L5: Microbial Genetics I (BIO-440) PDF

L5: Microbial genetics I BIO-440 Jan 31st, 2025 By Dr. Anny Cárdenas Assistant Professor, Department of Biology 1 Content Topic 1: Genetic Elements and DNA Replication DNA and Genetic Information Flow Genetic Elements: Chromosomes and Plasmids DNA r...

L5: Microbial genetics I BIO-440 Jan 31st, 2025 By Dr. Anny Cárdenas Assistant Professor, Department of Biology 1 Content Topic 1: Genetic Elements and DNA Replication DNA and Genetic Information Flow Genetic Elements: Chromosomes and Plasmids DNA replication Topic 2: Transcription Transcription in Bacteria Transcription in Archaea Topic 3: Translation Translation and the Genetic Code The Mechanism of Protein Synthesis Content Topic 4: Protein Processing, Secretion, and Targeting Assisted Protein Folding and Chaperones Protein Secretion: The Sec and Tat Systems Protein Secretion: Gram-Negative Systems Topic 5: Microbial Regulatory Systems DNA-Binding Proteins Transcription Factors and Effectors Repression and Activation Central dogma of molecular biology CentraldogmaDNA RNA protein Central dogma of molecular biology DNA Polymearse haveBhymearse Pff replicated ebinds Pairs DNA nappers bosome Single mRNA molecule may encode several different proteins in Bacteria and Archaea  Coupled transcription and translation in prokaryotic cells. A fundamental difference in Bacteria and Archaea vs Eukaryotes is that transcription and translation in can occur simultaneously in a process known as coupled transcription and translation duetononucleus h appenatsame can everything time Genetic elements: Chromosomes and plasmids Genetic elements: Structures containing genetic material (DNA in cells but RNA in some viruses) Chromosomes: Main genetic element in bacteria and archaea, circular, generally one containing most genes. Plasmids: circular, replicate separately from the chromosome. Much smaller. Transposable elements: Sequences of DNA that are inserted into other DNA molecules but can move from one site on the DNA molecule to another, either within the same molecule or on a different DNA molecule (more about this next class!) Chromosomes Most bacterial genomes fall within the 3-5 Mb range. Median size is 3.65 Mb The average bacterium contains around 3,000 genes 4,288 open reading sones frames (genes) Plasmids: Many Bacteria and Archaea contain plasmids in addition to their chromosome(s) carrying accessory genes (nonessential) notessentialtolive About less than 5% of the size of the chromosome IS: Insertion sequences Some bacteria contain several different plasmids May be present in different copy number be could for used resistance antibiotic  Lactococcus lactis produce bacteriocins encoded in plasmids Nisin Modified from: Duarte, S. O., & Monteiro, G. A. (2021). International Journal of Molecular Ismael, M., et al (2024). Applications in Promoting Gastrointestinal Health. Foods, 13(23), 3887. Sciences, 22(3), 1379. DNA Replication at the replication fork continous was stints replicate573 DNAPolymearsecanonly strand soyouneedlaggingeleading Replication of circular DNA iiiiiiiii.is ii w a leadingelaggingstrand Transcription: RNA synthesis from a DNA template mRNAs encode genetic rRNAs play both a tRNAs function as the information from the genome structural and a functional carriers of amino acids to for the synthesis of proteins role in ribosomes the ribosome for protein synthesis RNA polymerases from bacteria have the simplest structure have4subunitsbetheybindto only Sigmafactorerelyonanothite in  Bacterial RNA polymerase and promoter -10 -35 Start of the Element transcription element (Pribnow) Bacterial promoters are recognized by sigma In 351 10boxtheysignalthatthesigma factor seavenceequivalentto shouldbindbeof thepromoter tag is ftp.fphffg  BRE TATA Archaeal promoter ArchaeaRNA Polymearse ismore c omplex to compared bacteria They haveaTata box INIT eTBPeTFB bindunion allows RNA polymease tobindtotranscribetranscription TATA-binding protein (TBP) Transcription factor B (TFB) Transcription in bacteria 1 Sigma binds to the promoter region and then RNA core polymerase binds to begin transcription Transcription in bacteria 2 RNA polymerase moves down the DNA chain, temporarily opening the double helix (creating a transcription bubble) and transcribing one of the DNA strands. Transcription in bacteria 3 Transcription continues until specific sequences called transcription terminators are reached. Transcription in bacteria 4 Polymerase and RNA are released Termination of transcription: Loop formation In bacteria, a common termination signal is a GC-rich sequence containing an inverted repeat with a central nonrepeating segment. ABCD DCBA g Termination of transcription: Rho dependent Rho causes both the RNA and RNA Rhea sniissiseiimite polymerase to be released from the DNA, thus terminating transcription. A ribosomal rRNA transcription unit from Bacteria Operons: cluster of genes under the control of a single promoter that are transcribed together as a single mRNA (polycistronic mRNA) in prokaryotes (bacteria and archaea). A ribosomal rRNA transcription unit from Bacteria Translation AUG: encodes N-formylmethionine at the beginning of polypeptide chains of Bacteria Translation Bacteria and Archaea have 30S and 50S ribosomal subunits 5S