Microbial Genetics Lecture 1-2024 PDF

MICROBIAL GENETICS Dr. Amira El-Ganiny Professor of Microbiology & Immunology Lecture 1 Definitions Genetics: branch of biology that study Genes, Genetic variation and Heredity in living organisms  Classical genetics: old branch of genetics based only...

MICROBIAL GENETICS Dr. Amira El-Ganiny Professor of Microbiology & Immunology Lecture 1 Definitions Genetics: branch of biology that study Genes, Genetic variation and Heredity in living organisms  Classical genetics: old branch of genetics based only on visible results of reproductive acts  based on experiments of Mendel  Molecular genetics New field of genetics that studies structure & function of Nucleic acids at molecular level  Chromosomes structures, replication, gene expression  Molecular GENETICS The nucleic acids  DNA and RNA are the carrier of genetic material in all living organisms. Nucleic acids have two important functions Ability to express genetic Ability to duplicate itself  traits through  transfer to progeny  by  transcription to mRNA chromosomal replication  translation into proteins.  The Structure of DNA  DNA consists of nucleotides  3 parts  backbone of alternating phosphate & sugar (2-deoxyribose)  Nitrogenous base  Purines : adenine (A), guanine (G)  Pyrimidines: cytosine (C), thymine (T) A paired with T (2 hydrogen bonds) G paired with C (3 hydrogen bonds)  nucleotides are joined by  phospho- diester bond  DNA is double helix  2 chains of nucleotides coiled around each other  the 2 standards are complementary  The 2 chains are antiparallel (i.e., their sugar-phosphate backbones are oriented in opposite directions)  5' to 3' direction  3' to 5' direction  In prokaryotes  DNA is circular, super-coiled molecule associated with basic proteins (histone-like)  In eukaryotes  DNA is more organized  contain histone proteins & coiled into repeating units known as nucleosomes.  RNA RNA is single strand  coil back on itself RNA composed of the sugar ribose  instead of 2- deoxyribose Uracil (U)  instead of thymine (T) There are 3 main types of RNA Ribosomal (rRNA) Transfer (tRNA) Messenger (mRNA)  Types of RNA 1. Messenger RNA (mRNA)  Carry message  direct protein synthesis in ribosomes  mRNA  formed by transcription of DNA  Transcription depend on RNA polymerase  initiates RNA synthesis at promoter site on gene stop when termination codon reached. 2. Ribosomal RNA (rRNA) rRNAs are components of ribosomes They are made from large precursors  Enzymatically cleaved to  16s, 23s and 5s rRNA 3. Transfer RNA (tRNA) Carry amino acids  during protein synthesis tRNAs can distinguish different amino acids Each type of tRNA is covalently bound to one of the 20 aa. Each tRNA has a triplet of nucleotides called 'anticodon‘  binds to triplet nucleotides on mRNA, called codon during protein synthesis. When aa is attached to tRNA  tRNA is said to be charged.  DNA REPLICATION Each strand of DNA serves as template for production of another complementary strand This pattern of replication is responsible for maintaining (conserving) proper sequence of bases on DNA molecule The pattern of DNA replication is described as 'semiconservative‘  produce 2 copies of DNA molecule  each copy contained one original strand and one new strand Replication starts at a point called 'origin of replication' by separation of the two strands  The replication origin  specific segment of DNA molecule consisting of about 245 bp. Replication fork  area of DNA molecule where strand separation occurs and the synthesis of new DNA takes place. A replicon  consists of origin of replication and DNA that is replicated from that origin  Bacterial chromosome has single replicon (one bubble).  Eukaryotes have multiple replicons (several bubbles exist) to efficiently replicate the relatively large molecules within a reasonable time  Polymerization of nucleotides occur in 5' to 3' direction  for original strand Leading strand (3'  5‘) A challenge in DNA replication is  how to achieve 5‘ to 3' polymerization in the opposite direction from the template strand which is itself is from 5'-3' direction (lagging strand) This problem is solved by having different modes of polymerization for the two growing strands  Leading strand Lagging strand Formed continuously Formed from 5‘3' end in small from the 5' to 3'end fragments  Okazaki fragments  linked together by ligase enzyme  DNA replication DNA replication  carried out using DNA polymerases  cells in all organisms contain multiple highly specialized DNA polymerases Bacteria  5 DNA polymerases  I, II, III, IV, V  Yeast  8 DNA polymerases humans  at least 15 DNA polymerases The rate of DNA synthesis 750-1000 bp/second  in Prokaryotes  50-100 bp/second  in Eukaryotes  Polymerase III  main polymerase in DNA replication process  To initiate replication DNA polymerase require presence of primer  short strand of RNA to which growing polynucleotide chain is covalently attached  Polymerase I has exonuclease activity to remove mismatched nucleotide & adds correct nucleotide  repair process  Proofreading  removal of incorrect nucleotides immediately after they are added to growing DNA during replication process.  The proofreading function of DNA polymerase I improves fidelity of replication to one error in every 109 -1010 bp  Steps of DNA replication in E. coli (1) DnaA protein binds to origin of replication (Ori C)  DNA replication starts by separation of the two strands. (2) Helicase(dnaB) bind replication fork to unwind the 2 DNA strands at same time topoisomerases (DNA gyrase) relieve the tension caused by unwinding process (3) Single-stranded DNA binding proteins (SSBs)  keep the single stranded region of the template DNA apart (4) Primase (dnaG)  synthesize small RNA molecule (~10 nucleotides)  act as primer for DNA synthesis (5) DNA polymerase III synthesizes complementary strand of DNA according to base-pairing rules The direction of DNA synthesis is from 5' to 3' end of the newly formed strand On one strand (leading strand), synthesis is continuous on the other (lagging strand) discontinuous synthesis (Okazaki fragments) are generated DNA synthesis is bi-directional  2 replication forks in opposite directions from origin of replication (6) DNA polymerase I  removes primer and fills gaps that result from RNA deletion. (7) DNA ligases join DNA fragments to form a complete DNA strand DNA replication is extraordinary complex; at least 30 proteins are required to replicate the E. coli chromosome.