOLOGY:

CENTRAL DOGMA OF MOLECULAR BIOLOGY A PRESENTATION BY CTECK SBS Expert Education Endeavor in Science

PowerPoint Presentation:

CTECK SBS

DNA REPLICATION :

DNA REPLICATION PREREQUISITES OF DNA REPLICATION- 4-deoxy nucleoside triphosphates – dGTP, dCTP,dATP and dTTP. Primer: template junction. Initiator proteins involved in Replication. CTECK SBS

PRIMER:TEMPLATE JUNCTION:

PRIMER:TEMPLATE JUNCTION DNA PM adds nucleotide to the template DNA Incoming nucleotide base pairs with the next available bases. Mg +2 and Zn+2 forms the next phosphodiester bonds. CTECK SBS

INITIATOR PROTEINS:

INITIATOR PROTEINS 1: DNA HELIX MUST BE UNWOUND This is achieved using Helicases. Are Hexameric proteins and uses ATP. Its Dna A and B in the case of Prokaryotes CTECK SBS

2.SUPERCOILS NEED TO BE REMOVED:

2.SUPERCOILS NEED TO BE REMOVED In the case of eukaryotes the supercoils created by helicases are removed by topoisomerases while Gyrases are employed in the case of prokaryotes CTECK SBS

3.PREVENTION FROM RECOILING:

3.PREVENTION FROM RECOILING Its done by using single stranded binding proteins- ssb’s CTECK SBS

4:FORMATION OF PRIMER:

4:FORMATION OF PRIMER Initiated by primase. Prokaryotes: primase+helicase= Primosome Eukaryotes:primase is intrinsic to polymerase alpha CTECK SBS

DNA POLYMERASES:

DNA POLYMERASES EUKARYOTES TYPES- 1.POLYMERASE ALPHA 2.PLOYMERASE DELTA 3.POLYMERASE EPSILON 4.POLYMERASE BETA 5.POLYMERASE THETA 6.POLYMERASE GAMMA PROKARYOTES TYPES 1.POLYMERASE I 2.POLYMERASE II 3.POLYMERASE III 4.POLYMERASE IV 5.POLYMERASE V CTECK SBS

PowerPoint Presentation:

THE REPLICATION FORK CTECK SBS

PROBLEMS IN THE FORMATION OF OKAZAKI FRAGMENTS:

PROBLEMS IN THE FORMATION OF OKAZAKI FRAGMENTS 1.HOW THE PRIMERS ARE FORMED? 2.HOW THE PRIMER IS REMOVED AFTER OKAZAKI FRAGMENTS SYNTHESIS? CTECK SBS

OVERVIEW OF DNA REPLICATION:

OVERVIEW OF DNA REPLICATION CTECK SBS

TRANSCRIPTION :

TRANSCRIPTION RNA primer doesnot need any primer so transcription is initiated denovo. Enzyme displaces the RNA a few nucleotides behind from where the ribonucleotides are added which is critical for the RNA to be translated. Its less accurate than Replication. CTECK SBS

RNA POLYMERASE-:

RNA POLYMERASE- PROKARYOTES: RNAP is a relatively large molecule. The core enzyme has 5 subunits (~400 kDa ): α2: The two α subunits assemble the enzyme and bind regulatory factors. Each subunit has two domains: αCTD (C-Terminal domain) binds the UP element of the extended promoter, and αNTD (N-terminal domain) binds the rest of the polymerase. This subunit is not used on promoters without an UP element. β: this has the polymerase activity (catalyzes the synthesis of RNA), which includes chain initiation and elongation. β': helps binding RNA polymerase to the DNA template ω: restores denatured RNA polymerase to its functional form in vitro CTECK SBS

PowerPoint Presentation:

EUKARYOTES RNA polymerase I synthesizes a pre-rRna 45S, which matures into 28S, 18S and 5.8S rRNAs which will form the major RNA sections of the ribosome. RNA polymerase II synthesizes precursors of mRNAs  and most snRNA and microRNAs.This is the most studied type, and due to the high level of control required over transcription a range of transcription factors are required for its binding to promoters. RNA polymerase III synthesizes tRNAs, rRNA 5S and other small RNAs found in the nucleus and cytosol. RNA polymerase IV synthesizes siRNA in plants. RNA polymerase V synthesizes RNAs involved in siRNA-directed heterochromatin formation in plants. CTECK SBS

STEPS OF TRANSCRIPTION:

STEPS OF TRANSCRIPTION INITIATION ELONGATION TERMINATION CTECK SBS

PROKARYOTIC INITIATION AND ELONGATION :

PROKARYOTIC INITIATION AND ELONGATION CTECK SBS

EUKARYOTIC TRANSCRIPTION:

EUKARYOTIC TRANSCRIPTION REQUIRES TRANSCRIPTION FACTORS TBP,TFIID,TFIIA,TFIIB,TFIIF,TFIIE,TFIIH,TFIIJ REQUIRES TATA BOX. REQUIRES ENHANCERS REQUIRES ATP CTECK SBS

PowerPoint Presentation:

CTECK SBS

TRANSLATION:

TRANSLATION CTECK SBS

PowerPoint Presentation:

CTECK SBS

Protein: Post-translational Modification:

Protein: Post-translational Modification CTECK SBS

TYPES OF PTM:

TYPES OF PTM Gene encoding region (ORF) ↓ transcription mRNA ↓ translation Protein ( nascent protein , precursor protein ) ↓ protein processing , PTM Mature protein ↓ folding Biological active protein CTECK SBS

Post-translational Modification:

Post-translational Modification 1. numerous and diverse 2. change the charge, conformation or size of protein molecule CTECK SBS

Effects of Post-translational Modification:

Effects of Post-translational Modification 1. stability of protein 2. biochemical activity (activity regulation) 3. protein targeting (protein localization) 4. protein signaling (protein-protein interaction,cascade amplification) CTECK SBS

Why protein post-translational modifications are made? (Biological functions):

Why protein post-translational modifications are made? (Biological functions) CTECK SBS

A. Regulation (interconvertable modifications) :

A. R egulation ( interconvertable modifications) Monocyclic cascade (allosteric effectors) -P/de-P receptor-associated Tyr-Kinase coupled cascade Cyclic cascade ADP-ribosylation / poly-ADP-ribosylation Coordinated Glycogen phosphorylase / Glycogen synthase Unidirectional cascade Proteolytic activation CTECK SBS

B. Cross-links:

B. Cross-links Disulfide bond-cross links (Cys-Cys) Isopeptide (N-(γ-Gln)-Lys Transamidation (Gln→Lys or Ornithine-α-NH 2 ) (Transglutaminases) Blood clotting factor VIII – coagulation Tissue Transglutaminase reaction Cell proliferation, aging, endocytosis, secretion, differentiation, apotosis, programmed cell death Stabilize or fix certain folded str. (Cofactors covalent binding) CTECK SBS

C. Covalent Cofactors:

C. Covalent Cofactors Biotinyl lysine (Carboxylase, transcarboxylases) Cys-bound linear tetrapyrrole (phycobiliproteins light-harvesting system of photosynthetic microorganisms) FAD-linked His, Cys, or Tyr (DHase, Oxidase) FMN-linked Cys Heme-covalent bound (Cyt. C) CTECK SBS

D. Membrane Anchors:

D. Membrane Anchors α-NH 2 myristoyl Cys- fattyacyl thioether Ser- palmitate or other fatty acids esters Thr- C-terminal glycophospholipids C-terminal Cys – prenylated group Farnesyl C15, Geranylgeranyl C20 CTECK SBS

E. Signaling, Recognition and Structural Amplification:

E. Signaling, Recognition and Structural Amplification CTECK SBS

F. Protein Turnover (Protein Degradation):

F. Protein Turnover (Protein Degradation) Spontaneous Oxidation: Cys, His, Tyr, Met Ubiquitination: Lys CTECK SBS

PowerPoint Presentation:

CTECK SBS

PowerPoint Presentation:

CTECK SBS

G. Others :

G. Others Iodination: Tyr, (Thyroid hormones) Sulfation or methylation; Tyr / secreted proteins CTECK SBS

Types of Post-translational Modification:

Types of Post-translational Modification CTECK SBS

Modification Involving Peptide Bonds Cleavage (limited proteolysis):

Modification Involving Peptide Bonds Cleavage (limited proteolysis) Signal leader peptide removed by signal peptidase (both in prokaryotes and eukaryotes) Precursor protein → mature protein (Insulin) Zymogen → active enzyme Trypsinogen → Trypsin Pepsinogen → Pepsin Prohormone → Hormone Polyprotein → neuropeptides (peptide hormone ) conversion Peptide Bonds Cleavage (limited proteolysis, specific and well-regulated ) CTECK SBS

PowerPoint Presentation:

Ser → esters Cys → thioesters Asp or Asn → isoaspartate Prolyl peptide cis-trans isomerization (prolyl i somerase catalyzed) Peptide Bond Isomerization (Intramolecular) CTECK SBS

PowerPoint Presentation:

peptide bond splicing with peptide deletion and/or permutation Plant lectin – Concanvalin A 3. Peptide Bond Formation, Transpeptidation CTECK SBS

Modifications Involving Amino and Carboxyl Termini:

Modifications Involving Amino and Carboxyl Termini N-Formyl- (C1) N-Acetyl- (C2) N-Acyl- (C2, C4, C6, C8, C10) N-Lauroyl- (C12) N-Myristoyl- (C14) N-Tetradeca (mono and di)enoyl- (C14 : 1 ; C14 : 2) N-Aminoacyl- N-α-Ketoacyl- N-Methyl- N-Pyrrolidone carboxyl- N-Glucuronyl- N-Glycosyl- The N terminus : H 3 N+— CTECK SBS

PowerPoint Presentation:

Amide O-(ADP-ribosyl)- O-Methyl- -(N-Ethanolamine-glycan-phosphoinositides) -(N α -TyT) The C Terminus : CTECK SBS

