Lecture 10 2024 Information Flow in Cells (II) PDF

Information Flow in Cells (II) Prepared by: Alvin Hee, Ph.D and AP Dr. Faridah Qamaruz Zaman BGY 3002 Cell & Molecular Biology  Learning Outcomes Upon completing this lecture, you are expected to be able to: 1. Describe the models of DNA replication....

Information Flow in Cells (II) Prepared by: Alvin Hee, Ph.D and AP Dr. Faridah Qamaruz Zaman BGY 3002 Cell & Molecular Biology  Learning Outcomes Upon completing this lecture, you are expected to be able to: 1. Describe the models of DNA replication. 2. Explain the process of DNA replication in relation to the direction of replication and the various types of enzymes involved (DNA polymerase, primase, helicase, ligase, topoisomerase, telomerase, etc.) 3. Define the terms replication and replication fork. 4. Suggest possible reasons for DNA damage. 5. Describe the process of DNA repair using the nucleotide excision repair. 6. Explain the phases occurring in the eukaryotic cell cycle. 7. Define the term cytokinesis. 8. Compare and contrast between mitosis and meiosis.  Learning Outcomes Upon completing this lecture, you are expected to be able to: 9. Explain the central dogma of molecular biology. 10. Describe the overall process of how the genetic information stored in the DNA is expressed and regulated via the process of transcription and translation. 11. Describe the different types of RNA and their functions. 12. Distinguish the different RNA polymerases. 13. Identify the key enzymes involved in the transcription process. 14. Explain the stages occurring in the translation of genetic information.  DNA replication DNA replication takes place by separation of the strands of the double helix & synthesis of two daughter strands complementary to the two parental templates (preparation for cell division). 3 models of replication: 1. Semi-conservative 2. Conservative 3. Dispersive The original Watson-Crick proposal for the During replication, the replication of a double-helix unwinds, double-helical and each of the molecule of DNA parental strands serves as a template of the synthesis of a new complementary strand Each daughter duplex contains one strand from the parent structure! Only one of the daughter duplexes would contain only parental DNA! The daughter duplexes would contain strands that were composites of old and new DNA! Meselson & Stahl, 1958- DNA replication in bacteria is semi-conservative  Before a cell divides, an enzyme breaks the bonds between the complementary bases in short sections of the double-stranded DNA molecules, and the complementary strands separate from one another. This is known as unwinding or unzipping.  DNA replication Synthesis of DNA is bidirectional, occurring in two directions simultaneously. Replication fork refers to the point where synthesis begins. Thus, there are two Replication forks, and replication ends when the replication forks meet on the other side of the circular chromosome at the termination site, the ter (t) region with the assistance of the ter binding protein (TBP).  Mechanism of DNA replication Two replication forks move in opposite directions from single origin. When the replication forks meet at the opposite point on the circle, replication is terminated, the two replicated duplexes detach from one another. New DNA strands shown in red. Bidirectional DNA replication in E.coli DNA synthesis in E. coli is a complex process requiring several steps - Binding of protein dnaA to the strand of DNA Interaction with protein dnaA (coalesce) results in the opening ( “melting”) of the double helical DNA Binding of protein dnaB and dnaC (chaperone of dna B) removal of dnaA, opening of the DNA strand through the activity of helicase (dnaB)- unwinding of duplex DNA The events following the opening of OriC is difficult to illustrate as it involved many proteins. However, the figure below try to show what happens:. Involvement of protein SSB to stabilise the DNA which is open Entry of PRIMASE ( a product of the dnaG gene) to synthesize the primer Synthesis of DNA by DNA pol III DNA gyrase ( a type of DNA topoisomerase) is required to release the tension in the DNA (Topoisomerases prevent tangling of DNA strands) The way synthesis is carried out is influenced by the two properties of DNA polymerase (i) Enzyme requires a primer primer is synthesized by PRIMASE (a type of RNA polymerase) Primer is RNA and has to be removed (ii) Direction of synthesis of the new strand of DNA is from 5’ to 3’ only There is a leading strand and a lagging strand and synthesis of the leading strand occurs continuously while the lagging strand is synthesized discontinuously in fragments (called Okazaki fragments). Each Okazaki fragment begins with a RNA primer Removal of the primer and addition of DNA bases is carried out by pol I The strands are joined by DNA ligase.  