BCH100 DNA RNA Protein Synthesis (2024-1) PDF

Central dogma DNA RNA and Protein Synthesis Thanet Sophonnithiprasert Biochemistry Unit Department of Medical Sciences Faculty of Sciences Rangsit University What is life made o...

Central dogma DNA RNA and Protein Synthesis Thanet Sophonnithiprasert Biochemistry Unit Department of Medical Sciences Faculty of Sciences Rangsit University What is life made of? Fundamental Information Body Cells DNA Bodies are made up of cells All cells run on a set of instructions spelled out in DNA Fundamental Information DNA Proteins Cells Body  DNA has the information to build proteins called “gene” DNA Bodies Proteins Cells Fundamental Information Cell organization  DNA  DNA is in the nucleus: gene → instructions for making proteins DNA as blueprint is locked in the vault Nucleus cytoplasm Fundamental Information Cell organization  Proteins  Chains of amino acids  Made by a ribosome (as protein factory) in cytoplasm Ribosome is a protein factory Nucleus cytoplasm Fundamental Information Passing on DNA information  Need to get DNA gene information from nucleus to cytoplasm  Need a copy of DNA → called “messenger RNA” mRNA Ribosome build protein from copy order Nucleus cytoplasm Fundamental Information Passing on DNA information  The flow of genetic information within a biological system is called “Central dogma”  It states that such information cannot be transferred back from protein to either protein or nucleic acid originally termed the sequence hypothesis by Francis Crick  Fundamental Information Passing on DNA information  The flow of genetic information within a biological system is called “Central dogma”  It states that such information cannot be transferred back from protein to either protein or nucleic acid @ DNA RNA Protein # @ Can be found in RNA retrovirus; # Can be found in RNA virus (lytic); require reverse-transcriptase enzyme require RNA-dependent RNA polymerase  Learning Check Point About genetic information transfer: Which are found in cells? DNA RNA DNA Protein RNA DNA ✓found   not found  found ✓not found  ✓found   not found RNA Protein Protein RNA ✓found   not found  found ✓not found  Fundamental Information Thus  Proteins assign the specify character of cells  Proteins constructed from genetic code on DNA  So, DNA must be copied or replicated before cell division (S phase of cell cycle)  Each new cell will then have an identical copy of the DNA Fundamental Information Thus  Proteins assign the specify character of cells  Proteins constructed from genetic code on DNA  So, DNA must be copied or replicated before cell division (S phase of cell cycle)  Each new cell will then have an identical copy of the DNA DNA replication Three hypotheses: DNA replication The Meselson–Stahl experiment: Click for Animation DNA replication The Meselson–Stahl experiment: Mechanism of DNA replication DNA gyrase relieve helix structure Click for Animation Drug target on DNA replication Quinolones  Broad-spectrum bactericidal: ciprofloxacin, levaquin  Act by inhibiting bacterial DNA gyrase (Topoisomerase II)  Learning Check Point About DNA replication: Which are found work in step of DNA replication?  Helicase  Gyrase  Primase  DNA polymerase  RNA polymerase  ssDNA binding protein  ribonucleotide Central Dogma of Gene Expression Localization In nucleus In cytoplasm Transcription process 1. Initiation: After RNA polymerase binds to the promoter, the DNA strands unwind, and the polymerase initiates RNA synthesis at the start point on the template strand. 2. Elongation: The polymerase moves downstream, unwinding the DNA and elongating the RNA transcript 5’→ 3’. In the wake of transcription, the DNA strands re-form a double helix. 3. Termination: Eventually, the RNA transcript is released, and the polymerase detaches from the DNA. RNA synthesis: Initiation step  RNA polymerase bind to sigma factor or transcription factor  RNA polymerase attach DNA at promoter region  RNA Polymerase unwind DNA double helix, while the DNA exiting RNA polymerase rewinds to form double helix. Promoter -35 region pribnow box CAAT box TATA box GC box (Goldberg-Hogness box) (sometime present) RNA polymerase in the process Prokaryotic vs Eukaryotic RNA polymerases Prokaryotic Eukaryotic Single RNA polymerase RNA pol 1: rRNA (nucleolus), except 5S rRNA RNA pol 2: mRNA RNA pol 3: tRNA, 5S rRNA Requires sigma factor to No sigma, but