and 23S rRNAs and 31 proteins 16S rRNA and 21 proteins Translation Bacteria and Archaea have 30S and 50S ribosomal subunits Initiator tRNA + N-formylmethionine Translation Protein processing, secretion, and targeting 1 Chaperones: catalyze a variety of macromolecular folding events  Protein processing, secretion, and targeting 2 Sec (secretion) systems: exports unfolded proteins across the cytoplasmic membrane or integrates them into the membrane. Most common protein secretion pathway in prokaryotes! Signal sequence: Amino acid sequence or 15-20 residues Protein processing, secretion, and targeting 3 Tat (Twin-Arginine Translocation) system: transports previously folded proteins through the cytoplasmic membrane. E.g. iron–sulfur proteins, cytochromes, and other respiratory enzymes Signal sequence contains a pair of arginine residues Protein processing, secretion, and targeting Gram negative secretion system: Used by gram negative bacteria to 4 insert proteins or other small molecules known as effectors into the outer membrane of other Bacteria, or to secrete them outside of the cell. Known as types I through VI Type I, II, and V: Secrete proteins out of the cell T2SS isthat ABC transporters: are a large family of proteins highly specific move substances across cell membranes. (e.g. Use cholera toxin). Transports from periplasm energy from ATP hydrolysis to move substances to extracellular environment Outer membrane pore T5SS are the simplest structurally. The protein to Membrane fusion protein be secreted is fused to a transmembrane protein bindsdomain essential specifically totothe the Cytoplasmatic membrane protein’s protein to be transport secretedacross and transporter the outer membrane. (E.g. requires ATP to initiate adhesion to host cells ) transport to the outside of the cell. T2SS and T5SS are two-step translocases T1SS systems secrete proteins in a single that depend on either step. E.g. bacteriocin) produced by E. coli the Sec or Tat system  Type I, II, and V: Secrete proteins out of the cell T5SS are the simplest structurally. The protein to be secreted is fused to a transmembrane protein domain essential to the protein’s transport across the outer membrane. (E.g. adhesion to host cells ) T2SS and T5SS are T2SS is highly specific (e.g. two-step translocases cholera toxin). Transports from that depend on either periplasm to extracellular the Sec or Tat system environment Type III, IV and VI: Inject proteins into a host cell T3SS commonly used by pathogenic bacteria to inject these molecules directly into eukaryotic host cells. Composed by over 100 proteins. Also called “injectisome” for its similarity to a syringe in both structure and function. Purified type III injectisomes from Salmonella enterica Type III, IV and VI: Inject proteins into a host cell T4SS are the most common in bacteria and archaea. Its primary role is to transfer DNA to other cells though the process of conjugation Type III, IV and VI: Inject proteins into a host cell T6SS is cytoplasmic and forms a needle-like protein with a pore- forming protein that contracts all the way through the donor cell’s two membranes and directly into a host cell once a substrate molecule is recognized E.g. used by Pseudomonas aeruginosa to inject a toxin into competing bacterial cells. Similarly, Vibrio cholerae uses it to deliver toxins to human intestinal cells during a cholera infection. Microbial Regulatory Systems Transcriptional Translational regulation regulation Transcriptional regulation: DNA-binging proteins DNA sequences that interact with proteins have inverted sequences, which are sequence followed downstream by its inverted complement Transcriptional regulation: DNA-binging proteins Helix-turn-helix structures: consists of two segments of polypeptide chain that have an - helix secondary structure connected by a short sequence the recognition helix interacts with DNA and the stabilizing helix stabilizes forming the “turn. the first helix by interacting with it by way of hydrophobic interactions Transcriptional regulation: Transcription factors Transcription factors: Proteins that control the rate of gene transcription by binding to specific DNA sequences Tactile no Repressor Activator  Transcriptional regulation: Effector molecules Effectors: Control transcription factors. Typically, cell metabolites such as substrates or end products of the pathways that they regulate. repressor Repressor finds Corepressor operator to binds of Activator toactivator binds + Inducer Transcriptional regulation: Repression A product of the biosynthetic pathway is the effector molecule Growth continues but production of the (corepressor) for that pathway enzymes for arginine synthesis stops. abscenciatne operator binds first binds opfa.gg activatesrepressor Transcriptional regulation: Induction (derepression) The addition of lactose to the medium An enzyme is made only when its substrate is present specifically induces synthesis of the enzyme iii Transcriptional regulation: Activation Some operons are transcribed only if a specific activator protein is first bound to the DNA An enzyme is made only when its substrate is present signal sends stiff tobetoo common if Transcriptional regulation: Activation a j Regulons: A series of operons controlled as a unit