Cell cycle Succession of events from one cell division to another. Consists of the M phase and interphase. - M phase includes the process of mitosis and cytokinesis (30-60 minutes) - Cytokinesis - Division of the cytoplasm of a parent cell into two daughter cells, usually follows mitosis. - Interphase (majority of cell cycle lasts longer than M phase). - Both synthesis of macromolecules and DNA replication occur during interphase.  Interphase includes G1, S and G2 phases (periods). G1 (Gap 1) is the period between the end of mitosis and the beginning of DNA replication (length most variable). DNA replication occurs in the S phase. G2 occurs between the end of DNA synthesis and the beginning of mitosis. Entry of the cell into M phase is triggered by the activation of a protein kinase known as the Maturation-Promoting Factor (MPF).  MPF consists of two subunits: a kinase & a regulatory subunit, cyclin. Increased [cyclin] activates the kinase. Stages of Mitosis  Cytokinesis in Animal Cells Starts with an indentation of the cell surface, lying on the same plane as chromosomes of the metaphase plate. Contractile ring theory - a thin cortical band composed of actin & myosin filaments generates the force to cleave the cell. - Sliding of actin filaments pulls the cortex and attaches plasma membrane to the centre of the cell. - Cortical ring is assembled rapidly prior to cytokinesis and dismantled immediately after it.  Operation of the contractile ring during cytokinesis Cytokinesis in Plant Cells- Formation of the Cell Plate Plant cells build a cell membrane and cell wall in the cell centre. The cell plate begins with the appearance of a microtubular phragmoplast. Material for the cell wall is brought to the phragmoplast by Golgi vesicles. Cell plate formation proceeds laterally from the centre of the cell. Formation of cell plate between two daughter plant cells during cytokinesis Formation of cell plate between two daughter plant cells during cytokinesis  Meiosis During meiosis, chromosome number is halved and haploid cells are formed. In vertebrates, formation of spermatozoa and eggs takes place by gametic meiosis. Meiosis consists of two subsequent divisions: - First division - homologous chromosomes pair and then segregate ensuring that daughter cells receive a full haploid complement of chromosomes. - Second division - the two chromatids are separated. - Genetic recombination occurs during pairing of homologues. Expression of Genetic Information Expression of Genetic Information The Central Dogma of Molecular Biology  rRNA: ribosomal RNA that forms part of the ribosome structure. tRNA: transfer RNA that joins with the amino acids, tagging them with the information necessary for the ribosome to recognize them & charging them with the energy needed to promote the synthesis of the peptide bonds. mRNA: messenger RNA, copies the genetic code in DNA and specify the assembly of the sequence of amino acids in a peptide. Genetic code in triplets (3 nucleotides) i.e. codon: 64 codons including start (AUG) and stop codons, UAA, UAG & UGA. Common terms used to define the flow of information from DNA to RNA (transcription) The template strand of DNA is used for RNA synthesis. It is read in the 3’ to 5’ direction. The RNA transcript has a base sequence complementary to the template strand but identical to the DNA coding strand. The gene region on the template strand begins at base +1 and proceeds downstream (increasing + numbers). RNA polymerase catalyzes the synthesis of RNA transcript.  Types of RNA polymerases Prokaryotes: One type only, but with different initiation (σ) factors. Eukaryotes use 3 different RNA polymerases: - RNA pol I: synthesizes larger species rRNA, - RNA pol II: synthesizes the mRNA precursor (hnRNA), - RNA pol III: synthesizes small RNAs, including tRNA & smallest rRNA (5S) and snRNA that participates in splicing. Transcription - Process of RNA synthesis Step 1: Initiation: RNA polymerase binds to DNA molecule at promoter. In prokaryotes- Pribnow or TATA box (TATAAT) at -10 region, and the -35 region that has the sequence TTGACA upstream. Transcription - Process of RNA synthesis Polymerase complex unwinds transcription bubble (region of about 17 base pairs). Polymerase copies sense or template strand only. Ơ sigma subunit Transcription - Process of RNA synthesis Step 2: Elongation: Synthesis proceeds in the same direction as DNA synthesis (5’ to 3’) i.e. adding to the 3’-OH. Genes are transcribed from the upstream end to the downstream end. Transcription - Process of RNA synthesis Step 3: Termination: In prokaryotes (E. coli), involves base sequences having termination signals or factors (ρ (rho) protein). In eukaryotes, RNA polymerases uses nucleotide triphosphates, clipping off terminal two phosphates as the phosphodiester bond is formed. Post-transcriptional Modification of RNA (Prelude to transport from the nucleus) Newly synthesized RNA molecules called primary transcripts are usually biologically inactive. Must be processed into mature, biologically functional molecules. Depends on type of cell and kind of RNA.  