C. Modifications Involving Individual Amino Acid (Side Chains):

C. Modifications Involving Individual Amino Acid (Side Chains) N ω -(ADP-ribosyl)- N ω -Methyl- N ω -Dimethyl- N ω -N ω ’ -Dimethyl- Ornithine Citrulline N ω -Phosphoryl- Arginine : CTECK SBS

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N-Glycosyl- Aspartate N-Methyl- N ε -(β-Aspartyl)lysine erythro -β-Hydroxy- N-(ADP-ribosyl)- As paragine : CTECK SBS

PowerPoint Presentation:

D-Asp (racemization) β-Carboxy- erythro -β-Hydroxy- β-Methylthio- O-Phosphoryl- O-Methyl- 3. Aspartate : CTECK SBS

PowerPoint Presentation:

Cystine S-γ-Glutamyl- S-(2-Histidyl)- S-(3-Tyr) S-( sn -1-Glyceryl)- S-( sn -1-Diacylglyceryl)- S-( sn -1-{2,3,-Di-O-[3’ ,7’ ,11’ .15’- tetramethylhexadecyl]}glyceryl)- S-Palmitoyl- S-Farnesyl- S-Geranylgeranyl- S-Heme S-Phycocyanobilin S- p -Coumaroyl S-(6-Flavin [FMN]) S-(8 α -Flavin [FAD]) S-Coenzyme A S-(ADP-ribosyl)- S-Glycosyl- Dehydroalanine Lysinoalanine Lanthionine Selenocysteine 4. Cysteine : HS-CH 2 - CTECK SBS

PowerPoint Presentation:

O-(ADP-ribosyl) γ-Carboxy- O-Methyl- N α -( γ -Glutamyl)-Glu 1-5 N α -( γ -Glutamyl)-Glu 3-34 N- (γ-Glutamyl)ethanolaminephosphate) S-γ-Glutamyl-Cys is listed under Cys Glutamate : CTECK SBS

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Glutamate N ε -( γ -Glutamyl)lysine N-( γ -Glutamyl)-L-ornithine N-( γ -Glutamyl)polyamine N,N-(Bis- γ -glutamyl)polyamine N 5 -Methyl- Glutamine : CTECK SBS

PowerPoint Presentation:

Diphthamide N τ -(ADP-ribosyl)diphthamide N-Phosphoryl- N π -Methyl- 4-Iodo-and diiodo- N τ - and N π –(8 α -flavin [FAD]) N π -(8 α -Flavin[FMN]) Histidine : CTECK SBS

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N ε -Acetyl- N ε -( N α -Monomethylalanyl)- N ε -Murein (peptidoglycan) N ε -Lipoyl- N ε -Biotinyl- N ε -Ubiquitinyl- N ε -Phosphoryl- N ε -Phosphopyridoxyl- N ε -Retinyl- N ε -Glycosyl- N ε -Mono-, di- , trimethyl- Hypusine : N ε -(4-amino-2-hydroxybutyl)- Allysine δ-Hydroxy- δ-Hydroxyallysine Cross-links (desmosines, syndesines, pyridinolines) δ-Glyxosyloxy- Lysine : CTECK SBS

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Sulfoxide 9. Methionine : Phenylalanine : β-Glycosyloxy- CTECK SBS

PowerPoint Presentation:

3-Hydroxy- 4-Hydroxy- 3,4-Dihydroxy- O 4 -Arabinosylhydroxy- O 4 -Galactosylhydroxy- O 4 -Glucosylhydroxy- 11. Proline : CTECK SBS

PowerPoint Presentation:

Selenocysteine O-Phosphoryl- O-Pantetheinephosphoryl- O-(GlcNAc-1-phosphoryl)- O-(Glycerol-1-phosphoryl)- O-Methyl- O-Glycosyl- Alanino( τ - or π -histidine) Lanthionine O-Acetyl- O-Fatty acyl- Serine : HO-CH 2 CTECK SBS

How modifications are made ?:

How modifications are made ? CTECK SBS

A. Nonenzymatic Reaction:

A. Nonenzymatic Reaction deamidation : Asn, Gln racemization : Asp, Ser dehydroalanine : Cys, phosphor-Ser slow oxidation : Cys, His, Met slow cleavage and permutation of peptide bonds reducing sugar reaction with NH 2 -group of aa ’ s or side chains (Lys) : Maillard reaction (Browing reaction) ; Schiffs base reaction. CTECK SBS

B. Enzymatic Reaction:

B. Enzymatic Reaction N-linked glycosylation Carboxyl methylation S-isoprenylation-Cys 1. Irrversible, Unidirectional Reaction (permanently modified) CTECK SBS

PowerPoint Presentation:

Phosphorylation (protein kinase) / Dephosphorylation (phosphatase) : Ser, Tyr, Thr. Uridylyl and adenylyl transfer in bacterial glutamine synthetase 2. Irrversible, Bi-directional Reaction. (Signal Amplificaion) CTECK SBS

PowerPoint Presentation:

RS-SR + R ’ -SH ↹ R ’ -S-S-R + RSH(disulfide isomerase) Coupled with protein-folding process 3. Reversible Reaction CTECK SBS

PowerPoint Presentation:

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