transcription factors (TFIID) initiates at a promoter bind before RNA polymerase Sometimes requires rho No rho required to terminate Inhibited by Rifampin, RNAP2 inhibited by a-Amanitin (mushrooms), RNA polymerase in the process Eukaryotic RNA polymerases Drug target on RNA synthesis Rifamycin  Antibiotics that inhibit bacterial RNA polymerase  Act by binding to RNA polymerase (b Subunit at allosteric site): block Transcription of DNA to RNA  Antituberculosis RNA polymerase inhibitor Alpha-amanitin  Strong inhibitor (toxin) from “death cap”  Inactivates RNA pol II and can kill a person  RNA pol I and III are less affected by toxin  RNA synthesis: Elongation step  RNA polymerase move along the gene adding new ribonucleotide  RNA polymerase use antisense strand as template  RNA Polymerase adding nucleotide in the 5’ to 3’ direction  RNA Polymerase works at up to 60 nucleotide/second  RNA synthesis: Termination step  RNA Polymerase reaches the terminator region of the gene  3’ synthesized RNA form hairpin loop structure, then RNA polymerase (and factors) are released  Learning Check Point About transcription: Which are found work in step of transcription?  Helicase  promoter  DNA polymerase  terminator  RNA polymerase  ssDNA binding protein  deoxyribonucleotide  ribonucleotide  codon Post-Transcriptional modification RNA processing :  Adding CAP (7-methylguanosine) at 5 end (5-Capping)  Adding poly-A (Adenylate) at 3 end (3-Polyadenylate)  Split intron (Exon splicing or RNA splicing)  Post-Transcriptional modification The procedure of RNA processing for protein genes Source: http://www.web-books.com/MoBio/Free/Ch5A.htm 5´- CAPPING structure function The 5' cap has 4 main functions: 1. Regulation of nuclear export. 2. Prevention of degradation by exonucleases. 3. Promotion of translation 4. Promotion of 5' proximal intron excision.  3´ Polyadenylation Mainly functions: - increase stability of mRNA (Slowing down the destruction of RNA) Source: http://www.web-books.com/MoBio/Free/Ch5A.htm Splicing mechanism snRNPs – Spliceosome – lariat form Recognition sequence: GU-AG Spliceosome Eukaryotic Genes are Fragmented  Learning Check Point About RNA processing: Match the process to the action: A remove intron from ….. 5’-Capping immature mRNA ….. 3’-polyadenylation B remove exon from immature mRNA ….. Exon splicing C remove adenine from immature mRNA F add intron into immature D add adenine into immature mRNA mRNA G add 7-methylguanosine into E remove 7-methylguanosine immature mRNA from immature mRNA Localization  In prokaryote  In eukaryote Translation process Protein translation: Initiation step  Ribosome bind to 5’ CAP of mRNA (Eukaryote) or ribosome binding site (Prokaryote)  Start codon AUG and anticodon with Methionine (Eukaryote) or formyl-Methionine (Prokaryote) bind a P site  A site is open and ready to receive new tRNAs Ribosome in the process Prokaryotic vs Eukaryotic ribosome Drug target on protein translation Inhibition of protein synthesis  Structure of prokaryotic ribosome acts as target for many antimicrobials of this class - Differences in prokaryotic and eukaryotic ribosomes responsible for selective toxicity  Drugs of this class include: - Aminoglycosides - Tetracyclins - Macrolids - Chloramphenicol Protein translation: Elongation step  Adding new amino acid: codon recognition and peptide bond formation in the A site  Translocation: ribosome moves along mRNA, aminoacyl tRNA shifts from A site to P site Protein translation: Elongation step Protein translation: Termination step  Occurs when a stop codon (UAA, UAG, UGA) is revealed in the A site  A release factor enters the A site and promotes the hydrolysis of the bond linking the tRNA in the P-site with its polypeptide. Polyribosome - translation Genetic Code  The genetic code is the set of rules by which information encoded in genetic material is translated into protein by living cells  The information in DNA is in the form of triplet codons; every triplet codon in the DNA specifies one amino acid in the protein  Genetic code is redundant, also called “Codon degeneracy” Codon degeneracy Genetic Code In case of codon degeneracy Aspatate GAU GAC High frequency used High frequency used in in species I species II Each organism prefer a different set of codons over other, called “Codon Bias” Genetic Code Each organism prefer a different set of codons over other, called “Codon Bias”