Eukaryotic mRNA processing Prokaryotic mRNA requires little or no post- transcriptional alteration before it is translated to protein structures. Eukaryotic mRNA are chemically modified in the nucleus before transport to the ribosomes for translation.  Eukaryotic mRNA processing mRNA from hnRNA (heterogeneous nuclear RNA) 1) GTP cap added (also in prokaryotes) 2) Poly A tail added enzymatically on most messages 3) Splicing- intron (non-coding DNA region) removed, exon attached for proper reading. Accomplished by spliceosome, a complex of enzymes and snRNA.  Translation of Genetic Information Ribosomes carry out polypeptide synthesis. Ribosomes as particles consist of rRNA and protein that reside in the mitochondrial matrix and chloroplast stroma (in eukaryotes). Binding Sites on a Ribosome Translation of Genetic Information Amino acid activation- Attachment of amino acid to tRNA Amino acid attach to tRNA via high energy bond. Aminoacyl tRNA synthetase- enzyme that catalyzes formation of an ester bond between the carboxyl group of an aa and the 3’-OH of the appropriate tRNA, generating an aminoacyl tRNA.  Initiation of Translation in Bacteria  Polypeptide Chain Elongation in Bacteria  Termination of Translation  DNA Damage & Repair DNA as the ‘instruction manual’ for the cell, containing all the information necessary to make proteins that perform cellular functions. Imperative that cells maintain integrity of their DNA. Errors committed during replication of DNA must be repaired so that they are not passed on to future generations. DNA damaged by extreme environmental conditions must be restored to its original form.  DNA Damage Induced mutations- exposure to environmental agents (mutagens) - Ionizing radiation- production of ions and highly reactive free radicals; removal of hydrogen atom from DNA, forming H2O and reactive DNA radical leading to broken DNA strand (single strand readily repaired; double-strand more difficult- leading to cell death or cancer). - Chemical mutagens (e.g. Ethidium bromide) as intercalating agents- inserting between stacked base pairs in DNA leading to structural distortions.  Ultraviolet radiation Primary source: solar exposure (sunlight) Affecting the bonds between adjacent pyrimidines located on the same or opposite DNA strands. E.g. Formation of thymine-thymine dimer in DNA. T-T dimer is known as cyclobutyl thymine. UV radiation can cause adjacent thymine bases to bind to each other (T-T) resulting in a mutation. If not repaired, it may initiate events associated with skin cancer. Individuals suffering from the disease Xeroderma pigmentosum are extremely sensitive to UV radiation and develop skin cancer, due to mutation of the genes involved in DNA repair. The cross-linking of the pyrimidine and thymine is not repaired.  The girl who lives in the dark Wan Lao Yang is a 9-year-old girl from China suffering from Xeroderma pigmentosum. This is a genetic disorder which makes the skin unable to repair itself after exposure to UV. light. Lao Yang lives on top of the hills, being even closer to the UV rays of the sun that harms her body. Due to this, her face was covered with cancer tumors. Formation of thymine dimers UV light can affect the DNA leading to the formation of thymine dimers (dimers involving cytosine and uracil can also occur but with less frequency) A child with Xeroderma pigmentosum playing outdoors. Space suit invented by NASA  The repair system recognizes distortion in the DNA double helix and uses a number of enzymes to excise the damaged region and fill in the resulting gap with the correct base sequence. Nucleotide excision repair by uvrABC endonucleases is used to repair pyrimidine dimers Cells from individuals with Xeroderma pigmentosum are usually unable to carry out excision repair. Nucleotide Excision Repair  Damage recognition in the global pathway is mediated by an XPC protein, whereas recognition in transcription-coupled pathway by stalled RNA polymerase in conjunction with a CSB protein DNA strand separation (by XPB & XPD proteins, two helicase subunits of TFIIH). TFIIH- a huge protein that also participates in initiation of transcription. Incision by XPG on 3’ side and the XPF-ERCC1 complex on the 5’ side. Excision DNA repair synthesis by DNA polymerase Ligation by DNA ligase